ANTI-BTLA ANTIBODY PHARMACEUTICAL COMPOSITION AND USE THEREOF
20230192854 · 2023-06-22
Inventors
- Hongchuan Liu (Suzhou, CN)
- Peixiang Liu (Suzhou, CN)
- Jing ZHANG (Suzhou, CN)
- Yuehua Zhou (Suzhou, CN)
- Hui Liu (Suzhou, CN)
- Xueru Chen (Suzhou, CN)
- Jing Wang (Suzhou, CN)
- Hui Feng (Suzhou, CN)
- Sheng YAO (Suzhou, CN)
Cpc classification
A61P29/00
HUMAN NECESSITIES
A61P31/00
HUMAN NECESSITIES
A61K47/22
HUMAN NECESSITIES
C07K2317/94
CHEMISTRY; METALLURGY
C07K2317/33
CHEMISTRY; METALLURGY
A61K47/26
HUMAN NECESSITIES
C07K2317/73
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
A61P37/06
HUMAN NECESSITIES
C07K2317/71
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C07K16/28
CHEMISTRY; METALLURGY
A61K47/22
HUMAN NECESSITIES
A61K47/26
HUMAN NECESSITIES
Abstract
The present invention provides a stable pharmaceutical composition comprising an anti-BTLA (B and T lymphocyte attenuator) antibody and use thereof in medicines. The pharmaceutical composition comprises an anti-BTLA antibody and a buffer, further comprises at least one stabilizer, and optionally further comprises a surfactant.
Claims
1. A pharmaceutical composition, comprising: (1) a buffer; and (2) an anti-BTLA antibody or an antigen-binding fragment thereof, wherein the anti-BTLA antibody or the antigen-binding fragment thereof has an HCDR1, an HCDR2 and an HCDR3 having amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and an LCDR1, an LCDR2 and an LCDR3 having amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
2. The pharmaceutical composition according to claim 1, wherein the buffer is selected from an acetic acid buffer, a citric acid buffer and a histidine buffer.
3. The pharmaceutical composition according to claim 2, wherein the buffer has a concentration of about 10 mM to 30 mM, and/or the buffer has a pH of about 5.0-6.5.
4. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises a stabilizer selected from one or more of sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol and trehalose.
5. The pharmaceutical composition according to claim 4, wherein the stabilizer is sodium chloride at a concentration of about 50-200 mM; or the stabilizer is mannitol at a concentration of about 100-300 mM; or the stabilizer is sorbitol at a concentration of about 100-300 mM; or the stabilizer is sucrose at a concentration of about 100-300 mM; or the stabilizer is trehalose at a concentration of about 100-300 mM; or the stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM mannitol; or the stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM sorbitol; or the stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM sucrose; or the stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM trehalose.
6. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises a surfactant selected from polysorbate 80, polysorbate 20 and poloxamer 188.
7. The pharmaceutical composition according to claim 6, wherein the surfactant has a concentration of about 0.01%-0.1%.
8. The pharmaceutical composition according to claim 1, wherein the anti-BTLA antibody has a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region having an amino acid sequence set forth in SEQ ID NO: 8; or has a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region having an amino acid sequence set forth in SEQ ID NO: 9; or has a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region having an amino acid sequence set forth in SEQ ID NO: 10.
9. The pharmaceutical composition according to claim 1, wherein the anti-BTLA antibody has a heavy chain amino acid sequence set forth in SEQ ID NO: 11 and a light chain amino acid sequence set forth in SEQ ID NO: 12.
10. The pharmaceutical composition according to claim 1, wherein the anti-BTLA antibody or the antigen-binding fragment thereof has a concentration of about 5-100 mg/mL.
11. The pharmaceutical composition according to claim 1, comprising or consisting of components as shown in any one of (1) to (13) below: (1) (a) about 10 mg/mL to 50 mg/mL of the anti-BTLA antibody or the antigen-binding fragment thereof; (b) about 10-30 mM histidine buffer at a pH of about 5.0-6.0; (c) a combination of about 30 mM to about 100 mM sodium chloride and about 50-200 mM trehalose; and (d) about 0.01%-0.05% of polysorbate 80; (2) (a) about 10 mg/mL to 50 mg/mL of the anti-BTLA antibody or the antigen-binding fragment thereof; (b) about 10-30 mM histidine buffer at a pH of about 5.0-6.0; (c) a combination of about 30 mM to about 100 mM sodium chloride and about 50-200 mM mannitol; and (d) about 0.01%-0.05% of polysorbate 80; (3) (a) about 10 mg/mL to 50 mg/mL of the anti-BTLA antibody or the antigen-binding fragment thereof; (b) about 10-30 mM histidine buffer at a pH of about 5.0-6.0; (c) a combination of about 30 mM to about 100 mM sodium chloride and about 50-200 mM sucrose; and (d) about 0.01%-0.05% of polysorbate 80; (4) (a) about 10 mg/mL to 50 mg/mL of the anti-BTLA antibody or the antigen-binding fragment thereof; (b) about 10-30 mM histidine buffer at a pH of about 5.0-6.0; (c) a combination of about 30 mM to about 100 mM sodium chloride and about 50-200 mM sorbitol; and (d) about 0.01%-0.05% of polysorbate 80; (5) (a) about 10 mg/mL to 50 mg/mL of the anti-BTLA antibody or the antigen-binding fragment thereof; (b) about 10-30 mM histidine buffer at a pH of about 5.0-6.0; (c) about 100 mM to about 300 mM trehalose; and (d) about 0.01%-0.05% of polysorbate 80; (6) (a) about 10 mg/mL to 50 mg/mL of the anti-BTLA antibody or the antigen-binding fragment thereof; (b) about 10-30 mM citric acid buffer at a pH of about 5.5-6.5; (c) about 100 mM to about 300 mM trehalose; and (d) about 0.01%-0.05% of polysorbate 80; (7) (a) about 10 mg/mL to 50 mg/mL of the anti-BTLA antibody or the antigen-binding fragment thereof; (b) about 10-30 mM citric acid buffer at a pH of about 5.5-6.5; (c) a combination of about 30 mM to about 100 mM sodium chloride and about 50-200 mM mannitol; and (d) about 0.01%-0.05% of polysorbate 80; (8) (a) about 10 mg/mL to 50 mg/mL of the anti-BTLA antibody or the antigen-binding fragment thereof; (b) about 10-30 mM acetic acid buffer at a pH of about 5.0-6.0; (c) a combination of about 30 mM to about 100 mM sodium chloride and about 50-200 mM mannitol; and (d) about 0.01%-0.05% of polysorbate 80; (9) (a) about 20 mg/mL of the anti-BTLA antibody or the antigen-binding fragment thereof; (b) about 20 mM histidine buffer at a pH of about 6.0; (c) a combination of about 54 mM sodium chloride and about 132 mM trehalose; and (d) about 0.02% of polysorbate 80; (10) (a) about 20 mg/mL of the anti-BTLA antibody or the antigen-binding fragment thereof; (b) about 20 mM histidine buffer at a pH of about 5.5; (c) about 220 mM trehalose; and (d) about 0.02% of polysorbate 80; (11) (a) about 20 mg/mL of the anti-BTLA antibody or the antigen-binding fragment thereof; (b) about 20 mM histidine buffer at a pH of about 5.5; (c) a combination of about 50 mM sodium chloride and about 140 mM mannitol; and (d) about 0.02% of polysorbate 80; (12) (a) about 20 mg/mL of the anti-BTLA antibody or the antigen-binding fragment thereof; (b) about 20 mM histidine buffer at a pH of about 5.5; (c) a combination of about 50 mM sodium chloride and about 140 mM trehalose; and (d) about 0.02% of polysorbate 80; and (13) (a) about 20 mg/mL of the anti-BTLA antibody or the antigen-binding fragment thereof; (b) about 20 mM histidine buffer at a pH of about 5.0-6.0; (c) a combination of about 50 mM sodium chloride and about 140 mM trehalose; and (d) about 0.02% of polysorbate 80.
12. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises or consists of: a histidine-histidine hydrochloride buffer at a pH of 5.5±0.3 and with a concentration of 20±5 mM, 40-80 mM sodium chloride, 120-150 mM trehalose, 0.02-0.04% (w/v) of polysorbate 80, and 15-25 mg/mL of the anti-BTLA antibody or the antigen-binding fragment thereof; wherein the anti-BTLA antibody has a heavy chain amino acid sequence set forth in SEQ ID NO: 11 and a light chain amino acid sequence set forth in SEQ ID NO: 12.
13. (canceled)
14. The pharmaceutical composition according to claim 2, wherein the buffer is a histidine-hydrochloride buffer, or is a histidine-histidine hydrochloride buffer.
15. The pharmaceutical composition according to claim 3, wherein the buffer has a pH of about 5.0-6.0.
16. The pharmaceutical composition according to claim 4, wherein the stabilizer is a combination of trehalose and sodium chloride.
17. The pharmaceutical composition according to claim 7, wherein the surfactant has a concentration of about 0.01%-0.05%.
18. The pharmaceutical composition according to claim 10, wherein the anti-BTLA antibody or the antigen-binding fragment thereof has a concentration of about about 10-50 mg/mL or has a concentration of 15-25 mg/mL.
19. A method for treating or preventing BTLA-mediated diseases comprising administering to a subject or patient in need thereof a therapeutically effective amount of the pharmaceutical composition according to claim 1.
20. The method according to claim 19, wherein the diseases are tumors, infectious diseases or autoimmune diseases.
21. The method according to claim 20, wherein the tumors include melanoma, breast cancer, renal cancer, prostate cancer, colon cancer, lung cancer, pancreatic cancer, bone cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, small intestine cancer, cervical cancer, vaginal cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, cancer of the endocrine system, thyroid cancer, carcinoma of adrenal gland, soft tissue cancer, urethral cancer, chronic or acute leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumor, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, neoplasms of the central nervous system, primary central nervous system lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma and environmentally induced cancers; the autoimmune diseases include organ-specific autoimmune diseases and systemic autoimmune diseases, wherein the organ-specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis; and the systemic autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia and ulcerative colitis.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0064]
[0065]
[0066]
DEFINITIONS AND DESCRIPTION
[0067] In order to facilitate the understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. It should be understood that the present invention is not limited to particular methods, agents, compounds, compositions or biological systems, which can, of course, be modified. It should also be understood that the terms used herein are for the purpose of illustrating particular embodiments only, and are not intended to be limiting. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents, unless otherwise specified. Thus, for example, reference to “a polypeptide” includes a combination of two or more polypeptides and the like.
