COMPOSITION AND FORMULATION COMPRISING RECOMBINANT HUMAN IDURONATE-2-SULFATASE AND PREPARATION METHOD THEREOF
20180112199 ยท 2018-04-26
Inventors
- Thong-Gyu Jin (Seoul, KR)
- Yo Kyung Chung (Yongin-si, KR)
- Sang Hoon Paik (Yongin-si, KR)
- Yoo Chang Park (Yongin-si, KR)
- Jinwook Seo (Yongin-si, KR)
- Yong Woon Choi (Yongin-si, KR)
- Jong Mun Son (Yongin-si, KR)
- Yong-Chul Kim (Yongin-si, KR)
Cpc classification
A61K9/0019
HUMAN NECESSITIES
A61K38/465
HUMAN NECESSITIES
International classification
A61K47/26
HUMAN NECESSITIES
Abstract
A composition comprising recombinant iduronate-2-sulfatase (IDS) and a method for producing a purified recombinant IDS are provided. The glycosylation pattern and formylglycine content of the IDS composition are different from those of ELAPRASE and have superior pharmaceutical efficacy and are safer than the conventional agent and thus can be effectively used for the therapy of Hunter Syndrome.
Claims
1. A method for treating Hunter syndrome in a subject in need thereof, the method comprising administering to said subject a pharmaceutical composition comprising an effective amount of a purified recombinant iduronate-2-sulfatase (I2S) having the amino acid sequence of SEQ ID NO: 1 and a carrier, wherein the purified recombinant I2S comprises at least 75% conversion of the cysteine residue corresponding to Cys59 of SEQ ID NO:1 to Ca-formylglycine (FGly), and wherein the purified recombinant I2S has a purity of at least 99.9% as measured using size exclusion high performance liquid chromatography (SE-HPLC).
2. The method of claim 1, wherein the purified recombinant I2S comprises at least 80% conversion of the cysteine residue corresponding to Cys59 of SEQ ID NO:1 to Ca-formylglycine (FGly).
3. The method of claim 1, wherein the pharmaceutical composition is administered intravenously.
4. The method of claim 3, wherein the pharmaceutical composition is administered by intravenous injection.
5. The method of claim 3, wherein the pharmaceutical composition is administered at a dose of 0.5-1.0 mg purified recombinant I2S/kg body weight.
6. The method of claim 5, wherein the dose is 0.5 mg purified recombinant I2S/kg body weight.
7. The method of claim 5, wherein the dose is 1.0 mg purified recombinant I2S/kg body weight.
8. The method of claim 1, wherein administration of the pharmaceutical composition results in a reduction of glycosaminoglycans within lysosomes in the subject.
9. The method of claim 1, wherein the purified recombinant I2S is safe and efficacious.
10. The method of claim 1, wherein the purified recombinant I2S is 99.9% pure or higher as characterized by silver stain SDS-PAGE.
11. The method of claim 1, wherein the purified recombinant I2S is 99.9% pure or higher as characterized by SYPRO stain SDS-PAGE.
12. The method of claim 1, wherein the purified recombinant I2S is 100% pure as measured using size exclusion high performance liquid chromatography (SE-HPLC), wherein analytes were loaded onto a TSK G3000SWx1 column linked to a TSK SWXL guard column.
13. A method for treating Hunter syndrome in a subject in need thereof, the method comprising administering to said subject a pharmaceutical composition comprising an effective amount of a purified recombinant iduronate-2-sulfatase (I2S) having the amino acid sequence of SEQ ID NO: 1 and a carrier, wherein the purified recombinant I2S comprises at least 75% conversion of the cysteine residue corresponding to Cys59 of SEQ ID NO:1 to Ca-formylglycine (FGly), and wherein the purified recombinant I2S has a Ku.sub.ptake value of 18nM or less.
14. The method of claim 13, wherein a Lineweaver-Burk plot of the purified recombinant I2S has an x-intercept of approximately 0.25, wherein the x-intercept is the negative reciprocal of the K.sub.uptake.
15. The method of claim 13, wherein the purified recombinant I2S has approximately 3.0 moles of mannose-6-phosphate (M6P) per mole of purified recombinant I2S.
16. The method of claim 13, 14, or 15 wherein the pharmaceutical composition is administered intravenously.
17. The method of claim 16, wherein the pharmaceutical composition is administered by intravenous injection.
18. The method of claim 16, wherein administration of the pharmaceutical composition results in a reduction of glycosaminoglycans within lysosomes in the subject.
