METHODS FOR IDENTIFYING AND ANALYZING AMINO ACID SEQUENCES OF PROTEINS
20210255194 · 2021-08-19
Inventors
Cpc classification
International classification
Abstract
The disclosure provides methods for determining the biosimilarity of a test protein in relation to a target biologic in which the test protein is digested by two distinct proteases, the resultant digested peptide fragments are analyzed by column chromatography-tandem mass spectrometry to achieve 100% of the amino acid sequence coverage and 100% amino acid sequence accuracy of the test protein.
Claims
1. A method for determining the biosimilarity of a test protein in relation to a target biologic, the method comprising the steps of: (a) digesting a first sample of a test protein for a first incubation time using a first protease and digesting a second sample of the test protein for a second incubation time using a second protease, wherein the first sample and the second sample are physically separated; (b) applying column chromatography and tandem mass spectroscopy to the first sample under conditions sufficient to enhance binding of small peptides to the column, and generating a sequence of the test protein in the first sample; (c) applying column chromatography and tandem mass spectroscopy to the second sample under conditions sufficient to enhance binding of small peptides to the column, and generating the sequence of the test protein in the second sample, wherein the first sample and second sample are physically separated; (d) identifying the test protein as biosimilar to the target biologic when the test protein comprises 100% sequence identity to the target biologic; and (e) identifying the test protein as not biosimilar to the target biologic when the test protein does not comprise 100% sequence identity to the target biologic.
2. The method of claim 1, wherein the monoclonal antibody comprises Adalimumab.
3. The method of claim 1, wherein the first protease is Trypsin.
4. The method of claim 1, wherein the second protease is Chymotrypsin.
5. The method of claim 1, wherein the first digestion period is about 0.1 to about 1.0 hour.
6. The method of claim 5, wherein the first digestion period is about 0.1 to about 0.5 hour.
7. The method of claim 5, wherein the first digestion period is about 0.6 to about 1.0 hour.
8. The method of claim 5, wherein the first digestion period is about 0.5 hours.
9. The method of claim 1, wherein the second digestion period is about 0.1 to about 2.0 hours.
10. The method of claim 9, wherein the second digestion period is about 0.1 to about 1.5 hours.
11. The method of claim 9, wherein the second digestion period is about 1.5 to about 2.0 hours.
12. The method of claim 9, wherein the second digestion period is about 1.5 hours.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Additional aspects, features, and advantages of the disclosure, both as to its methods and use, will be understood and become more readily apparent when the disclosure is considered in light of the following description of illustrative embodiments made in conjunction with the accompanying drawings, wherein:
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DETAILED DESCRIPTION
[0045] As used herein, the term “biologic” (singular or plural) refers to peptide and protein products. For example, biologics include naturally-derived or recombinant products expressed in cells, such as, e.g., proteins, glycoproteins, fusion proteins, growth factors, vaccines, blood factors, thrombolytic agents, hormones, interferons, interleukin-based products, monospecific (e.g., monoclonal) antibodies, therapeutic enzymes. Biologics may be approved under a biologics license application (BLA), under Section 351(a) of the Public Health Service (PHS) Act, whereas biosimilar and interchangeable biologics referencing a BLA as a reference product are licensed under Section 351(k) of the PHS Act. Section 351 of the Public Health Service (PHS) Act is codified as 42 U.S.C. 262. Other biologics may be approved under Section 505(b)(1) of the Federal Food and Cosmetic Act, or as abbreviated applications under Sections 505(b)(2) and 505(j) of the Hatch Waxman Act, wherein Section 505 is codified as 21 U.S.C. 355.
[0046] As used herein, the term “isoform” (singular or plural) refers to any of several different forms of the same protein, arising from either single nucleotide polymorphisms, differential splicing of mRNA, or post-translational modifications (e.g., sulfation, glycosylation, etc.).
[0047] As used herein, the term “antibody” (singular or plural) refers, in the broadest sense, to monoclonal antibodies (including full length monoclonal antibodies) of any of the classes IgG, IgM, IgD, IgA, and IgE, as well as antibody fragments that exhibit a desired biological activity. The phrase “antibody fragments” refers to a portion of a full-length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multi-specific antibodies formed from antibody fragments.