[0068] The term “pharmaceutical composition” or “formulation” refers to a mixture containing one or more compounds described herein or a pharmaceutically acceptable salt or prodrug thereof, and other components, such as physiologically acceptable carriers and excipients. The pharmaceutical composition is intended to promote the administration to an organism and facilitate the absorption of the active ingredient, thereby exerting bioactivity.
[0069] The term “liquid formulation” refers to a formulation in liquid state and is not intended to refer to a resuspended lyophilized formulation. The liquid formulation of the present invention is stable during storage and its stability is independent of lyophilization (or other state change methods, such as spray drying).
[0070] The term “aqueous formulation” refers to a liquid formulation using water as the solvent. In some embodiments, the aqueous formulation is a formulation that requires no lyophilization, spray drying and/or freezing to maintain its stability (e.g., chemical and/or physical stability and/or bioactivity).
[0071] The term “excipient” refers to an agent that may be added to a formulation to provide a desired property (e.g., consistency and improved stability) and/or to adjust osmotic pressure. Examples of commonly used excipients include, but are not limited to, sugars, polyols, amino acids, surfactants and polymers. As used herein, when referring to measurable values (e.g., amounts and durations), “about” is intended to encompass variations of ±20% or ±10% based on the particular value, including ±5%, ±1% and ±0.1%, as such variations are suitable for implementing the disclosed methods.
[0072] The term “buffer at a pH of about 5.0-6.5” refers to such an agent that, through the action of its acid/base conjugate components, renders a solution containing the agent resistant to pH changes. The buffer used in the formulation of the present invention may have a pH in a range of about 5.0 to about 6.5, or a pH in a range of about 5.5 to about 6.5, or a pH in a range of about 5.0 to about 6.0.
[0073] Examples of “buffer” that control the pH within this range herein include acetate (e.g., sodium acetate), succinate (e.g., sodium succinate), gluconic acid, histidine, histidine hydrochloride, methionine, citrate, phosphate, citrate/phosphate, imidazole, acetic acid, acetate, citrate and a combination thereof, and other organic acid buffers.
[0074] The “histidine buffer” is a buffer containing histidine ions. Examples of histidine buffer include histidine and a salt of histidine such as histidine hydrochloride, histidine acetate, histidine phosphate and histidine sulfate, for example, a histidine buffer containing histidine and histidine hydrochloride; the histidine buffer of the present invention also includes a histidine buffer containing histidine and acetate (e.g., sodium salt or potassium salt).
[0075] The “citrate buffer” is a buffer containing citrate ions. Examples of citrate buffer include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate and the like. The preferred citrate buffer is a citric acid-sodium citrate buffer.
[0076] The “acetate buffer” is a buffer containing acetate ions. Examples of acetate buffer include acetic acid-sodium acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate and the like. The preferred acetate buffer is an acetic acid-sodium acetate buffer.
[0077] The term “stabilizer” refers to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during manufacture, storage and use. The stabilizers include, but are not limited to, sugars, amino acids, salts, polyols and metabolites thereof as defined below, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, arginine, glycine, alanine (α-alanine, β-alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ-aminobutyric acid (GABA), opines (alanopine, octopine, strombine) and trimethylamine N-oxide (TMAO), human serum albumin (hsa), bovine serum albumin (bsa), α-casein, globulin, α-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin and RNAaseA. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol and sucrose, may also serve to control osmotic pressure. The stabilizer specifically used in the present invention is selected from one, two and more of polyols, salts and sugars. The preferred salts are sodium chloride, the preferred sugars are sucrose and trehalose, and the preferred polyols are sorbitol and mannitol. The preferred stabilizers are sodium chloride, mannitol, sorbitol, sucrose, trehalose, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose and sodium chloride-trehalose, more preferably sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose and sodium chloride-trehalose, more preferably sodium chloride-trehalose.
[0078] The term “surfactant” generally includes agents that protect proteins, such as antibodies, from gas/liquid interface-induced stress and liquid/solid-induced stress to reduce aggregation of the antibodies or minimize the formation of particulate matters in the formulation. Exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycol, polyoxyethylene stearate, polyoxyethylene alkyl ethers such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamers, Pluronics) and sodium dodecyl sulfate (SDS).
[0079] The term “isotonic” refers to a formulation having substantially equivalent osmotic pressure to human blood. An isotonic formulation generally has an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured by a vapor pressure osmometer or cryoscopic osmometer.
[0080] The term “stable” formulation is a formulation in which the antibody substantially retains its physical and/or chemical stability and/or bioactivity during the manufacturing process and/or upon storage. A pharmaceutical formulation can be stable even if the contained antibody fails to retain 100% of its chemical structure or biological function after a certain period of storage. In certain instances, a pharmaceutical formulation that retains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of antibody structure or function after a certain period of storage may also be considered “stable”. Various analytical techniques for measuring protein stability are available in the art and are described in Peptide and Protein Drug Delivery, 247-301, Vincent Lee ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A., (1993) Adv. Drug Delivery Rev. , 10:29-90, both of which are incorporated herein for reference.
[0081] After a certain period of storage at a certain temperature, the stability of the formulation can be measured by determining the percentage of remaining native antibody (and by other methods). Except other methods, the percentage of native antibody can be measured by size-exclusion chromatography (e.g., size-exclusion high-performance liquid chromatography (SEC-HPLC)), and “native” refers to unaggregated and undegraded. In some embodiments, the stability of a protein is determined by a percentage of monomeric protein in a solution having a low percentage of degraded (e.g., fragmented) and/or aggregated protein. In some embodiments, the formulation is stable at room temperature, about 25-30° C., or 40° C. for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or longer, and the formulation has no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of antibody in aggregated form.
[0082] Stability can be measured by determining the percentage of migrating antibody in a fraction that is more acidic (“acidic form”) than the main fraction of the antibody (“main charged form”) during ion exchange (and can be measured by other methods), wherein the stability is inversely proportional to the percentage of acidic form of antibody. The percentage of “acidified” antibody can be measured by, except other methods, ion exchange chromatography (e.g., cation exchange high-performance liquid chromatography (CEX-HPLC)). In some embodiments, an acceptable stability means that the acidic form of the antibodies that can be detected in the formulation is no more than about 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% after a certain period of storage at a certain temperature. The certain period of storage prior to measuring stability can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. When the stability is assessed, the certain temperature at which the pharmaceutical formulation is allowed to be stored may be any temperature in a range of about −80° C. to about 45° C., e.g., at about −80° C., about −30° C., about −20° C., about 0° C., about 2-8° C., about 5° C., about 25° C. or about 40° C.
[0083] An antibody in pharmaceutical composition “retains its physical stability” if the antibody does not substantially show signs of, such as aggregation, precipitation and/or denaturation, during visual inspection of color and/or clarity or by UV light scattering or measurement by pore-exclusion chromatography. Aggregation is a process in which individual molecules or complexes associate, covalently or non-covalently, to form aggregates. Aggregation can proceed to the point where a visible precipitate is formed.
[0084] Stability, e.g., physical stability, of a formulation can be assessed by methods well known in the art, including measuring the apparent extinction (absorbance or optical density) of a sample. Such extinction measurement correlates with the turbidity of the formulation. Turbidity of a formulation is, in part, an inherent property of proteins dissolved in a solution and is generally measured by nephelometry and is measured in nephelometric turbidity unit (NTU).
[0085] Turbidity levels that vary with, for example, the concentration of one or more components in a solution (e.g., protein and/or salt concentration) are also referred to “opacification” or “opacified appearance” of a formulation. Turbidity levels can be calculated with reference to a standard curve based on suspensions of known turbidity. The reference standards for determining the turbidity level of a pharmaceutical composition may be based on the standard of the European Pharmacopoeia, 4.sup.th edition, Directorate for the Quality of Medicine of the Council of Europe (EDQM), Strasbourg, France. According to the standard of European Pharmacopoeia, a clear solution is defined as a solution having a turbidity lower than or equal to that of a reference suspension according to the standard of European Pharmacopoeia having a turbidity of about 3. Turbidity measurement by nephelometry can measure Rayleigh scattering in the absence of association or non-ideal effects, which generally varies linearly with concentration. Other methods for assessing physical stability are well known in the art.
[0086] An antibody “retains its chemical stability” in a pharmaceutical composition if the chemical stability of the antibody at a given time point allows the antibody to retain its bioactivity as defined hereinafter. Chemical stability can be assessed, for example, by detecting or quantifying the form of chemical changes in the antibody. Chemical changes can include size changes (e.g., truncation), which can be assessed by, for example, size-exclusion chromatography, SDS-PAGE and/or matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI/TOF MS). Other types of chemical changes include charge changes (e.g., occurring as a result of deamidation or oxidation), which can be assessed by, for example, ion exchange chromatography.
[0087] An antibody in a pharmaceutical composition “retains its bioactivity” if it is biologically active for its intended purpose. For example, the formulation described herein is considered stable if, after a certain period of storage (e.g., 1 to 12 months) at a certain temperature, e.g., 5° C., 25° C. or 45° C., the binding affinity of the anti-BTLA antibody in the formulation for BTLA is at least 90%, 95% or greater of that of the antibody prior to storage. Binding affinity can also be determined by, for example, ELISA or plasmon resonance.
[0088] In the context of the present invention, a “therapeutically effective amount” or “effective amount” of an antibody, pharmacologically refers to an amount that is effective in preventing, treating or alleviating the symptoms of a disorder that the antibody can effectively treat. In the present invention, “therapeutically effective amount” or “therapeutically effective dose” of a medicament is any amount of the medicament that, when used alone or in combination with an additional therapeutic agent, protects a subject from the onset of a disease or promotes the regression of a disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free phase, or the prevention of injury or disability resulting from the affliction of the disease. The ability of a medicament to promote the regression of a disease can be assessed using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems that predict human efficacy, or by determining the activity of the agent in an in vitro assay. A therapeutically effective amount of a medicament includes a “prophylactically effective amount” which is any amount of a medicament that, when administered alone or in combination with other therapeutic drugs to a subject at risk of developing diseases or a subject having disease recurrence, inhibits the development or recurrence of diseases.