19. A method for treating Hunter syndrome in a subject in need thereof, the method comprising administering to said subject a pharmaceutical composition comprising an effective amount of a purified recombinant iduronate-2-sulfatase (I2S) having the amino acid sequence of SEQ ID NO: 1 and a carrier, wherein the purified recombinant I2S comprises at least 75% conversion of the cysteine residue corresponding to Cys59 of SEQ ID NO:1 to Ca-formylglycine (FGly), and wherein the purified recombinant I2S has a specific activity of 19-55 nmol/min/pg as determined by an in vitro fluorescent assay using 4-methylumbelliferyl-L-iduronide-2-sulfate Na2 (MU-IdoA-2S) as a substrate.
20. A method for treating Hunter syndrome in a subject in need thereof, the method comprising administering to said subject a pharmaceutical composition comprising an effective amount of a purified recombinant iduronate-2-sulfatase (I2S) having the amino acid sequence of SEQ ID NO: 1 and a carrier, wherein the purified recombinant I2S comprises at least 75% conversion of the cysteine residue corresponding to Cys59 of SEQ ID NO:1 to Ca-formylglycine (FGly), and wherein the purified recombinant I2S has a specific activity of 30-70 nmol/min/g as determined by an in vitro fluorescent assay using 4-methylumbelliferyl-L-iduronide-2-sulfate Na2 (MU-IdoA-2S) as a substrate.
21. A method for treating Hunter syndrome in a subject in need thereof, the method comprising administering to said subject a pharmaceutical composition comprising an effective amount of a purified recombinant iduronate-2-sulfatase (I2S) having the amino acid sequence of SEQ ID NO: 1 and a carrier, wherein the purified recombinant I2S comprises at least 75% conversion of the cysteine residue corresponding to Cys59 of SEQ ID NO:1 to Ca-formylglycine (FGly), and wherein the purified recombinant I2S has an isoelectric point of 3.5 or less.
22. The method of claim 21, wherein the purified recombinant I2S contains on average at least 16 sialic acids per molecule.
Description
DESCRIPTION OF DRAWINGS
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MODE FOR INVENTION
[0087] A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as limiting the present invention.
[0088] According to the method below, human iduronate-2-sulfatase (IDS) was prepared by DNA recombination method. The method of preparation is briefly described in
TABLE-US-00001 TABLE 1 Category GC1111 Elaprase Manufacturer Green Cross Corp. Shire (GCC) Generic name Idursulfase beta Idursulfase Amino acid 525 AAs, 525 AAs, identical to human identical to human IDS, IDS, glycoprotein glycoprotein Formulation/Dose Liquid, 6 mg/3 mL/vial Liquid, 6 mg/3 mL/vial Host cell CHO-DG44 Human cell line HT- 1080 Expression vector pJK-dhfr-Or2-IDS pXI2S 1 MCB/WCB preparation Serum-free Bovine serum used Culture method Suspension culture, Continuous culture, fed-batch, serum-free bovine serum used Distillation process 2 UF processes, 2 UF processes, 4 column processes 6 column processes Virus inactivation Yes No process M6P content 3.0 mol/mol 2.0 mol/mol (cellular uptake) Formylglycine content 80 15% 50% (substrate degradation) Purity 99.9% or higher 99.9% or higher Column, SDS-PAGE Column, (silver, SYPRO) SDS-PAGE characterization, spatial conformation
[0089] Purity >99.9%: The degree of purity of GC1111 is expected to be higher than that of Elaprase and, thus, it is predictable that the stability related with adverse effects due to the presence of impurities and the overall effectiveness thereof will be enhanced.
[0090] Based on the criteria and the testing methods of purity analysis, characterization and the study of crystallization for spatial conformation, the absolute purity of the GC1111 is expected to be at least 99.9%.
EXAMPLE 1
Preparation of IDS
[0091] <1-1> Gene Acquisition
[0092] Peripheral blood mononuclear cells (PBMC) were isolated from human blood as described previously [S. Beckebaum et al., Immunology, 2003, 109:487-495]. Total RNA was extracted from the PBMC according to a protocol described previously [M. J. Holland et al., Clin. Exp. Immunol., 1996, 105:429-435]. In order to construct a cDNA library from the total RNA, single-stranded cDNA was synthesized using oligo-(dT) primer with the aid of a single-strand synthesis kit (Boehringer mannheim). In this regard, DEPC-treated distilled water was added to an eppendorf tube containing 1 g of the total RNA so as to form a final volume of 12.5 L. Then, 1 L of a 20 pmol oligo(dT) primer was added to the tube, followed by incubation at 70 C. for 2 min and cooling. To this reaction mixture were added 4 L of a reaction buffer, 1 L of dNTP, 1 L of an RNase inhibitor, and 1 L of reverse transcriptase which were then reacted at 42 C. for one hour to synthesize single stranded cDNA. PCR was performed on the cDNA as a template in the presence of primers of SEQ ID NOS: 2 to 4 to amplify a human IDS gene. In this context, each primer was designed to contain a restriction enzyme recognition site for use in gene cloning.