[0048] As used herein, the term “monoclonal antibody” (singular or plural) refers to antibodies that are highly specific, being directed against a single antigenic epitope. Alternatively, the term “monoclonal antibody” refers to an antibody produced from a single spleen cell clone. In a non-limiting example, a monoclonal antibody can be a fully humanized antibody, i.e., both its variable and constant region are derived from a human source.
[0049] As used herein, the term “approval” refers to the procedure by which a regulatory entity, e.g., the USFDA, approves a candidate for therapeutic or diagnostic use in humans or animals. As used herein, a primary approval process is an approval process which does not refer to a previously approved protein, e.g., it does not require that the protein being approved have structural or functional similarity to a previously approved protein, e.g., a previously approved protein having the same primary amino acid sequence or a primary amino acid sequence. In embodiments, the primary approval process is one in which the applicant does not rely, for approval, on data, e.g., clinical data, from a previously approved product. Exemplary primary approval processes include, in the United States, a Biologics License Application (BLA), or supplemental Biologics License Application (sBLA), a new drug application (NDA) under Section 505(b)(1) of the Federal Food and Cosmetic Act, and, in Europe, an approval in accordance with the provisions of Article 8(3) of the European Directive 2001/83/EC, or an analogous proceeding in other countries or jurisdictions.
[0050] As used herein, the term “glycoprotein” refers to an amino acid sequence that includes one or more oligosaccharide chains (e.g., glycans) covalently attached thereto. Exemplary amino acid sequences include peptides, polypeptides, and proteins. Exemplary glycoproteins include glycosylated antibodies and antibody-like molecules (e.g., Fc fusion proteins). Exemplary antibodies include monoclonal antibodies and/or fragments thereof, polyclonal antibodies and/or fragments thereof, and Fc domain containing fusion proteins (e.g., fusion proteins containing the Fc region of IgG1, or a glycosylated portion thereof). A glycoprotein preparation is a composition or mixture that includes at least one glycoprotein.
[0051] As used herein, the phrase “target biologic”, e.g., target protein, refers to a commercially available, or approved, biologic which defines or provides the basis against which a test biologic is measured or evaluated. In embodiments, a target biologic is commercially available for therapeutic use in humans or animals. In other embodiments, the target biologic is approved for use in humans or animals by a primary approval process. In further embodiments, the target biologic is a reference listed drug for a secondary approval process. An exemplary target protein is an antibody, e.g., humanized or human antibody. Other target proteins include glycoproteins, cytokines, hematopoietic proteins, soluble receptor fragments, and growth factors.
[0052] As used herein, the term “evaluating” refers to reviewing, considering, determining, assessing, measuring, and/or detecting the presence, absence, level, and/or ratio of one or more parameters in a test protein and/or target biologic to provide information pertaining to the one or more parameters. In some instances, evaluating a glycoprotein preparation includes detecting the presence, absence, level, or ratio of one or more points of similarity between a test protein and a target biologic.
[0053] As used herein, the term “analyzing” refers to performing a process that involves a physical change in a sample or another substance, e.g., a starting material. Exemplary changes include making a physical entity from two or more starting materials, shearing or fragmenting a substance, separating or purifying a substance, combining two or more separate entities into a mixture, or performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond. Analyzing a sample can include performing an analytical process which includes a physical change in a substance, e.g., sample, analyte, or reagent (sometimes referred to herein as “physical analysis”), performing an analytical method, e.g., a method which includes one or more of the following: separating or purifying a substance, e.g., an analyte, or a fragment or other derivative thereof, from another substance; combining an analyte, or fragment or other derivative thereof, with another substance, e.g., a buffer, solvent, or reactant; or changing the structure of an analyte, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom of the analyte; or by changing the structure of a reagent, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom of the reagent.
[0054] As used herein, the phrase “input value” refers to a value associated with a parameter of a test biologic. The value can be qualitative, e.g., present, absent, intermediate, or the value can be qualitative, e.g., it can be a numerical value such as a single number, or a range, for a parameter.