[0089] The term “subject” or “patient” is intended to include mammalian organisms. Examples of subjects/patients include human and non-human mammals such as non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats and transgenic non-human animals. In one specific embodiment of the present invention, the subject is human.
[0090] The terms “administering”, “giving” and “treating” refer to introducing a composition comprising a therapeutic agent into a subject using any one of a variety of methods or delivery systems known to those skilled in the art. Routes of administration of the anti-PD-1 antibody include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as injection or infusion. “Parenteral administration” refers to modes of administration apart from enteral or local administration, typically by injection, including but not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation.
[0091] Anti-BTLA Antibody
[0092] The term “antibody” as used herein should be construed including intact antibody molecules and antigen-binding fragments thereof. The term “antigen-binding moiety” or “antigen-binding fragment” of an antibody (or simply “antibody moiety” or “antibody fragment”) as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to human BTLA (B and T lymphocyte attenuator) or an epitope thereof.
[0093] The term “full-length antibody” or “intact antibody molecule” as used herein refers to an immunoglobulin molecule comprising four peptide chains, including two heavy (H) chains (about 50-70 kDa in total length) and two light (L) chains (about 25 kDa in total length) linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of 3 domains CHL CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further divided into complementarity determining regions (CDRs) with high variability and more conservative regions called framework regions (FRs) that are spaced apart by the CDRs. Each VH or VL region consists of 3 CDRs and 4 FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including the binding of various cells of the immune system (e.g., effector cells) to the first component (Clq) of classical complement system.
[0094] As used herein, the term “CDR” refers to the complementarity determining regions within an antibody variable sequence. There are 3 CDRs in each variable region of the heavy and light chains, which are named HCDR1, HCDR2 and HCDR3, and LCDR1, LCDR2 and LCDR3 for heavy and light chain variable regions. Exact boundaries of the CDRs may vary among different systems.
[0095] The precise amino acid sequence boundaries of the variable region CDRs of the antibodies disclosed herein can be determined using any of a number of well-known schemes, including Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al., (1989) Nature 342:877-883; Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273:927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4.sup.th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (1999 Nucleic Acids Research, 27, 209-212), and North CDR definition based on the affinity propagation clustering using a large number of crystal structures. The boundaries of the CDRs of the antibodies disclosed herein can be determined by one skilled in the art according to any scheme (e.g., different assignment systems or combinations) in the art.
[0096] “Antigen-binding fragment” as used herein includes an antibody fragment or a derivative thereof, generally including at least one fragment of an antigen-binding region or variable region (e.g., one or more CDRs) of a parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab′, F(ab′).sub.2 and Fv fragments; a diabody; a linear antibody; a single-chain antibody molecule, such as sc-Fv; a nanoclay and multispecific antibody formed by antibody fragments. A binding fragment or a derivative thereof generally retains at least 10% of the antigen-binding activity of the parent antibody when the binding activity of the antibody is expressed on a molar concentration basis. Preferably, the binding fragment or the derivative thereof retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen-binding affinity of the parent antibody. It is also contemplated that an antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly alter its bioactivity (referred to as “conservative variants” or “function-conservative variants” of the antibody).
[0097] The anti-BTLA antibody or the antigen-binding fragment thereof described herein include any of the anti-BTLA antibody described in application No. CN201810870514.0, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the CDR sequences of the antibody used in the methods and compositions of the present invention comprise the CDR sequences of antibody hu18 described in CN201810870514.0. In some embodiments, the CDR sequences of the antibody used in the methods and compositions of the present invention comprise the CDR sequences of antibody hu17 described in CN201810870514.0. In some embodiments, the CDR sequences of the antibody used in the methods and compositions of the present invention comprise the CDR sequences of antibody hul9 described in CN201810870514.0.
[0098] The non-limiting and exemplary antibody used in the examples herein is selected from hu17, hu18 and hu19 described in CN201810870514.0, which are all full humanized antibodies that specifically bind to human BTLA, wherein antibodies hu17, hu18 and hu19 all have amino acid sequences an HCDR1, an HCDR2 and an HCDR3 set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and amino acid sequences an LCDR1, an LCDR2 and an LCDR3 set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; preferably, antibody hul7 has a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region having an amino acid sequence set forth in SEQ ID NO: 8; the antibody hu18 has a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region having an amino acid sequence set forth in SEQ ID NO: 9; the antibody hu19 has a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region having an amino acid sequence set forth in SEQ ID NO: 10; preferably, antibody hu18 has a heavy chain amino acid sequence set forth in SEQ ID NO: 11 and a light chain amino acid sequence set forth in SEQ ID NO: 12. The CDR amino acid sequences of the above humanized antibody are defined by the IMGT system.
[0099] Pharmaceutical Formulation
[0100] The pharmaceutical composition provided by the present invention is a highly stable pharmaceutical composition containing a humanized antibody specifically binding to BTLA. In particular, the present inventors have found that the combination of trehalose with sodium chloride may significantly improve the stability of the pharmaceutical composition.
[0101] The present invention provides a pharmaceutical composition comprising: (1) a buffer; and (2) an anti-BTLA antibody or an antigen-binding fragment thereof.
[0102] The antibody in the pharmaceutical composition of the present invention may be a murine antibody, a chimeric antibody and a humanized antibody, preferably a humanized antibody, and may have an HCDR1, an HCDR2 and an HCDR3 set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and an LCDR1, an LCDR2 and an LCDR3 set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. Preferably, the antibody in the pharmaceutical composition of the present invention has a heavy chain variable region set forth in SEQ ID NO: 7 and a light chain variable region set forth in SEQ ID NO: 8; or has a heavy chain variable region set forth in SEQ ID NO: 7 and a light chain variable region set forth in SEQ ID NO: 9; or has a heavy chain variable region set forth in SEQ ID NO: 7 and a light chain variable region set forth in SEQ ID NO: 10; more preferably, the antibody in the pharmaceutical composition of the present invention has a heavy chain amino acid sequence set forth in SEQ ID NO: 11 and a light chain amino acid sequence set forth in SEQ ID NO: 12.
[0103] The anti-BTLA antibody or the antigen-binding fragment thereof in the pharmaceutical composition of the present invention has a concentration of about 1-200 mg/mL, preferably about 5-100 mg/mL, preferably about 10-50 mg/mL, and more preferably 15-25 mg/mL. As non-limiting examples, the anti-BTLA antibody or the antigen-binding fragment thereof has a concentration of about 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL or 50 mg/mL; preferably about 10 mg/mL, 20 mg/mL or 40 mg/mL.
[0104] The buffer in the pharmaceutical composition of the present invention may be selected from an acetic acid buffer, a citric acid buffer and a histidine buffer to provide a pH of 5.0 to 6.5, preferably 5.0 to 6.0, to the pharmaceutical composition of the present invention. In another aspect, the buffer used in the pharmaceutical composition of the present invention may have a pH of 5.0 to 6.5, preferably 5.0 to 6.0.
[0105] A particularly preferred buffer in the pharmaceutical composition of the present invention is a histidine buffer. Preferably, the histidine buffer used in the present invention has a pH of 5.0 to 6.0, more preferably 5.5 ±0.3, preferably about 5.5. Preferably, the histidine buffer comprises a histidine-hydrochloride buffer or a histidine-acetate buffer, preferably a histidine-hydrochloride buffer. More preferably, the histidine-hydrochloride buffer consists of histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. In some embodiments, the histidine buffer is prepared by 1-20 mM L-histidine and 1-20 mM L-histidine monohydrochloride. In some embodiments, the histidine buffer consists of histidine and histidine hydrochloride in a molar ratio of 1:1-1:4. In some embodiments, the histidine buffer consists of histidine and histidine hydrochloride in a molar ratio of 1:1. In some embodiments, the histidine buffer consists of histidine and histidine hydrochloride in a molar ratio of 1:3. In some embodiments, the histidine formulation is a histidine buffer at pH 5.5 prepared by 4.5 mM L-histidine and 15.5 mM L-histidine monohydrochloride. In some embodiments, the histidine formulation is a histidine buffer at pH 6.0 prepared by 15 mM L-histidine and 15 mM L-histidine monohydrochloride.
[0106] The histidine buffer in the pharmaceutical composition of the present invention may be a histidine-acetic acid buffer preferably in a molar ratio of 1:1 to 1.5:1; preferably such a buffer has a pH of 5.5 ±0.3, preferably about 5.5; preferably such a buffer contains 15-20 mM histidine and 12-15 mM acetic acid.
[0107] Accordingly, the pharmaceutical composition of the present invention may comprise: a histidine-histidine hydrochloride buffer at a pH of 5.0-6.0 and with a concentration of 10-30 mM in the pharmaceutical composition; and 15-25 mg/mL of the anti-BTLA antibody or the antigen-binding fragment thereof of any one of the embodiments above, particularly hu17, hu18, or hu19 or antigen-binding fragment thereof described herein.
[0108] In some embodiments, the pharmaceutical composition of the present invention further comprises a stabilizer. Preferably, the stabilizer is selected from one or more of sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol and trehalose. Preferably, the stabilizer in the pharmaceutical composition comprises at least sodium chloride, optionally one or more of mannitol, sorbitol, sucrose and trehalose. For example, the pharmaceutical composition may comprise sodium chloride and mannitol, sodium chloride and sorbitol, sodium chloride and sucrose, or sodium chloride and trehalose. When comprising the above compositions, the stabilizer in the pharmaceutical composition has a concentration of about 20 mM to 300 mM, preferably 50 mM to 300 mM, and more preferably 120 mM to 250 mM. In some embodiments, the stabilizer is sodium chloride at a concentration of about 50-200 mM; or the stabilizer is mannitol at a concentration of about 100-300 mM; or the stabilizer is sorbitol at a concentration of about 100-300 mM; or the stabilizer is sucrose at a concentration of about 100-300 mM; or the stabilizer is trehalose at a concentration of about 100-300 mM; or the stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM mannitol; the stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM sorbitol; the stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM sucrose; the stabilizer is a combination of about 30-100 mM sodium chloride and about 50-200 mM trehalose.