[0093] <1-2> Construction of Expression Vector
[0094] A. Construction of pJK-dhfr-IDS-S1 Vector
[0095] A light chain signal sequence of an antibody (derived from a part of the human IgG light chain) as a non-coding sequence was introduced into the 5-terminus of the IDS gene acquired by Example <1-1> before PCR. After the PCR product obtained thereby was run on gel by electrophoresis, the human IDS gene was isolated using a gel extraction kit. The isolated IDS gene and the pJK-dhfr-Or2 vector (Aprogen) were digested with EcoRV and ApaI and ligated to each other at 16 C. for 20 hours. The recombinant vector thus constructed was transformed into E. coli (DH5) which was then spread over an LB plate containing 50 g/mL ampicillin and incubated overnight. Colonies grown on the plates were selected and cultured so as to isolate the plasmid therefrom (
[0096] B. Construction of Recombinant Human IDS Expression Plasmid
[0097] In order to change the non-coding sequence of the plasmid constructed above to a signal sequence, the recombinant human IDS was subcloned to a pJK-dhfr-or2 vector. To this end, the pJK-dhfr-IDS-S1 vector was digested with EcoRV and ApaI to give a partial IDS gene (1233 bp) which was then inserted into the pJK-dhfr-Or2 vector previously treated with the same restriction enzymes, to construct a pJK-dhfr-IDS-S2 vector. In order to introduce a non-coding sequence and a signal sequence to the 5-terminus, an IDS N1 forward primer (SEQ ID NO: 5) and an IDS 4 reverse primer (SEQ ID NO: 7) were used for PCR with the pJK-dhfr-IDS-S1 vector serving as a template. After starting at 94 C. for 5 min, PCR was performed with 30 cycles of 94 C. for 1 min, 55 C. for 30 sec and 72 C. for 40 sec and finished by extension at 72 C. for 10 min.
[0098] The PCR amplification afforded a partial IDS gene that was 448 bp. This gene was used as a template for the PCR which was performed again in the presence of an IDS N2 forward primer (SEQ ID NO: 6) and an IDS 4 reverse primer (SEQ ID NO: 7) under the same conditions as described above. This resulted in the synthesis of a DNA fragment 476 bp long.
[0099] Subsequently, the pJK-dhfr-IDS-S2 vector and the recombinant human IDS gene fragment (476 bp) were separately digested with EcoRV. The digests were separated on gel by electrophoresis to obtain the vector and the 476 bp-long IDS fragment. These vector and insert were ligated at 16 C. for 12 hours in the presence of T4 DNA ligase to construct pJK-dhfr-Or2-IDS plasmid. These procedures are illustrated in
[0100] To confirm the construction of the IDS expression plasmid, DH5 was transformed with pJK-dhfr-Or2-IDS and cultured for 24 hours on an LB plate containing ampicillin (50 g/mL). From the colonies thus formed, a plasmid was isolated and digested to measure the size of the insert. Also, base sequencing was conducted using a T7 primer (SEQ ID NO: 8).
[0101] <1-3> Selection of Recombinant Human IDS Expression Cell Line
[0102] A. Transfection of CHO-DG44
[0103] CHO-DG44 was used as a host cell for expressing the IDS of the present invention. The mutant Chinese hamster ovary cell CHO-DG44 carries a double deletion for the endogenous dhfr (dihydrofolate reductase) gene which encodes DHFR enzyme. The DHFR enzyme is involved in the conversion of folate through dihydrofolate (FH2) into tetrahydrofolate (FH4) which is involved in the de novo synthesis of nucleic acids. The level of dhfr in the cells is dependent on the concentration of MTX. MTX, which is structurally similar to folic acid, a substrate of DHFR, competes with folic acid for binding dihydrofolate reductase, so that most dihydrofolate reductase loses its activity in the presence of MTX. Hence, if cells do not amplify a sufficient amount of dhfr, they die because they cannot synthesize nucleic acids necessary for their life. In contrast, if the amplification is sufficient, the cells can survive under a high concentration of MTX because they are relatively abundant in dhfr. This system may be applied to animal cells to select a transfected cell line which can amplify the dhfr gene and thus a structural gene of interest.
[0104] To this end, a dhfr gene was introduced as an amplifiable marker into the IDS expression vector pJK-dhfr-Or2-IDS, constructed in Example 1-2, and gene amplification was conducted using MTX and the dhfr gene.