General Method of the Disclosure
[0055] The methods of the disclosure can be used for analytically determining similarity of a recombinant protein (e.g., test protein) to a parent innovator biologic product (e.g., target protein) throughout the development and manufacture of biosimilar therapeutic molecules.
[0056] Non-limiting applications of the method include use in determining the similarity of recombinant proteins to biologic products including, but not limited to, Adalimumab (Humira®), Bevacizumab (Avastin®), Denosumab (Xgeva®), Cetuximab (Erbitux®); Rituximab (Rituxan®); Mabthera®; Campath®; Herceptin®; Xolair®; Prolia®; Vectibix®; ReoPro®; Zenapax®; Simulect®; Synagis®; Remicade®; Mylotarg®; Campath®; Raptiva®; Zevalin®; Erbitux®; Tysabri®; Lucentis®; Soliris®; Cimzia®; Ilaris®; Arzerra®; Bexxar®; Simponi®; Actemra®; Benlysta®; Adcetris®; and Yervoy®, as well as other biologics.
[0057] The method provides an analysis for evaluating the primary structure of a test protein, either for analyzing a test protein or a target protein, and/or for analyzing the test protein in comparison to a target protein. This method provides up to 100% amino acid sequence coverage and accuracy.
[0058] In an embodiment, a test protein, such as a recombinant protein that can include variants, can be analyzed. In a non-limiting alternative embodiment, a test protein can optionally be initially purified by column chromatography, e.g., HPLC, to separate the biologic from basic and acidic variants of the biologic and/or any other by-products of manufacture of the biologic, e.g., enzymes, cells, and cellular debris, etc. The biologic, its variants, and related manufacturing by-products can be processed through a chromatographic system, e.g., cation-exchange column, that is capable of high-efficiency, high resolution separation of closely eluting proteins.
[0059] In a further non-limiting example, the disclosure provides methods for identifying and confirming the primary structure of the test protein—a monoclonal antibody (e.g., ONS-3010 a biosimilar to the monoclonal antibody Adalimumab (Humira®))—for characterization.
[0060] In accordance with the general methods of the disclosure, the test protein and/or target protein, e.g., monoclonal antibody (mAb), is denatured, reduced, alkylated, and spun down through a 10 kDa centrifuge filter. This involves optimal digestion and complete sequence coverage by solubilization of the test protein, denaturation of the test protein, and disulphide bond reduction. Discrete peptides are selectively fragmented by using trypsin (Try) and chymotrypsin (Chy). This peptide mixture is injected onto a reverse-phase ultra-high performance liquid chromatography (RP-UPLC) system to obtain a unique profile (peptide map). The exact mass charge ratios (m/z) of the peptides are determined by full scan on a high resolution mass spectrometer. The peptide is then broken into ion fragments for MS/MS amino acid sequence analysis. The MS/MS data can be analyzed by using, for example, Proteome Discoverer software against an Adalimumab amino acid sequence database to identify peptide sequence. The similarity of amino acid sequences between reference product or target product, and test product is reported.
[0061] With its application of identifying sequences, the Proteome Discoverer software extracts relevant MS/MS spectra from the “.raw” file and determines the precursor charge state and the quality of the fragmentation spectrum. The SEQUEST search algorithm correlates experimental MS/MS spectra through comparisons to theoretical MS/MS spectra from protein databases. The Proteome Discoverer uses a probability-based scoring system to rate the relevance of the best matches found by the SEQUEST algorithm. The algorithm color codes the amino acid table to show the portion of the corresponding peptide sequence that is identified. Green, yellow, and pink indicate high, medium, and low confidence, respectively. No color means no hit on the peptide. The Protein Results View highlights the fragment ions in a peptide MS/MS spectrum that match predicted fragment masses. Specifically in ), a long broken line (
), and a short broken line (......) instead of color, respectively.
[0062] In a specific example, two separate aliquots of the monoclonal antibody (target protein, e.g., Adalimumab and/or bioequivalents) are prepared at a concentration of ≥3.0 mg/mL by transferring water into a 1.5 mL polypropylene centrifuge tube and adding 300 μg of sample into the tube. Negative controls (e.g., formulation buffer or HPLC-grade water) and positive controls (e.g., reference standard) are also prepared as a reference. The peptide standard mixture used as an instrument system suitability control is a 20 μg/mL HPLC peptide standard mixture prepared by adding 2.5 mL of Mobile Phase A (see below) to one vial of standard mixture (Sigma, Cat #H2016-1VL).