[0109] Thus, in some embodiments, the pharmaceutical composition of the present invention comprises: a histidine-histidine hydrochloride buffer at a pH of 5.0-6.0 and with a concentration of 10-30 mM in the pharmaceutical composition; 15-25 mg/mL of the anti-BTLA antibody or the antigen-binding fragment thereof of any one of the embodiments, particularly hu17, hu18, or hu19 or antigen-binding fragment thereof described herein; and 20-300 mM stabilizer, preferably, the stabilizer at least comprises sodium chloride, optionally comprises one of mannitol, sorbitol, sucrose and trehalose, preferably 50-150 mM sodium chloride, or 40-80 mM sodium chloride and 120-150 mM mannitol, or 40-80 mM sodium chloride and 120-150 mM sorbitol, or 40-80 mM sodium chloride and 120-150 mM sucrose, or 40-80 mM sodium chloride and 120-150 mM of trehalose. In some embodiments, the stabilizer is 200-300 mM trehalose.
[0110] In some embodiments, the pharmaceutical composition of the present invention further comprises a surfactant. The preferred surfactant is selected from polysorbate 80, polysorbate 20 and poloxamer 188. The most preferred surfactant is polysorbate 80. The surfactant in the pharmaceutical composition of the present invention has a concentration of about 0.001%-0.1%, preferably about 0.01%-0.05%, and preferably about 0.02%-0.04% (w/v). As non-limiting examples, the surfactant in the pharmaceutical composition of the present invention has a concentration of about 0.02%, 0.03% or 0.04%, preferably 0.02%.
[0111] Thus, in some embodiments, the pharmaceutical composition of the present invention comprises: a histidine-histidine hydrochloride buffer at a pH of 5.0-6.0 and with a concentration of 10-30 mM in the pharmaceutical composition; 15-25 mg/mL of the anti-BTLA antibody or the antigen-binding fragment thereof of any one of the embodiments, particularly hu17, hu18, or hul9 or antigen-binding fragment thereof described herein; 20-300 mM stabilizer, preferably, the stabilizer is trehalose, or at least comprises sodium chloride, optionally comprises one of mannitol, sorbitol, sucrose and trehalose, preferably 200-300 mM trehalose, or 50-150 mM sodium chloride, or 40-80 mM sodium chloride and 120-150 mM mannitol, or 40-80 mM sodium chloride and 120-150 mM sorbitol, or 40-80 mM sodium chloride and 120-150 mM sucrose, or 40-80 mM sodium chloride and 120-150 mM trehalose; and 0.02%-0.04% (w/v) of polysorbate 80. In a particularly preferred embodiment, the pharmaceutical composition of the present invention comprises or consists of: a histidine-histidine hydrochloride buffer at a pH of 5.5 ±0.3 and with a concentration of 20±5 mM; 40-80 mM sodium chloride; 120-150 mM trehalose, 0.02-0.04% (w/v) of polysorbate 80; 15-25 mg/mL of an antibody hu18 or an antigen-binding fragment thereof.
[0112] The pharmaceutical composition of the present invention may be a liquid formulation or a lyophilized formulation. It should be understood that the liquid formulation, in addition to the buffers, stabilizers, antibodies or antigen-binding fragments thereof and surfactants described herein, also comprises water for formulating the pharmaceutical composition.
[0113] Medical Uses and Methods
[0114] The present invention also provides the pharmaceutical composition according to any embodiment of the present invention for use in treating or preventing BTLA-mediated diseases, use of the pharmaceutical composition according to any embodiment of the present invention in the preparation of a medicament for treating or preventing BTLA-mediated diseases, and a method for administering a therapeutically effective amount of the pharmaceutical composition according to any embodiment of the present invention to a subject or patient in need thereof to treat or prevent BTLA-mediated diseases.
[0115] In the present invention, the BTLA-mediated diseases refer to diseases in which BTLA is involved in the development and progression of diseases, including, but not limited to, tumors, infectious diseases, and inflammatory or autoimmune diseases. In the present invention, tumors suitable for treatment and prevention with the pharmaceutical composition of the present invention include melanoma, breast cancer, renal cancer, prostate cancer, colon cancer, lung cancer, pancreatic cancer, bone cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, small intestine cancer, cervical cancer, vaginal cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, cancer of the endocrine system, thyroid cancer, carcinoma of adrenal gland, soft tissue cancer, urethral cancer, chronic or acute leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumor, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, neoplasms of the central nervous system, primary central nervous system lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of the cancers. The autoimmune diseases suitable for treatment and prevention with the pharmaceutical composition of the present invention include organ-specific autoimmune diseases and systemic autoimmune diseases; the organ-specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis and the like; and the systemic autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, ulcerative colitis and the like.
DETAILED DESCRIPTION
[0116] The present invention will be illustrated hereinafter by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. The methods and materials used in the examples are, unless otherwise indicated, conventional in the art.
EXAMPLE 1
Buffer System and pH Selection
[0117] In a liquid pharmaceutical composition, the buffer system and pH closely affect the stability of the antibody, and each antibody having unique physicochemical properties may have an optimum buffer and pH. This example is intended to select an optimal buffer system and pH to provide the anti-BTLA antibody disclosed herein with optimal stability for clinical application.
[0118] This example was performed with antibody hu18 at concentrations of about 10 mg/mL, 20 mg/mL and 40 mg/mL. Buffer concentration and exchange by ultrafiltration was performed on the samples using a VIVAFlow200, the samples after buffer exchange were added into a corresponding formula, and the samples were sealed in a centrifuge tube for buffer selection. An acetic acid buffer, a citric acid buffer and a histidine buffer were selected at a pH from 5.0 to 6.5 (as shown in Table 1). The samples were placed at 40° C. and removed for analysis at weeks 0, 2 and 4. The major degradation pathways of the protein were the formations of aggregates, cleavage products and charged variants. The percentages of native (protein monomer) and aggregated protein monomer were determined by size exclusion high-performance liquid chromatography (SEC-HPLC) and the percentages of acidic and basic antibody were determined by cation exchange high-performance chromatography (CEX-HPLC). The effect of different buffer systems and pH on the antibody hu18 stability was examined at baseline (0 w), two weeks (2 w) and four weeks (4 w). By linear fitting, straight lines were plotted according to the SEC-HPLC monomer content and the CEX-HPLC main peak content and the slopes (%/week) were calculated. The results are summarized in Table 2 and Table 3.
TABLE-US-00001 TABLE 1 Formula for buffer system and pH selection Formula No. pH Buffer system Excipient 1 Excipient 2 1 5.0 20 mM acetic acid buffer (acetic acid- 50 mM sodium 140 mM sodium acetate) chloride mannitol 2 5.0 20 mM acetic acid buffer (acetic acid- 140 mM sodium — sodium acetate) chloride 3 5.5 20 mM histidine buffer 50 mM sodium 140 mM (histidine-acetic acid) chloride mannitol 4 5.5 20 mM histidine buffer 140 mM sodium — (histidine-acetic acid) chloride 5 5.5 20 mM acetic acid buffer (acetic acid- 50 mM sodium 140 mM sodium acetate) chloride mannitol 6 5.5 20 mM acetic acid buffer (acetic acid- 140 mM sodium — sodium acetate) chloride 7 6.0 20 mM histidine buffer 50 mM sodium 140 mM (histidine-histidine hydrochloride) chloride mannitol 8 6.0 20 mM histidine buffer 140 mM sodium — (histidine-histidine hydrochloride) chloride 9 6.0 20 mM citric acid buffer 50 mM sodium 140 mM (citric acid-sodium citrate) chloride mannitol 10 6.0 20 mM citric acid buffer 140 mM sodium — (citric acid-sodium citrate) chloride 11 6.5 20 mM citric acid buffer 50 mM sodium 140 mM (citric acid-sodium citrate) chloride mannitol 12 6.5 20 mM citric acid buffer 140 mM sodium — (citric acid-sodium citrate) chloride Note: “—” represents no addition.
TABLE-US-00002 TABLE 2 Degradation rate of SEC-HPLC monomer content in buffer system and pH selection 10 mg/mL of 20 mg/mL of 40 mg/mL of protein protein protein Average Monomer Monomer Monomer decline of Formula decline decline decline monomer No. (%/week) (%/week) (%/week) (%/week) 1 0.27 0.38 0.52 0.39 2 0.46 0.53 0.71 0.57 3 0.17 0.27 0.38 0.27 4 0.23 0.36 0.48 0.36 5 0.22 0.32 0.46 0.34 6 0.36 0.45 0.58 0.46 7 0.16 0.21 0.35 0.24 8 0.14 0.24 0.37 0.25 9 0.23 0.33 0.44 0.33 10 0.21 0.31 0.41 0.31 11 0.27 0.37 0.52 0.38 12 0.24 0.35 0.46 0.35
TABLE-US-00003 TABLE 3 Degradation rate of CEX-HPLC charge heteroplasmon content in buffer system and pH selection 10 mg/mL of 20 mg/mL of 40 mg/mL of Average protein protein protein decline of Main peak Main peak Main peak major Formula decline decline decline peak No. (%/week) (%/week) (%/week) (%/week) 1 5.82 5.44 5.38 5.54 2 5.48 4.67 4.89 5.01 3 6.08 4.65 4.45 5.06 4 7.75 5.89 5.87 6.51 5 6.46 5.00 4.57 5.34 6 6.88 4.75 3.51 5.05 7 6.67 6.35 5.50 6.17 8 6.58 6.85 5.53 6.32 9 7.11 5.27 6.69 6.36 10 7.06 5.11 5.67 5.95 11 8.46 8.23 7.11 7.93 12 6.52 5.64 4.84 5.67
[0119] 5 As shown in Tables 2 and 3, in SEC-HPLC analysis, after the monomer was placed at a high temperature of 40° C. for 4 weeks, the monomer content declined at a high rate along with the increase of the protein concentration, while the monomer content declined at a low rate under a histidine buffer system with a pH of 5.5-6.0, and the average decline was less than or equal to 0.27%/week; in CEX-HPLC analysis, it could be seen that the main peak content degradation rate by CEX-HPLC had a certain correlation with pH, the higher the pH was, the higher the main peak content degradation rate was, the main peak content degradation rate was relatively low in a buffer system with a pH of 5.0-6.0, and the correlation between the main peak content degradation rate and the protein concentration was not significant.