[0105] In this regard, the DG44 cell line (obtained from Dr. Chaisin, Columbia University) was suspended in 10 mL of DMEM/F12 (supplemented with nucleotides and nucleosides, and 10% fetal bovine serum (FBS)) and harvested by spinning at 1000 rpm for 5 min. The cells were inoculated into 50 mL of a culture medium in a T-175 flask and incubated at 371 C. in a 51% CO.sub.2 incubator. One day before transfection, the culture medium for DG44 cells was removed from the T-175 flask and the cells were washed twice with PBS and detached by trypsinization. Then, they were seeded at a density of 510.sup.5 cells into a T-25 flask and cultured at 371 C. for 24 hours in a 51% CO.sub.2 incubator. Bacterial or fungal contamination was examined under an optical microscope while PCR-ELISA was performed to examine whether the cells were contaminated with mycoplasma.
[0106] The germ-free DG-44 cells were transfected with the IDS expression vector pJK-dhfr-Or2-IDS, constructed in Example 1-2, using a Lipofectamine kit. In this regard, 5 g of the expression vector and 50 L of Lipofectamine were separately diluted in 800 L of Opti-MEM I, mixed carefully so as not to form bubbles, and left at room temperature for 15 min. Meanwhile, DG44 cells were washed once with sterile PBS and three times with Opti-MEM I. To the DG44 cells were carefully added the DNA-lipofectamine mixture and then 6.4 mL of Opti-MEM before incubation at 371 C. for 5 hours in a 51% CO.sub.2 incubator. Thereafter, the incubation was conducted for an additional 48 hours in the medium supplemented with 8 mL of DMEM/F12 and 1.6 mL of FBS to promote the recovery of cell membranes and the growth of cells.
[0107] B. Selection of Geneticin(G418)-Resistant Cell Line
[0108] The cultured cells were detached with 0.25% trypsin, counted, and seeded at a density of 510.sup.3 cells/well into 96-well plates containing 100 L of MEM-alpha medium (supplemented with 10% dialyzed FBS and 550 g/mL G418) per well. Next day, the same medium was added in an amount of 100 L/well and the cells were cultured for 2-3 weeks to form colonies. When the cells grew to 50% confluency, the medium was replaced with a fresh one. After maintenance for 3 days, the culture media were collected for enzyme analysis.
[0109] The medium was replaced with 200 L of a fresh medium every three days. On day 3-4 after culturing, non-transfected cells, that is, cells that were not resistant to geneticin started to detach from the bottom of the 96-well plates when observed with an optical microscope. The selected clones were cultured while being sequentially transferred from the 96-well plates to 24-well plates, 6-well plates and 100-mm dishes in the order. When the cells grew to 8090% confluency in 100-mm dishes, the expression level was measured again. The cells were detached with 0.25% trypsin, counted and plated at a density of 510.sup.5 cells/well/3 mL into 6-well plates, maintained for 3 days and counted. The expression level of the protein was quantitatively analyzed. According to the analysis results, 15 clones were selected.
[0110] C. Selection of IDS Expression Cell Line with High Productivity
[0111] The 15 selected clones were cultured at an increased concentration of MTX to select cell lines in which IDS was amplified.
[0112] In this context, the cells were inoculated at a density of 110.sup.6 cells/100 mm dish/10 mL of a medium containing MTX and cultured to 8090% confluency. One tenth of the volume of the cell culture was inoculated again into 100 mm dish/10 mL. This sub-culturing process was repeated twice. The cells were allowed to undergo at least three passages so that they were sufficiently adapted to increased MTX concentrations. The concentration of MTX was increased, from 5 nM for the clones selected after conducting an analysis for the first three days, to 20 nM. In each step, the clones adapted to the increased MTX concentration were cultured for three days to measure cell growth rates. IDS expression levels were measured to select cell lines in which the amplification of the IDS gene took place, that is, cell lines in which the recombinant IDS was expressed at a high rate. Of the selected cell lines, NI4 was used in subsequent experiments because it had the highest expression level.
[0113] D. Selection of Single cell by Limiting Dilution
[0114] There was the possibility that the cell line NI4 might have become mixed with other cell lines. Hence, the cell line was separated into a single cell line. The N14 clones which survived 20 nM MTX were subcloned through limiting dilution so as to select a desired cell line.
[0115] First, NI4 was inoculated at a density of 0.5 cells/well into IMDM medium (Gibco BRL, Cat #12200) in 96-well plates and cultured with the medium replenished every three days. On day three, the plates were observed under a microscope to exclude the wells in which two or more colonies had been formed per well. The wells in which only one colony had formed per well were selected and continued to be cultured. After culturing for 15 days, the cells were sub-cultured to 96-well plates and when cells had grown to 90% confluency, the medium was freshly replenished.