[0063] Each aliquot of monoclonal antibodies is denatured by adding a mixture of 500 μL of 8N Guanidine HCl (Fisher, Cat #24115), 40 μL of 2.5 M Tris base (3.03 g of Tris base in HPLC water to a final volume of 10 mL), and 20 μL of 1N HCl (Fisher Scientific, Cat #SA48-1 or equivalent) into each tube.
[0064] A stabilizing reagent, e.g., Dithiothreitol (DTT), is added to each sample of the denatured protein under conditions that promote the disruption of disulfide bonds of the denatured protein. Each sample of the denatured monoclonal antibodies is then reduced by adding 20 μL of 25 mg/mL of Dithiothreitol (DTT) (Bio-Rad, Cat #161-0611) (e.g., 25 mg of DTT in HPLC Grade water (Fisher Scientific, Cat #W5-4) to a final volume of 1.0 mL) to each sample. The samples are incubated separately at about 37±2° C. for about 0.5 hour.
[0065] At the end of the incubation, each sample undergoes alkylation by adding 8 μL of 200 mg/mL sodium iodoacetate (Sigma, Cat #12512) (e.g., 200 mg of sodium iodoacetate mixed with HPLC water to a final volume of 1 mL) to each sample, and then the samples are incubated in the dark at ambient temperature for about 15 minutes.
[0066] At the end of the alkylation incubation, each sample is desalted. The desalting process involves washing each sample in a Millipore Biomax-10 kDa Ultrafree 0.5 Centrifuge filter. The filter is initially wetted by centrifuging about 300 μL ammonium bicarbonate centrifuged at 10,000 rpm for 5 minutes. Each sample is transferred to the surface of a pre-wetted filter and is then washed with 300 μL of 0.1 M ammonium bicarbonate and centrifuged at 10,000 rpm for 10 minutes. The wash step can be repeated up to 2 or more times. The final wash involves centrifugation for about 10-13 minutes at 10,000 rpm so that each sample has a final volume of about 100 μL.
[0067] The desalted samples are then enzymatically digested by adding a different protease, e.g., trypsin and chymotrypsin, to the samples at optimized incubation conditions that include a reduced time frame for digestion, e.g., up to 0.5 hour for trypsin and up to 1.5 hours for chymotrypsin. The reduced incubation times are shorter in duration than traditional incubations times, e.g., 2-4 hours and even up to 18 hours. The shorter digestion time period provides more instances of specific miscleavage of amino acids so that the glycoprotein produced by digestion comprises longer peptides, rather than shorter peptides produced by longer digestion time. The digestion occurs in two desalted samples individually, namely, one sample is digested using trypsin that is quenched after passage of a first incubation time period, e.g., 0.5 hour, and then a second sample is digested using chymotrypsin that is quenched after passage of a second incubation time, e.g., 1.5 hours. This use of chymotrypsin protease digestion supports the coverage for a small peptide EAK in light chain and a small peptide SLR in heavy chain to archive 100% sequence coverage. During the digestion, the polypeptide chain of the denatured protein is cut into shorter fragments as the enzymes split peptide bonds that link amino acid residues in the denatured protein.
[0068] The first sample undergoes proteolytic digestion with trypsin. For example, trypsin (sequence grade modified, Promega, Cat #V5111, 20 μg) can be reconstituted in 20 μL of reconstitution buffer. 15 μL of reconstituted trypsin is added to each sample to reach a ratio of trypsin-to-sample ratio of about 1:20. The sample with trypsin added is incubated at 37° C. for 0.5 hour and then about 5 μL of 10% Formic acid (v/v) (Thermo, Product #28905 or equivalent, e.g., 100 μl of formic acid in 900 μl of HPLC grade water) is added to each sample to quench the enzymatic digestion in preparation for the second digestion.