[0120] After the product specification and the target quality attribute (polymer content level) of the product were comprehensively considered, a histidine buffer with the protein concentration of 20 mg/mL and a pH of 5.5-6.0 was selected for the selection of excipient (stabilizer) formula.
EXAMPLE 2
Stabilizer Selection
[0121] In order to further explore the effect of different excipients on the stability of the antibody, a formulation of one or a combination of sodium chloride, mannitol, sorbitol, sucrose or trehalose was selected for comparative testing. The above various excipients or combinations thereof were added into 20 mM histidine buffer or citric acid buffer comprising about 20 mg/mL of antibody hu18, and the specific formula is shown in Table 4. The formulations were aliquoted, then placed at 40° C., and removed for analysis at weeks 0, 2 and 4. Changes in antibody hu18 content were determined by size-exclusion high-performance liquid chromatography (SEC-HPLC), and the charged main peak content of antibody hu18 was determined by cation exchange high-performance liquid chromatography (CEX-HPLC). The results are shown in Table 5.
[0122] According to the results of stability investigation, after the formulation samples of different excipients were placed at a high temperature of 40° C. for 2 weeks, the antibody had stronger thermostability.
[0123] After comprehensive data analysis, it could be found from comparing F13-F17 with F18-F21 that the excipient combination of mannitol, sorbitol, sucrose, trehalose and sodium chloride was better than a single excipient of mannitol, sorbitol, sucrose and trehalose, which was manifested in that the degradation rates of the monomer content by SEC-HPLC and the main peak content by CEX-HPLC were lower. In the excipient comprising trehalose, a histidine buffer system with a pH of 5.5 was the most stable, the decline of antibody monomer purity was as low as 0.11%/week, and the decline of antibody main charge was only 3.72%/week, which was significantly lower than those in a buffer system with a pH of 6.0.
[0124] Therefore, the excipient combination of the trehalose and the sodium chloride with a pH of 5.5 was more favorable for the stability of the product. It could also be seen that a histidine buffer system with a pH of 6.0 (F17) was significantly better than a citrate buffer system with a pH of 6.0 (F23) for degradation of the monomer content by SEC-HPLC.
TABLE-US-00004 TABLE 4 Formulas in excipient selection Formula No. Buffer PH Excipient 1 Excipient 2 Polysorbate 80 13 20 mM histidine buffer 6.0 135 mM sodium — 0.02% (histidine-histidine chloride 14 hydrochloride) 240 mM mannitol — 0.02% 15 240 mM sorbitol — 0.02% 16 220 mM sucrose — 0.02% 17 220 mM trehalose — 0.02% 18 54 mM sodium 144 mM mannitol 0.02% chloride 19 54 mM sodium 144 mM sorbitol 0.02% chloride 20 54 mM sodium 132 mM sucrose 0.02% chloride 21 54 mM sodium 132 mM trehalose 0.02% chloride 22 20 mM histidine buffer 5.5 220 mM trehalose — 0.02% (histidine-acetic acid) 23 20 mM citric acid buffer 6.0 220 mM trehalose — 0.02% (citric acid-sodium citrate) Note: “—” represents no addition.
TABLE-US-00005 TABLE 5 Summary of results of excipient selection SEC-HPLC CEX-HPLC Monomer Main peak decline decline Formula No. (%/week) (%/week) 13 0.13 4.66 14 0.12 5.11 15 0.12 5.13 16 0.12 5.08 17 0.13 5.27 18 0.12 4.98 19 0.11 4.92 20 0.12 4.99 21 0.12 4.81 22 0.11 3.72 23 0.19 4.99
EXAMPLE 3
Surfactant Selection
[0125] Surfactants are commonly used in liquid formulations as an agent for protecting proteins such as antibodies from gas/liquid-induced stress, liquid/solid-induced stress during storage to reduce aggregation of the antibodies or minimize the formation of particulate matter in the formulation, which facilitates the stability of the physicochemical properties of the antibodies. Polysorbate 20 or polysorbate 80 at different concentrations was added into the formulations comprising 20 mM histidine buffer (in a molar ratio of histidine to histidine hydrochloride of 1:1, pH 6.0) and 20 mg/mL of antibody hu18, and the mixture was analyzed after placed at 40° C. for 4 weeks. The results are shown in Table 6.
[0126] After comprehensive data analysis, it was shown from the selection of surfactants (F17, F24, F25 and F26) that the addition of different concentrations of polysorbate 80 or polysorbate 20 had no significant effect on the monomer content by SEC-HPLC and the main peak content by CEX-HPLC.
TABLE-US-00006 TABLE 6 Results of surfactant selection CEX-HPLC (%) SEC-HPLC (%) Main charged Formula No. Excipient Surfactant content Monomer (%) form (%) 17 220 mM trehalose 0.02% of polysorbate 80 0.13 5.27 24 0.04% of polysorbate 80 0.13 4.95 25 0.02% of polysorbate 20 0.12 4.98 26 0.04% of polysorbate 20 0.13 4.99
[0127] In summary, the stability of the recombinant humanized anti-BTLA monoclonal antibody hu18 is explored and researched by examining different buffer systems, different pH conditions, different antibody concentrations and different excipients and surfactant compositions, and the formula of optimal liquid formulation is determined. A histidine buffer was selected for adjusting the pH of antibody hu18, trehalose and sodium chloride were selected for adjusting the osmotic pressure of formulation, and polysorbate 80 was added to increase the solubility of the formulation.
EXAMPLE 4
Long-Term Stability Study
[0128] Liquid pharmaceutical products comprising therapeutic antibodies generally require a temperature of 2-8° C. for storage, which is important for the stability of the formulation over a long-term storage. Based on the above selection results, formula No. 27 on the basis of the previous 26 formulas was designed to perform long-term stability study of the formulation.
[0129] Four batches of stock solutions were selected for formulating the formulations of formula No. 27 as shown in Table 7, then the formulations were stored in transparent vials, and the formulation samples were analyzed after placed at 2-8° C. for 6 months. Stability was assessed by the following parameters: (a) appearance; (b) insoluble microparticles by light-blockage method (OD: 405 nm); (c) pH; (d) molecular weight of the antibody by CE-SDS (capillary electrophoresis sodium dodecyl sulfate); (d) contents of antibody monomer (specification: ≥97.0%), polymers (specification: ≤3.0%) or fragments (specification: ≤1.0%) by SEC-HPLC; (e) main charged form (specification: ≥70.0%), acidic forms (specification: ≤30.0%) or basic forms (specification: ≤15.0%) of the antibody by CEX-HPLC; (f) antibody binding activity by ELISA (specification: 70%-130% of reference standard); and (g) protein content (specification: 18-22 mg/mL).
[0130] The results showed that the appearance, pH, insoluble microparticles, protein content, purity (determined by SEC-HPLC (size-exclusion high-performance liquid chromatography), CEX-HPLC (cation exchange high-performance liquid chromatography), R-CE-SDS (reduced electrophoresis) and NR-CE-SDS (non-reduced electrophoresis)) and biological activity of 4 batches of stock solutions in the formulations of formula No. 27 were not significantly changed, and the specific results are shown in Table 8. The results show that the stock solutions of the 4 batches in the formulations of formula No. 27 have very good stability during storage for 0-24 months at 2-8° C.
TABLE-US-00007 TABLE 7 Formula for long-term stability study of formulation Formula No. pH Composition 27 5.5 20 mM histidine buffer, 50 mM sodium chloride, 140 mM trehalose, 0.02% of polysorbate 80 and 20 mg/mL of antibody hu18 Note: the histidine buffer is prepared by a molar ratio of histidine to histidine hydrochloride being 1:3.