[0116] A total of 263 single cell lines were identified from the N14cell line. Of them, cell line S46 was found to have the highest IDS activity and named NI4-S46.
[0117] <1-4> Cell Culture
[0118] A. Shake flask Culture
[0119] The NI4-S46 cell line was cultured on a large scale to produce the IDS of the present invention. The cell line was inoculated into an EX-cell 302 serum-free medium (containing glutamine, dextran sulfate, and poloxamer 188 in 125 mL culture flasks and cultured at 371 C. in a 51% CO.sub.2 incubator. Subsequently, the cells were passaged at a ratio of 1:11:8 every two to three days using shake flasks. Upon the passage, the culture volume was gradually increased to approximately 2,400 mL. In many shake flasks, the cells were cultured to a level sufficient to be inoculated into a bioreactor.
[0120] B. Culture in 30 L Bioreactor (Working Volume 20 L)
[0121] When the density of the cells in the shake flasks reached 1.310.sup.6 cells/mL, they were inoculated into a 30 L bioreactor. During cell culturing, the culture conditions were kept at a dissolved oxygen content of 10% or higher, a culture temperature of 371 C. and a pH of 7.00.2. If necessary, cell samples were taken and observed under a microscope. The cell culture was examined to analyze cell count, cell viability, pH, glucose concentration and glutamine concentration. On the basis of the analysis results, when it was decided that the cells were sufficiently grown, the cells were inoculated into a 150 L bioreactor.
[0122] C. Culture in 150 L Bioreactor (Working Volume 100 L)
[0123] When the cells in a 30 L bioreactor reached a density of 0.910.sup.6 cells/mL or higher, they were inoculated into a 150 L bioreactor. During cell culturing, the culture condition was kept at a dissolved oxygen content of 10% or higher, a culture temperature of 371 C. and a pH of 7.00.2. If necessary, cell samples were taken and observed under a microscope. The cell culture was examined to analyze cell count, cell viability, pH, glucose concentration and glutamine concentration. On the basis of the analysis results, when it was decided that the cells were sufficiently grown, the cells were inoculated into a 650 L bioreactor.
[0124] D. Culture in 650 L Bioreactor (Working Volume 500 L)
[0125] When the cells in a 150 L bioreactor reached a density of 0.910.sup.6 cells/mL or higher, they were inoculated into a 650 L bioreactor. During cell culturing, the culture condition was kept at a dissolved oxygen content of 10% or higher, a culture temperature of 341 C. and a pH of 6.90.2 for three days and then, at a culture temperature of 321 C. and a pH of 6.90.2. If necessary, cell samples were taken and observed under a microscope to analyze cell counts, cell viability, pH, glucose concentrations and glutamine concentrations. Depending on the analysis result, glucose and glutamine concentrations were adjusted to continue cell growth. During the culturing, a hydrolysate was added to increase the formylglycine conversion.
[0126] <1-5> Purification of IDS
[0127] IDS was isolated from the cell culture using a series of the following four chromatographic processes.
[0128] A. Harvest and Filtration of Culture Medium
[0129] When the cell viability remained in the range of 8085% 10 days after inoculation into the 650 L bioreactor, culturing was stopped. The cells were harvested from the culture using the Millipore POD filter system and DOHC filter (Millipore) at a pressure of 0.9 bar or less. After the cells were removed, the supernatant was filtered through a pre-filter (Millipore, 0.50.2 m) and a 0.450.2 m filter and recovered in a disposable sterile bag. The harvested culture solution was stored at 2-8 C.
[0130] B. Concentration and Diafiltration
[0131] The filtrate recovered in A was about 10-fold concentrated using an ultrafiltration system (Tangential Flow Filtration Membrane System). The membrane (cutoff: 30K, Pall) installed inside the ultrafiltration system was washed with WFI (water for injection) at a flow rate of 2025 L/min and then equilibrated with a buffer (pH 7.00.3) containing 20 mM sodium phosphate (sodium dihydrogen phosphate monohydrate and sodium hydrogen phosphate heptahydrate). After equilibration, the filtrate was fed into the membrane while recovering the fractions that did not pass the membrane. Once the recovered volume became about 1/10 of the initial volume of the filtrate, the concentration procedure was stopped. The buffer was consecutively exchanged in a volume three to four times as large as that of the concentrate. If the conductivity and the pH fell within the criteria, the process was stopped. [criteriaconductivity: 5.0 mS/cm, pH 7.00.2.