[0069] The second sample undergoes proteolytic digestion with chymotrypsin. For example, chymotrypsin (Promega Chymotrypsin Sequencing Grade, 25 μg, Cat #V1062) can be reconstituted into 20 μL of 1 mM of HCl in HPLC-grade water (Fisher Scientific, Cat #SA48-1 or equivalent) reconstitution buffer. About 15 μL of reconstituted chymotrypsin is added to each sample at a chymotrypsin-to-sample ratio of about 1:16. The sample with chymotrypsin added is incubated at 37° C. for 1.5 hours and then about 5 μL of 10% Formic acid (v/v) is added to each sample to quench the enzymatic digestion.
[0070] The samples of digested monoclonal antibodies are then run separately through UPLC-MS/MS for analysis under conditions that promote adsorption of the peptides to the column including smaller chain peptides that are less likely to be bound under normal conditions. For example, the UPLC column can be an UPLC column (Waters BEH300 C18, 2.1×100 mm, 1.7 μm, Cat #186003555) having a pre-column (VanGuard, BEH300 C18, 2.1×5 mm, 1.7 μm, Cat #186003975). Samples are injected into the column at a volume of about 10 μL having a protein concentration of about 3 μg/μL at a temperature of about 45° C. After the sample is loaded on the column, a gradient consisting of Mobile Phase A (0.1% TFA in water (Optima LC/MS, Fisher Scientific, Cat #LS119)) and Mobile Phase B (0.085% TFA in 95% Acetonitrile (ACN) (v/v)) (HPLC grade, Fisher Scientific, Cat #A-998-4 or equivalent), and are passed through the column at a flow rate of about 400 μL/min. The UPLC system parameters are as follows: the autosampler is set at about 4° C.; data rate 20 pts/sec; PMT gain at 1; and PDA wavelength 210 nm and 280 nm.
[0071] The gradient for the peptide standard mixture is run as follows:
TABLE-US-00001 Time (min) % Mobile Phase A % Mobile Phase B 0 100 0 7 67 33 7.5 59 41 8 30 70 8.5 30 70 9 100 0 10 100 0
[0072] The gradient for a sample is run as follows:
TABLE-US-00002 Time (min) % Mobile Phase A % Mobile Phase B 0 100 0 10 100 0 15 98 2 75 67 33 85 60 40 90 30 70 95 30 70 96 100 0 98 100 0
[0073] The sample batch may be set as follows:
TABLE-US-00003 Description Method # Injections Equilibration Run Equilibration 1 Water Peptide std. 2 Peptide Standard Peptide std. 2 Water Peptide std. 2 Trypsin-Buffer Blank Peptide 98 min run-MS/MS 1 Water Peptide std. 2 Trypsin-ONS3010- Peptide 98 min run-MS/MS 1 Ref Std. Water Peptide std. 2 Trypsin-ONS2010- Peptide 98 min run-MS/MS 1 Sample Water Peptide std. 2 Chymotrypsin-Buffer Peptide 98 min run-MS/MS 1 Blank Water Peptide std. 2 Chymotrypsin- Peptide 98 min run-MS/MS 1 ONS3010-Ref Std. Water Peptide std. 2 Chymotrypsin- Peptide 98 min run-MS/MS 1 ONS3010- Sample Water Peptide std. 2 Peptide Standard Peptide std. 1 Water Peptide std. 2 Water Shutdown 1
[0074] Amino Acid Sequence Coverage Search Proteome Discoverer 1.3 was used for data analysis of each UPLC run.
[0075] Data acceptance, record, and report included exact mass calibration followed by application of the peptide standard layout of Xcalibur onto peptide standard injections. Record 5 peaks of RT of peptide standard injection on MS chromatograms, calculate % RSD of each peak RT individually. Record exact mass of selected peptide at m/z 532 and calculate the mass accuracy for all injections.
[0076] The peptide standard layout was as follows:
EXAMPLES
Example 1: ONS-3010/ONS-1045
[0077] ONS-3010 (lot #X1302-BDS-O) and ONS-1045(lot #1407104501) were prepared in accordance with the disclosed methods by implementing the reduced digestion time (0.5 hour for trypsin) and by testing the digested peptides with mass spectroscopy using the 98 minute methodology to allow time for peptides to bind to the column. Purified samples of ONS-3010 and ONS-1045, which were analyzed using an 88 minute methodology, were analyzed using the 98 minute methodology, and the results were compared against results obtained using the 88 minute methodology. A summary of the improved sequence coverage is provided in Table 1.