TABLE-US-00008 TABLE 8 Long-term stability of formulations Test Time Result item (month) Batch 1 Batch 2 Batch 3 Batch 4 Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 2 Colorless and NA NA NA clear liquid with slight opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid slight slight slight opalescence opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with almost clear slight slight slight liquid opalescence opalescence opalescence 18 Colorless and Colorless and Colorless and NR clear liquid with clear liquid with clear liquid with slight slight slight opalescence opalescence opalescence 24 Colorless and Colorless and Colorless and NR clear liquid with clear liquid with clear liquid with slight slight slight opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.7 NA NA 5.6 2 5.6 NA NA NA 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.6 5.7 9 5.6 5.6 5.6 5.9 12 5.6 5.7 5.6 5.6 18 5.6 5.6 5.6 NR 24 5.6 5.6 5.6 NR SEC-HPLC 0 99.7% 99.7% 99.7% 99.7% monomer 1 99.7% NA NA 99.7% content (%) 2 99.7% NA NA NA 3 99.5% 99.6% 99.6% 99.7% 6 99.7% 99.6% 99.6% 99.6% 9 99.7% 99.6% 99.6% 99.6% 12 99.7% 99.5% 99.5% 99.6% 18 99.7% 99.6% 99.5% NR 24 99.6% 99.5% 99.5% NR SEC-HPLC 0 0.3% 0.3% 0.0% 0.3% polymer 1 0.3% NA NA 0.3% content (%) 2 0.3% NA NA NA 3 0.5% 0.4% 0.0% 0.3% 6 0.3% 0.4% 0.0% 0.4% 9 0.3% 0.4% 0.4% 0.4% 12 0.3% 0.4% 0.5% 0.4% 18 0.3% 0.4% 0.5% NR 24 0.3% 0.4% 0.5% NR SEC-HPLC 0 0.0% 0.3% 0.0% 0.0% fragment 1 0.0% NA NA 0.0% content (%) 2 0.0% NA NA NA 3 0.0% 0.4% 0.0% 0% 6 0.0% 0.4% 0.0% 0% 9 0.0% <0.1% <0.1% <0.1% 12 0.0% <0.1% <0.1% <0.1% 18 0.0% <0.1% <0.1% NR 24 <0.1% <0.1% <0.1% NR CEX-HPLC 0 63.7% 62.6% 61.7% 63.1% main peak 1 61.5% NA NA 62.6% content (%) 2 61.7% NA NA NA 3 61.1% 65.4% 64.6% 64.3% 6 65.8% 67.5% 66.8% 66.7% 9 65.5% 68.8% 68.2% 68.5% 12 68.1% 71.2% 70.7% 69.3% 18 70.4% 70.8% 70.1% NR 24 69.9% 71.5% 71.1% NR CEX-HPLC 0 10.0% 13.1% 12.6% 12.0% acidic peak 1 11.0% NA NA 13.0% content (%) 2 10.9% NA NA NA 3 11.8% 13.9% 13.6% 13.3% 6 12.4% 14.2% 13.7% 15.2% 9 15.3% 14.5% 14.0% 15.7% 12 13.1% 14.5% 14.1% 16.0% 18 13.6% 17.3% 16.9% NR 24 15.3% 17.8% 17.6% NR CEX-HPLC 0 26.3% 24.3% 25.7% 24.9% basic peak 1 27.5% NA NA 24.4% content (%) 2 27.4% NA NA NA 3 27.1% 20.6% 21.8% 22.3% 6 21.8% 18.3% 20.0% 18.1% 9 19.2% 16.7% 17.8% 15.9% 12 18.7% 14.2% 15.2% 14.8% 18 16.0% 11.8% 13.0% NR 24 14.8% 10.7% 11.3% NR R-CE-SDS 0 99.9% 99.8% 99.7% 99.6% content (%) 1 99.8% NA NA 99.5% 2 99.8% NA NA NR 3 99.9% 99.5% 99.5% 99.4% 6 99.9% 99.4% 99.4% 99.6% 9 99.7% 99.2% 99.3% 99.5% 12 99.6% 99.2% 99.2% NR 18 99.6% 99.3% 99.2% NR 24 99.2% 99.4% 99.3% NR NR-CE-SDS 0 99.1% 98.4% 99.3% 96.4% content (%) 1 99.0% NA NA 97.5% 2 99.1% NA NA NR 3 98.7% 98.7% 98.4% 97.4% 6 98.0% 97.8% 97.8% 93.8% 9 98.6% 97.5% 97.6% 97.2% 12 98.2% 97.5% 97.2% NR 18 98.2% 98.2% 98.1% NR 24 98.4% 96.9% 96.8% NR Biological 0 107.4% 101% 100% .sup. 98% activity 1 NA NA NA .sup. 99% (by ELISA) % 2 NA NA NA NR 3 NA .sup. 95% 100% 101% 6 106.1% 100% 102% 105% 9 NA .sup. 96% .sup. 99% 103% 12 109.7% .sup. 95% 100% NR 18 NA 100% .sup. 98% NR 24 113% 103% 105% NR Protein content 0 21.3 mg/mL 20.9 mg/mL 20.9 mg/mL 20.3 mg/mL (ultraviolet 1 21.8 mg/mL NA NA 20.5 mg/mL spectrophotometry) 2 21.6 mg/mL NA NA NA 3 21.3 mg/mL 21.0 mg/mL 21.0 mg/mL 20.7 mg/mL 6 21.1 mg/mL 20.7 mg/mL 20.8 mg/mL 20.6 mg/mL 9 21.7 mg/mL 21.0 mg/mL 21.1 mg/mL 20.5 mg/mL 12 21.6 mg/mL 20.9 mg/mL 21.1 mg/mL 20.1 mg/mL 18 21.9 mg/mL 21.6 mg/mL 21.7 mg/mL NR 24 21.3 mg/mL 20.0 mg/mL 19.9 mg/mL NR Insoluble 0 ≥10 μm ≥10 μm ≥10 μm ≥10 μm microparticles microparticles: microparticles: microparticles: microparticles: 32 17 10 69 microparticles/ microparticles/ microparticles/ microparticles/ vial ≥25 μm vial ≥25 μm vial ≥25 μm vial ≥25 μm microparticles: microparticles: microparticles: microparticles: 2 6 0 7 microparticles/vial microparticles/vial microparticles/vial microparticles/vial 1 ≥10 μm NA NA ≥10 μm microparticles: microparticles: 67 12 microparticles/ microparticles/ vial ≥25 μm vial ≥25 μm microparticles: microparticles: 1 0 microparticle/vial microparticles/vial 2 ≥10 μm NA NA ≥10 μm microparticles: microparticles: 3 71 microparticles/vial microparticles/vial ≥25 μm ≥25 μm microparticles: microparticles: 0 3 microparticles/vial microparticles/vial 3 ≥10 μm ≥10 μm ≥10 μm ≥10 μm microparticles: microparticles: microparticles: microparticles: 14 3 4 55 microparticles/vial microparticles/vial microparticles/vial microparticles/vial ≥25 μm ≥25 μm ≥25 μm ≥25 μm microparticles: microparticles: microparticles: microparticles: 1 2 1 3 microparticle/vial microparticles/vial microparticle/vial microparticles/vial 6 ≥10 μm ≥10 μm ≥10 μm ≥10 μm microparticles: microparticles: microparticles: microparticles: 27 2 11 27 microparticles/vial microparticles/vial microparticles/vial microparticles/vial ≥25 μm ≥25 μm ≥25 μm ≥25 μm microparticles: microparticles: microparticles: microparticles: 2 0 2 4 microparticles/vial microparticles/vial microparticles/vial microparticles/vial 9 ≥10 μm ≥10 μm ≥10 μm ≥10 μm microparticles: microparticles: microparticles: microparticles: 39 7 4 38 microparticles/vial microparticles/vial microparticles/vial microparticles/vial ≥25 μm ≥25 μm ≥25 μm ≥25 μm microparticles: microparticles: microparticles: microparticles: 12 2 1 1 microparticles/vial microparticles/vial microparticle/vial microparticle/vial 12 ≥10 μm ≥10 μm ≥10 μm ≥10 μm microparticles: microparticles: microparticles: microparticles: 408 13 8 69 microparticles/vial microparticles/vial microparticles/vial microparticles/vial ≥25 μm ≥25 μm ≥25 μm ≥25 μm microparticles: microparticles: microparticles: microparticles: 28 5 2 7 microparticles/vial microparticles/vial microparticles/vial microparticles/vial 18 ≥10 μm ≥10 μm ≥10 μm NR microparticles: microparticles: microparticles: 71 41 12 microparticles/vial microparticles/vial microparticles/vial ≥25 μm ≥25 μm ≥25 μm microparticles: microparticles: microparticles: 48 34 5 microparticles/vial microparticles/vial microparticles/vial 24 ≥10 μm ≥10 μm ≥10 μm NR microparticles: microparticles: microparticles: 31 15 16 microparticles/vial microparticles/vial microparticles/vial ≥25 μm ≥25 μm ≥25 μm microparticles: microparticles: microparticles: 20 6 2 microparticles/vial microparticles/vial microparticles/vial Note: NA in Table represents the non-significant test item at the time point, no detection is performed at the time point, and NR indicates that the time point is not reached. The same applies hereinafter.
EXAMPLE 5
Accelerated Stability Study of Formulations
[0131] Four batches of stock solutions were selected for formulating the formulations of formula No. 27 as shown in Table 7, then the formulations were stored in transparent vials, and the formulation samples were analyzed after placed under the conditions of 25±2° C. and relative humidity (RH) 60±5% for 0-12 months.
[0132] As shown in Table 9, formulations of formula No. 27 have higher stability against protein degradation, and the resulting degradation kinetic parameters measured at 25±2° C. meet the requirements of storage in room temperature environment for up to 12 months.