[0132] C. Anion Exchange Chromatography
[0133] To remove media component and various impurities from the concentrate recovered in B, anion exchange chromatography was conducted on a column (GE Healthcare) filled with Q Sepharose resins (GE Healthcare). The column was equilibrated with equilibrium buffer (pH 7.00.3) containing 20 mM sodium phosphate (sodium dihydrogen phosphate monohydrate and sodium hydrogen phosphate heptahydrate). The concentrate obtained in B was filtered through a 0.450.2 m filter (Sartorius) and loaded at a flow velocity of 100120 cm/h into the equilibrated column. After the loading was completed, the column was primarily washed with the equilibrium buffer and then with washing buffer (pH 7.00.3) containing sodium chloride. Subsequently, a target protein was eluted with an eluting buffer (pH 7.00.3) containing sodium chloride.
[0134] D. Hydrophobic Chromatography
[0135] To remove the media component and impurities that remained after anion exchange chromatography, hydrophobic chromatography was performed on a column (GE Healthcare) filled with phenyl Sepharose resins (GE Healthcare). The column was equilibrated with equilibrium buffer (pH 6.00.3) containing sodium chloride. The eluate obtained in C was filtered through a 0.450.2 m filter (Sartorius) and loaded at a flow velocity of 70100 cm/h into the equilibrated column. After the loading was completed, the column was washed with the equilibrium buffer. Subsequently, a target protein was eluted with an eluting buffer (pH 5.50.2) containing glycerol.
[0136] E. Inactivation of Virus by Low pH
[0137] Viruses that may be derived from host cells or any material used in the processes carried out were inactivated by a low pH condition. In this regard, the eluate obtained in D was maintained for 2 hours at an acid condition (pH: 3.70.05) of which acidity was adjusted with 25% acetic acid. Thereafter, the pH of the eluate was increased to pH: 4.30.2 using 0.5 M sodium hydroxide for use in the next process. The inactivation by low pH was conducted at 122 C.
[0138] F. Cation Exchange Chromatography
[0139] IDS is glycoprotein with oligosaccharides, and exists as an isomer that has a different isoelectric point according to the content of sialic acid at the end of the Glycan chain. As oligosaccharides with a negative charge, sialic acid shows a difference in terms of the degree of binding to cation exchange resin according to the content of sialic acid. Using this characterization, cation exchange chromatography was conducted to obtain IDS showing high activity (a high content of formylglycine) with a high content of sialic acid and to remove other impurities [Product impurity (Aggregated IDS, processed IDS), process impurity (Host Cell protein)]. In detail, a column filled with cation exchange Capto MMC resins (GE Healthcare) was equilibrated with glycerol-added equilibration buffer (pH 4.30.2). The inactivated eluate obtained in E was filtered through a 0.450.2 m filter (Sartorius) and loaded at a flow velocity of 100120 cm/h onto the equilibrated column. Subsequently, the column was washed with the equilibration buffer, followed by elution with glycerol-added eluting buffer (pH 5.30.2) to give IDS with a high sialic acid content (isoelectric point 3.5 or less), high activity (formylglycine content: 8015%) and high purity (SE-HPLC, 98% or higher).
[0140] G. Affinity chromatography
[0141] Affinity chromatography (Blue SEPHAROSE, GE Healthcare) was conducted to remove the glycerol used in the cation exchange chromatography and to reduce the volume of the eluate. The eluate obtained in F was filtered through a 0.450.2 m filter (Sartorius) and loaded at a flow velocity of 100120 cm/h onto a Blue SEPHAROSE resin-filled column (GE Healthcare) that was previously equilibrated with glycerol-added equilibration buffer (pH 4.50.2). After completion of the loading, the column was washed with washing buffer (pH 4.50.2) and the target protein was eluted with eluting buffer (pH 6.20.2).
[0142] H. Concentration and Buffer Exchange
[0143] An ultrafiltration system (Tangential Flow Filtration Membrane System) was used to adjust the protein concentration of the eluate obtained in G and to exchange the buffer of the purified protein with formulation buffer. The membrane (cutoff: 10K, Pall) installed inside the ultrafiltration system was washed with WFI (water for injection) at a flow rate of 450650 mL/min and then equilibrated with a formulation buffer (2.25 g/L sodium dihydrogen phosphate monohydrate, 0.99 g/L sodium hydrogen phosphate heptahydrate, 8 g/L sodium chloride, pH 6.00.2,) without polysorbate 20, followed by concentrating the target protein. The buffer was consecutively exchanged in a volume three to four times as large as that of the concentrate. If the conductivity and the pH fell within the criteria, the process was stopped. [criteriaconductivity: 15.03.0 mS/cm, pH 6.00.2]. Adjust the content of the concentrated solution to 4.00.5 mg/mL.