TABLE-US-00004 TABLE 1 Sequence Coverage Improvements with 0.5 hour Trypsin Digestion and 98 Minute UPLC Method ONS-3010 Heavy Chain Light Chain 0.5 hour Trypsin digestion + 100.00% 98.60% 98 min instr method 1 hour Trypsin digestion + 95.79% 97.20% 88 min instr method ONS-1045 Heavy Chain Light Chain 0.5 hour Trypsin digestion + 100.00% 98.60% 98 min instr method 1 hour Trypsin digestion + 96.91% 98.60% 88 min instr method
[0078] Trypsin digested ONS-3010 heavy chain sequence coverage increased from 95.79% to 100% and trypsin digested ONS-1045 heavy chain sequence coverage increased from 96.91% to 100%.
[0079] ONS-1045 samples #1407104501 and #1408104502 were analyzed according to the methods of the disclosure using mass spectroscopy implementing the 98 minute UPLC methodology. Samples were frozen at −80° C. and re-injected on the same instrument with the same mobile phases, but with the 98 minute UPLC method, which includes an extra 10 minutes of 0.4 mL/min 100% mobile phase A at the beginning of the gradient. The prior method implemented an 88 minute run without an extra 10 minutes of mobile phase A at the start of the run. According to the results of reinjections, the 98 minute UPLC method increased amino acid sequence coverage of the trypsin digested samples. Furthermore, the peptide CK was detected as cleaved peptide CKVSNK, but was missed in the 88 minute method.
[0080] To evaluate the specificity of the assay, matrix, positive control (Humira), and negative control (Rituxan) were used for specificity determination. Matrix showed no hit from target sequence. Positive control had 100% sequence coverage against Adalimumab sequence on both heavy chain (HC) and light chain (LC) after combining the results of the analysis of the trypsin and chymotrypsin digested samples. Negative control showed no sequence coverage on Fab region against Adalimumab sequence. Some peptides were detected in negative control because the constant region amino acid sequence of different IgG1 is the same. Certain figures herein show either sequence coverage results from Proteome Discoverer or total ion chromotograms. The general sequence coverage of each sample is recorded in Table 2.
TABLE-US-00005 TABLE 2 Sequence Coverage Summary for Specificity Heavy Chain Light Chain Try Chy Overall Try Chy Overall Matrix 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% ONS-3010 100.0% 100.0% 100.0% 98.6% 89.3% 100.0% Ref Std. Positive 98.9% 84.1% 100.0% 98.6% 89.3% 100.0% Control - Humira (H35) ONS-3010 98.9% 87.8% 100.0% 98.6% 89.3% 100.0% Sample Negative 69.4% 50.8% 72.1% 48.6% 45.8% 50.0% Control - Rituxan (M6)
[0081] To evaluate the reproducibility of the assay, an ONS-3010 reference standard was tested in each run and repeated for 3 runs. The heavy chain and light chain sequence coverage of the reference standards and samples from both trypsin and chymotrypsin digestions are recorded in Table 3.
TABLE-US-00006 TABLE 3 Reproducibility for ONS-3010 Sequence Method Heavy Chain Light Chain Try Chy Overall Try Chy Overall #1 Ref. Std. 100.0% 86.3% 100.0% 98.6% 89.3% 100.0% #1 Sample 98.9% 87.8% 100.0% 98.6% 89.3% 100.0% #2 Ref. Std. 99.3% 81.2% 100.0% 98.6% 89.3% 100.0% #2 Sample 99.3% 86.3% 100.0% 98.6% 89.3% 100.0% #3 Ref. Std. 99.3% 81.2% 100.0% 98.6% 89.3% 100.0% #3 Sample 99.3% 81.2% 100.0% 98.6% 89.3% 100.0%
[0082] While the disclosure has been described above in conjunction with specific embodiments, alternatives, modifications, permutations, and variations will become apparent to a person of skill in the art in light of the foregoing description. Accordingly, it is intended that the present invention embraces all such alternatives, modifications, and variations as falling within the scope of the claims below.