TABLE-US-00009 TABLE 9 Accelerated stability data for formulations Test Time Result item (month) Batch 1 Batch 2 Batch 3 Batch 4 Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 0 99.7% 99.7% 99.7% 99.7% 1 99.7% 99.6% 99.7% 99.6% Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 2 99.7% 99.7% 99.7% 99.6% 3 99.7% 99.6% 99.6% 99.5% Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 SEC-HPLC 1 5.6 5.6 5.6 5.7 monomer 2 5.6 5.6 5.6 5.7 content (%) 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 6 99.6% 99.6% 99.5% 99.4% 12 99.6% 99.4% 99.3% NR Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 0 0.3% 0.3% 0.3% 0.3% 1 0.3% 0.4% 0.3% 0.3% Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 2 0.3% 0.3% 0.3% 0.4% 3 0.3% 0.3% 0.3% 0.4% Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 SEC-HPLC 1 5.6 5.6 5.6 5.7 polymer 2 5.6 5.6 5.6 5.7 content (%) 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 6 0.3% 0.4% 0.4% 0.5% 12 0.3% 0.5% 0.6% NR Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 0 0.0% 0.0% 0.0% 0.0% 1 0.0% 0.0% 0.0% <0.1% Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 2 0.0% 0.0% 0.0% <0.1% 3 0.0% 0.0% 0.0% 0.1% Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 SEC-HPLC 1 5.6 5.6 5.6 5.7 fragment 2 5.6 5.6 5.6 5.7 content (%) 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 6 0.1% 0.0% 0.0% <0.1% 12 0.1% 0.1% 0.1% NR Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 0 63.7% 62.6% 61.7% 63.1% 1 68.2% 73.5% 72.9% 69.2% Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 2 70.8% 71.6% 71.4% 69.6% 3 69.2% 69.0% 69.1% 67.3% Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 CEX-HPLC 1 5.6 5.6 5.6 5.7 main peak 2 5.6 5.6 5.6 5.7 content (%) 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 6 62.9% 63.8% 63.4% 58.4% 12 50.4% 50.7% 50.2% NR Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 0 10.0% 13.1% 12.6% 12.0% 1 15.2% 15.6% 15.2% 16.0% Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 2 16.9% 19.1% 18.6% 18.7% 3 19.8% 21.7% 21.4% 21.4% Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 CEX-HPLC 1 5.6 5.6 5.6 5.7 acidic peak 2 5.6 5.6 5.6 5.7 content (%) 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 6 26.9% 28.4% 28.2% 30.5% 12 38.8% 40.0% 39.8% NR Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 0 26.3% 24.3% 25.7% 24.9% 1 16.6% 10.9% 11.8% 14.8% Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 2 12.4% 9.3% 10.0% 11.6% 3 11.0% 9.3% 9.5% 11.3% Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 CEX-HPLC 1 5.6 5.6 5.6 5.7 basic peak 2 5.6 5.6 5.6 5.7 content (%) 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 6 10.2% 7.8% 8.4% 11.1% 12 10.8% 9.4% 10.0% NR Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR R-CE-SDS 0 99.9% 99.8% 99.7% 99.6% content (%) 1 99.8% 99.8% 99.8% 99.4% Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 2 99.9% 99.6% 99.7% 99.3% 3 99.6% 99.7% 99.6% 99.4% Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 6 99.4% 99.1% 99.1% 99.0% 12 98.1% 98.0% 97.8% NR Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR NR-CE-SDS 0 99.1% 98.6% 99.4% 96.4% content (%) 1 98.6% 97.7% 98.2% 97.4% Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 2 98.5% 97.4% 98.3% 97.3% 3 98.7% 98.3% 98.7% 96.6% Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 6 98.0% 97.7% 97.8% 92.4% 12 97.0% 96.2% 96.5% NR Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 0 107.4% 101% 100% .sup. 98% 1 NA .sup. 92% .sup. 92% 100% Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 2 NA .sup. 98% .sup. 97% 100% 3 NA .sup. 97% .sup. 98% 100% Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 Biological 1 5.6 5.6 5.6 5.7 activity 2 5.6 5.6 5.6 5.7 (by ELISA) % 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 6 104.0% .sup. 94% .sup. 95% .sup. 92% 12 97.4% .sup. 98% .sup. 94% NR Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR Protein content 0 21.3 mg/mL 20.9 mg/mL 20.9 mg/mL 20.3 mg/mL (ultraviolet 1 21.5 mg/mL 20.8 mg/mL 20.6 mg/mL 20.5 mg/mL Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 spectrophotometry) 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 2 21.9 mg/mL 20.8 mg/mL 20.7 mg/mL 20.5 mg/mL 3 21.7 mg/mL 20.9 mg/mL 20.8 mg/mL 20.6 mg/mL Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 6 21.9 mg/mL 20.8 mg/mL 20.6 mg/mL 20.7 mg/mL 12 21.2 mg/mL 20.3 mg/mL 21.0 mg/mL NR Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR Insoluble 0 ≥10 μm ≥10 μm ≥10 μm ≥10 μm microparticles microparticles: microparticles: microparticles: microparticles: 32 17 10 69 Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR microparticles/ microparticles/ microparticles/ microparticles/ vial ≥25 μm vial vial vial Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR microparticles: ≥25 μm ≥25 μm ≥25 μm 2 microparticles: microparticles: microparticles: 6 0 7 Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR microparticles/vial microparticles/vial microparticles/vial microparticles/vial Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 1 ≥10 μm ≥10 μm ≥10 μm ≥10 μm microparticles: microparticles: microparticles: microparticles: 27 11 8 21 Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR microparticles/ microparticles/ microparticles/ microparticles/ vial ≥25 μm vial vial vial Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR microparticles: ≥25 μm ≥25 μm ≥25 μm 2 microparticles: microparticles: microparticles: 8 1 1 Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR microparticles/ microparticles/ microparticle/ microparticle/ vial vial vial vi al Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 2 ≥10 μm ≥10 μm ≥10 μm ≥10 μm microparticles: microparticles: microparticles: microparticles: 108 2 2 26 Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR microparticles/ microparticles/ microparticles/ microparticles/ vial ≥25 μm vial vial vial Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR microparticles: ≥25 μm ≥25 μm ≥25 μm 88 microparticles: microparticles: microparticles: 0 0 7 Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR microparticles/vial microparticles/vial microparticles/vial microparticles/vial Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 3 ≥10 μm ≥10 μm ≥10 μm ≥10 μm microparticles: microparticles: microparticles: microparticles: 14 3 3 20 Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR microparticles/ microparticles/ microparticles/ microparticles/ vial ≥25 μm vial vial vial Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR microparticles: ≥25 μm ≥25 μm ≥25 μm 0 microparticles: microparticles: microparticles: 1 0 0 Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR microparticles/vial microparticle/vial microparticles/vial microparticles/vial Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 6 ≥10 μm ≥10 μm ≥10 μm ≥10 μm microparticles: microparticles: microparticles: microparticles: 109 11 12 25 Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR microparticles/ microparticles/ microparticles/ microparticles/ vial ≥25 μm vial vial vial Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR microparticles: ≥25 μm ≥25 μm ≥25 μm 66 microparticles: microparticles: microparticles: 4 5 7 Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR microparticles/vial microparticles/vial microparticles/vial microparticles/vial Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR 12 ≥10 μm ≥10 μm ≥10 μm NR microparticles: microparticles: microparticles: 92 12 25 Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR microparticles/ microparticles/ microparticles/ vial ≥25 μm vial vial Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR microparticles: ≥25 μm ≥25 μm 6 microparticles: microparticles: 5 7 Appearance 0 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 1 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 2 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 3 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 6 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence 9 Colorless and NA NA Colorless and clear liquid with clear liquid with slight slight opalescence opalescence 12 Colorless and Colorless and Colorless and Colorless and clear liquid with clear liquid with clear liquid with clear liquid with slight slight slight slight opalescence opalescence opalescence opalescence pH 0 5.6 5.6 5.6 5.7 1 5.6 5.6 5.6 5.7 2 5.6 5.6 5.6 5.7 3 5.6 5.4 5.4 5.6 6 5.6 5.6 5.5 5.6 9 5.6 NA NA NR 12 5.7 5.7 5.6 NR microparticles/vial microparticles/vial microparticles/vial Note: NA in Table represents the non-significant test item at the time point, no detection is performed at the time point, and NR indicates that the time point is not reached.
EXAMPLE 6
Detection of the Binding of Humanized Antibodies to hBTLA by ELISA
[0133] The binding specificity of the humanized antibodies hu17, hu18 and hu19 to hBTLA was detected by conventional ELISA. 0.5 μg/mL of hBTLA was immobilized on a 96-well plate and incubated at 37° C. for 60 minutes. The solution in wells was then discarded, and the wells were washed 3 times with a washing buffer and blocked for 60 minutes with PBS containing 2% BSA. After the wells were washed 3 times with washing buffer, gradiently-diluted antibody solutions were added (the components other than the antibody in the dilution were the same as those in formula No. 27), incubated at 37° C. for 60 minutes, and washed 3 times with washing buffer before addition of an HRP-labeled mouse anti-human IgG4 secondary antibody diluted at 1:10000. The mixture was incubated at 37° C. for 1 hour, washed three times with washing buffer, and added with 100 μL of TMB substrate solution for color development at room temperature for 30 minutes; then the reaction was stopped with 100 μL of 2 M hydrochloric acid solution and the absorbance was read at 450 nm.
[0134] The EC.sub.50 values are shown in Table 10 below. The results show that the humanized antibodies hu17, hu18 and hu19 can specifically bind to hBTLA.
TABLE-US-00010 TABLE 10 Data on the specific binding of humanized antibodies hu17, hu18 and hu19 to hBTLA Humanized antibody hu17 hu18 hu19 EC.sub.50(ng/mL) 6.3 5.4 6.6
EXAMPLE 7
Detection of the Binding of Humanized Antibodies to hBTLA on 293F Cells by FACS
[0135] The humanized anti-BTLA antibodies hu17, hu18 and hu19 were determined for their binding ability to hBTLA expressed on cells using cell-based flow cytometry (FACS) assay. 293F cells expressing hBTLA were centrifuged and resuspended in an FACS buffer after digestion, and then added into a 96-well round-bottom plate at a density of 2.5×10.sup.4/50 μL. Then the cells were mixed with 50 μL of antibody diluent at different concentrations (initial concentration: 10 μg/mL, 3-fold titration, the components other than the antibody in the diluent were the same as those in formula No. 27), and incubated at room temperature for 30 minutes. After being washed with an FACS buffer twice, the cells were added with 100 μL of goat anti-human IgG-PE antibody, incubated for 30 minutes away from light, and detected by FACS after two washes with an FACS buffer. The washed cells were resuspended in 4° C. buffer containing propidium iodide (PI) and 0.02% sodium azide that prevents receptor internalization and analyzed by flow cytometry. Viable cells were gated on the basis of PI positive cells being excluded from the FSC/SSC gate and their geometric mean fluorescence intensity was measured. The data were analyzed using a sigmoidal dose-response model within Prism™ software.
[0136] The EC.sub.50 values for each antibody are shown in Table 11 below. The results show that the humanized antibodies hu17, hu18 and hu19 effectively bind to hBTLA on 293F cells.
TABLE-US-00011 TABLE 11 hu17 hu18 hu19 EC.sub.50 (ng/mL) 96.74 89.34 95.06
EXAMPLE 8
Detection of Humanized Antibodies' Blocking of the Binding of BTLA to HVEM by FACS
[0137] The humanized antibodies hu17, hu18 and hu19 were determined for their ability to block the binding of hBTLA to hHVEM expressed on 293F cells using cell-based flow cytometry (FACS) assay. hHVEM-293F stable cells were centrifuged and resuspended in an FACS buffer after digestion, and then added into a 96-well round-bottom plate at a density of 2.5×10.sup.4 cells/50 μL. Then the cells were mixed with previously biotin-labeled hBTLA (1 μg/mL) protein, incubated at 4° C. for 15 minutes, followed by mixing with 50 μL of humanized antibody diluent (initial concentration: 5 μg/mL, 3-fold titration, the components other than the antibody in the diluent were the same as those in formula No. 27) at different concentrations, and incubating at room temperature for 30 minutes. After being washed with an FACS buffer twice, the cells were added with 100 μL of goat anti-human IgG-PE antibody, incubated for 30 minutes away from light, and detected by FACS after two washes with an FACS buffer. The washed cells were resuspended in 4° C. buffer containing propidium iodide (PI) and 0.02% sodium azide that prevents receptor internalization and analyzed by flow cytometry. Viable cells were gated on the basis of PI positive cells being excluded from the FSC/SSC gate and their geometric mean fluorescence intensity was measured. The data were analyzed using a sigmoidal dose-response model within Prism™ software.
[0138] The IC.sub.50 values for each antibody are shown in Table 12 below. The results show that the humanized antibodies hu17, hu18 and hu19 can effectively block the binding of BTLA to HVEM on the cell surface.