[0144] I. Nanofiltration
[0145] Using a nano filter (NFP, Millipore), nano filtration was performed to remove viruses that might have come from the host cells or any of the materials used. Integrity test for filter is performed after washing the nano filter with water for injection. Once the integrity test was passed, the nanofilter was equilibrated with 1 L of formulation buffer (2.25 g/L sodium dihydrogen phosphate monohydrate, 0.99 g/L sodium hydrogen phosphate, 8 g/L sodium chloride, pH 6.00.2) without polysorbate 20. After completion of equilibration, the concentrate obtained in H was passed through the filter at a pressure of about 2 bar to produce a nano-filtrate. After filtration was completed, the filter was washed with the formulation buffer (post wash solution). After combining the nano filtration solution and the post wash solution, protein content is measured.
[0146] J. Drug Substance
[0147] The protein concentration of the filtrate obtained in I was adjusted with formulation buffer without polysorbate 20. After the addition of polysorbate, the solution was filtered through a 0.2 m filter to produce a drug substance. The drug substance was aliquoted and stored in a deep freezer (7010 C.) until use.
[0148] K. Drug Product (Filling, labeling, Packaging)
[0149] The stock stored in a deep freezer was thawed in a water bath maintained at 281 C. and diluted to a protein concentration of about 2.050.2 mg/mL using formulation buffer (2.25 g/L sodium dihydrogen phosphate monohydrate, 0.99 g/L sodium hydrogen phosphate heptahydrate, 8 g/L sodium chloride, 0.23 g/L polysorbate 20, pH 6.00.3) Thereafter, the dilution solution was filtered through a 0.2 m filter to produce a final bulk solution. This final bulk solution was filled in 6 mL vial with approximately 3.3 g using auto filling. Once an vial inspection test was passed, the vials were packed to produce a drug product.
[0150] The procedure from cell line culturing to final product production is illustrated in
COMPARATIVE EXAMPLE 1
Preparation of ELAPRASE
[0151] ELAPRASE, commercially available recombinant IDS, was used as a comparative example.
EXPERIMENTAL EXAMPLE 1
Structural Analysis and Characterization of Inventive IDS
[0152] <1-1> Amino Acid SequencingInternal Sequencing
[0153] Deglycosylated IDS was separated by SDS-PAGE, followed by gel slicing. Then, digests resulting from treatment with various endoproteinases (trypsin, chymotrypsin, AspN, chymotrypsin/trypsin, AspN/trypsin, GluC and GluC/trypsin) were analyzed using MALDI-MS/MS and LC-ESI-MS/MS (
[0154] <1-2> Disulfide Bond Analysis
[0155] In a polypeptide, a disulfide bond is a covalent linkage, usually derived by the coupling of two SH groups of cysteine residues, playing an important role in stabilizing the higher structure of proteins. Theoretically, the 525 amino acids of IDS contain six cysteine residues, four of which form disulfide bonds. In this example, the location of cysteine residues responsible for the disulfide bonds of IDS was identified. First, IDS was deglycosylated by treatment with PNGase F to exclude the interference of sugars. In order to prevent the cysteine residues that do not take part in the formation of disulfide bonds from acting as an interfering factor, 4-vinylpyridine was used to convert IDS into a non-reduced sample so that the SH groups are restrained from randomly forming SS bonds. Meanwhile, the disulfide bonds were cleaved by DTT, followed by blocking with 4-vinylpyridine to give a reduced sample. Trypsin and AspN, selected on the result of Experimental Example 1-3, were applied to the non-reduced and the reduced sample. The peptide fragments thus obtained were separated by RP-HPLC. RP-HPLC chromatograms of the non-reduced and the reduced samples were compared so as to discriminate the peaks that were found in the non-reduced sample, but not in the reduced sample (
[0156] For more exact analysis, fractions at the discriminated peaks were reduced in size by additional treatment with endoproteinases, and the peaks containing disulfide bonds were analyzed using MALDI-MS (
[0157] Peaks with disulfide bonds were again sequence analyzed using MALDI-MS/MS (
[0158] <1-3> Analysis of Formylglycine Content
[0159] IDS degrades heparan sulfate and dermatan sulfate, both of which are a kind of glycosaminoglycan (GAG). This degradation activity is not acquired until the cysteine residue at position 59 in the active site (Cys59) is converted into formylglycine (FGly) by post-translational modification. Thus, the degradation activity of IDS was analyzed by examining the post-translational modification of Cys59 to FGly. For this analysis, AQUA (absolute quantification), a quantitative analysis method based on MS (Mass Spectroscopy), was used, in which a radio-labeled synthetic substrate (AQUA peptide) was spiked into a sample. To quantitatively analyze formylglycine at Cys59 position, a serial dilution of AQUA peptide was spiked into a sample and a calibration curve was drawn. Ratios of FGly-type peptide to Cys-type peptide were measured by LC-ESI-MS analysis, and applied to the AQUA calibration curve to calculate the content of formylglycine.