TABLE-US-00012 TABLE 12 hu17 hu18 hu19 IC.sub.50 (ng/mL) 142.7 172.2 161.5
EXAMPLE 9
Promotion of T Cell Activation by Humanized Anti-BTLA Antibodies
[0139] CHO cells stably expressing hPD-L1/hHVEM were spread on a 96-well plate at 5×10.sup.4 cells/well, and incubated overnight at 37° C., 7% CO.sub.2. After the cell supernatant was removed, each well was added with 40 μL of humanized anti-BTLA antibody diluent (initial concentration: 60 μg/mL, 3-fold gradient dilution, the components other than the antibody in the diluent were the same as those in formula No. 27), and 40 μL of Jurkat report cells capable of continuously expressing hPD-1/hBTLA/NFAT-luciferase, with a total cell number being 1×10.sup.5 cells. The mixture was incubated at 37° C. for 6 hours, 7% CO.sub.2, and added with luciferase reagent. The luminescence values were detected by a microplate reader.
[0140] The EC.sub.50 values for each antibody are shown in Table 13 below. The results show that the humanized anti-BTLA antibodies hu17, hu18 and hu19 can effectively promote T cell activation.
TABLE-US-00013 TABLE 13 hu17 hu18 hu19 EC.sub.50 (ng/mL) 256.7 277.3 290.4
EXAMPLE 10
Affinity of Humanized Anti-BTLA Antibodies for hBTLA
[0141] The assay was carried out using a Biacore T200 instrument (GE Healthcare). Series S CMS chip was loaded on the instrument and the system buffer used was HBS-EP+(10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20). The BTLA-Fc antigens were coupled in a chip detection channel: injecting a mixed solution of 400 mM EDC and 100 mM NHS at 10 μL/min for 420 s to activate the surface of the chip, diluting the BTLA-Fc antigens in a 10 mM sodium acetate/acetic acid (pH 5.5) buffer to a final concentration of 20 μg/mL and injecting at 10 μL/min for coupling, and injecting a 1 M ethanolamine-hydrochloric acid solution (pH 8.5) at 10 μL/min for 420 s for blocking.
[0142] Antibodies were diluted in a 2-fold gradient with Biacore system buffer for a total of 6 concentration points (the components other than the antibody in the diluent were the same as those in formula No. 27). Concentration gradients were 24 nM, 12 nM, 6 nM, 3 nM, 1.5 nM and 0.75 nM, with 24 nM being a replicate. Data were analyzed using Biacore T200 Evaluation Software (version no. 3.0, GE Healthcare). Data were fitted using a model of 1:1 Binding. The kinetic constants of the binding of the antibodies to the antigens, namely, the association rate constant ka (1/Ms), dissociation rate constant kd (1/s) and the affinity constant KD (M), were obtained by fitting, and the results are shown in Table 14.
TABLE-US-00014 TABLE 14 Affinity data for humanized antibodies ka(1/Ms) kd(1/s) KD(M) hu17 6.24E+05 1.16E−04 1.86E−10 hu18 6.39E+05 8.67E−05 1.36E−10
EXAMPLE 11
Characterization of Binding Kinetics of Humanized Antibodies to BTLAs of Different Species To detect cross-reactivity between chimeric antibodies and cynomolgus and murine BTLAs,
[0143] Fortebio assay was used. Briefly, human BTLA, cynomolgus BTLA or murine BTLA was coupled to an activated CM5 biosensor chip to achieve approximately 100-200 response units (RUs), and then the unreacted groups were blocked with IM ethanolamine. Humanized antibody samples (antibody in the samples was hu17, and other components therein were the same as those in formula No. 27) at increasing concentrations from 0.12 nM to 90 nM were injected at 30 times/min into SPR running buffer and binding responses at BTLAs of different species were calculated by subtracting RU from the blank flow cell.
[0144] The results are shown in
EXAMPLE 12
Inhibition of Tumor Growth in Mice by Humanized Antibodies (Formula No. 27)
[0145] The MC38-hHVME cell bank was created on MC38 cells (ATCC) by electroporating Hxp-hHVEM plasmids, the cells were subcloned by limiting dilution method, and the monoclones were screened by flow cytometry to acquire MC38-HVEM cells. The MC38-HVEM cells were inoculated into the right subcutaneous tissue of the humanized female B-HBTLA mice at 1×10.sup.6 cells/0.1 mL, and when tumors grew to approximately 118 mm.sup.3, the mice were randomly grouped, by tumor volume, into 5 groups (8 mice per group), namely G1 0.9% sodium chloride injection solvent control group, G2 KLH (anti-keyhole limpet hemocyanin antibody) (10 mg/kg) negative control group, G3 hu18 (1 mg/kg) group, G4 hu18 (3 mg/kg) group, and G5 hu18 (10 mg/kg) group. All groups were subjected to administration by intraperitoneal injection twice a week for 7 consecutive times, and the experiment ended 4 days after the last administration. Tumor volume and body weight of mice were measured and recorded twice a week. At the end of the experiment, animals were euthanized, tumors were taken, weighed and photographed, and the relative tumor inhibition (TGI%, TGI%=(1−(TT.sub.i−T.sub.0)/(V.sub.i−V.sub.0))×100%, wherein T.sub.i is the tumor volume at the end of administration of the treatment group, T.sub.0 is the tumor volume at the beginning of administration of the treatment group, V.sub.i is the tumor volume at the end of administration of the blank group, and V.sub.0 is the tumor volume at the beginning of administration of the blank group) was calculated.
[0146] The effect of each test sample on the tumor volume of MC38-hHVEM cell transplanted B-hBTLA mice is shown in Table 15 and
TABLE-US-00015 TABLE 15 Effect of hu18 on the tumor volume of MC38-hHVEM cell-transplanted B-hBTLA mice Tumor volume (mm.sup.3).sup.a 21 days after Before the first TGI Groups Test drug administration administration (%) P .sup.b G1 0.9% 118 ± 2 1029 ± 155 — — sodium chloride injection G2 KLH 118 ± 2 1560 ± 256 — — (10 mg/kg) G3 Antibody 118 ± 3 1073 ± 224 33.7 0.175 hu18 (1 mg/kg) G4 Antibody 118 ± 2 747 ± 268 56.4 0.046 hu18 (3 mg/kg) G5 Antibody 118 ± 3 868 ± 211 48.0 0.056 hu18 (10 mg/kg) Note: .sup.amean ± standard error; .sup.b the statistical comparison of the tumor volume of the dosing groups with tumor volumes of the KLH negative control group at 21 days after the administration, t-test.
[0147] The results of the effect of each test sample on the body weight of MC38-hHVEM cell-transplanted B-hBTLA mice are shown in Table 16 and
TABLE-US-00016 TABLE 16 Effect of test samples on the body weight of MC38-hHVEM cell-transplanted B-hBTLA mice Body weight (g) .sup.a Body weight 21 days change after (g) 21 Before the first days after Test admin- admin- admin- Groups drug istration istration P .sup.b istration G1 0.9% 18.4 ± 0.4 20.7 ± 0.4 — +2.3 sodium chloride injection G2 KLH 18.3 ± 0.5 20.8 ± 0.6 — +2.5 (10 mg/kg) G3 hu18 18.5 ± 0.4 20.8 ± 0.4 0.943 +2.3 (1 mg/kg) G4 hu18 18.5 ± 0.4 20.0 ± 0.6 0.341 +1.5 (3 mg/kg) G5 hu18 18.6 ± 0.3 20.5 ± 0.2 0.623 +1.9 (10 mg/kg) Note: .sup.a mean ± standard error; .sup.b body weights of the dosing groups compared statistically with that of the KLH negative control group 21 days after administration, t-test.
EXAMPLE 13
Humanized Antibody hu18 (Formula No. 27) Without ADCC Effector Function
[0148] ADCC is initiated when an antibody binds to a cell surface target protein and then links to an Fc γ receptor (FcγR) expressed on effector cells. It is clearly documented that human IgG1 has significantly higher binding affinity for FcγR than IgG4, particularly for FcγR-I and FcγR-IIIA, which correlates with the strength of IgG1 to activate ADCC. In conjunction with ADCC, CDC is activated when antibodies cross-link the cell surface target and C1q protein, followed by a cascade reaction of complement complex formation and target cell lysis. As a proxy for ADCC and CDC, the detection of antibody binding to FcγR and C1q may serve as basic indicators of ADCC and CDC. Thus, in the present invention, the kinetic binding affinity of monoclonal antibodies for major FcγRs was evaluated using biacore T200 (GE).
[0149] Anti-His antibody (GE) was immobilized on the sensor wafer. Various Fc receptors, including recombinant human FcγRIIIA (CD16a) V176, recombinant human FcγRIIA (CD32a) R167, recombinant human FcγRI (CD64) and recombinant human FcRn were captured, then a series of diluted recombinant human anti-BTLA antibodies (i.e., hu18) were injected, and the binding properties of the interactions were assayed and analyzed. hu18 is an IgG4 subtype, and hu18-IgG1 is an IgG1 subtype that shares the same Fab as hu18 and serves as a positive control.
[0150] As shown in Table 17, the binding of the recombinant human anti-BTLA antibody of the IgG4 subtype to Fc receptors was relatively weak compared to that of the control antibody of the IgG1 subtype, and the interaction of the hu18 antibody with FcγRIIIA (CD16a) V176 was 400-fold weaker compared to that of the control antibody of the IgG1 subtype. This suggests that hu18 has weak or no ADCC effect. As shown in Table 17, the hu18 antibody did not bind to C1q, while the hu18-IgG1 antibody was able to bind to C1q.
TABLE-US-00017 TABLE 17 Binding affinity of interaction between anti-BTLA antibodies and Fc receptors Fc receptor Antibody Ka (1/Ms) Kd (1/s) KD (M) FcγRIIIA (CD16a) hu18-IgG1 6.15E+04 8.85E−03 1.44E−07 V176 hu18 N/A N/A 6.02E−05 FcγRIIA (CD32a) hu18-IgG1 N/A N/A 5.90E−06 R167 hu18 N/A N/A 4.06E−05 FcγRI (CD64) hu18-IgG1 2.99E+05 1.20E−03 4.00E−09 hu18 2.10E+05 3.87E−03 1.84E−08 FcRn hu18-IgG1 1.69E+06 3.88E−02 2.29E−08 hu18 5.12E+05 7.08E−02 1.38E−07
TABLE-US-00018 TABLE 18 Binding affinity of interaction between anti-BTLA antibodies and human C1q Protein Antibody Conclusion C1q hu18-IgG1 Specific binding hu18 Non-specific binding