[0160] This analysis determined the conversion of Cys59 to FGly at a rate of 8015%. In consideration of the Cys59 to FGly conversion rate of about 50% in the commercially available agent ELAPRASE (Elaprase Science Discussion, EMEA, 2007; Genet Med 2006:8(8):465-473), the therapeutic composition comprising the IDS of the present invention and the formulation prepared with the composition is anticipated to have much higher therapeutic activity compared to ELAPRASE.
[0161] <1-4> Identification of Glycosylation Pattern
[0162] An assay was performed to examine whether the IDS of the present invention is glycosylated and to identify the glycosylation pattern if any. To this end, IDS was treated with various glycoside hydrolase enzymes, the digests were separated on by SDS-PAGE and their motility patterns were analyzed.
[0163] In detail, IDS samples were digested with combinations of the following four glycoside hydrolase enzymes and separated by SDS-PAGE.
TABLE-US-00002 TABLE 2 Properties of Sugar Cleaving Enzymes Function/Property PNGase F Cleaves a sugar moiety (N-glycan) from protein Asn at the cleavage site is converted into Asp Endo H Cleaves a sugar moiety (N-glycan) from protein unlike PNGase F, Endo H acts on oligosaccharides of high-mannose type and hybrid type O-Glycosidase Cleaves a sugar moiety (O-glycan) from protein Sialidase Cleaves terminal sialic acid residues of N-glycan or O-glycan
[0164] As can be seen in
[0165] <1-5> Analysis of Mannose-6-phosphate Content
[0166] Binding to a M6P receptor on cells, mannose-6-phosphate (M6P) allows IDS to be internalized into cells and thus to hydrolyze heparan sulfate or dermatan sulfate in lysosomes. In this Example, IDS was acid hydrolyzed with trifluoroacetic acid (TFA) and subjected to HPAEC-PAD (Bio-LC) to quantitatively analyze mannose-6-phosphate.
[0167] IDS was hydrolyzed with 6.75M TFA and the hydrolysate was analyzed using liquid chromatography (High Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection; HPAEC-PAD). M6P concentration of which was already known was analyzed under the same condition, and molar ratios of M6P to glycoprotein were obtained by comparison of the areas. Analysis was conducted in triplicate. M6P standard materials and M6P composition chromatograms of the IDS are shown in
TABLE-US-00003 TABLE 3 Analysis Results for Mannose-6-phosphate Content M-6-P Amount Amount Ratio M-6- Ret. time pmol/25 l pmol/25 l P/Protein Run No. (min) M-6-P Protein (mol/mol) 13 11.25 1320.59 428 3.09 14 11.23 1241.31 428 2.90 15 11.23 1245.83 428 2.91 Average 11.24 1269.25 428 2.97 CV 0.09% 3.51% 0.11
[0168] As is understood from the data of Table 3, there are approximately 3 moles of M6P per mole of IDS. From these results, it is inferred that the therapeutic composition comprising the IDS of the present invention and the formulation prepared with the composition have a high ability to catabolize GAG accumulated in lysosomes.
[0169] <1-6> Mass Analysis
[0170] Masses of glycosylated IDS and deglycosylated IDS were measured using MALDI-TOF-MS. Treatment of glycosylated IDS with PNGase F afforded deglycosylated IDS. MALDI-TOF-MS was performed using Voyager-DE PRO Biospectrometry (Applied Biosystems, USA) coupled with a delayed Extraction laser-desorption mass spectrometer. The instrument was normalized with bovine serum albumin and IgG1. Analysis results are summarized in Table 4, below.
TABLE-US-00004 TABLE 4 MALDI-TOF-MSMALDI-TOF-MS Analysis Results of IDS m/z Charge(z) Protein Mass (Da) Remark Glycosylated IDS 25646 3 76935 38708 2 77414 77360 1 77359 154533 1 77266 dimer Average 77244 210 Deglycosylated IDS 29767 2 59532 34655 PNGase F 59313 1 59312 118706 1 59353 dimer Average 59399 120 Sample Molecular Weight Theoretical 59298 Da Glycosylated 77244 210 Da Deglycosylated 59399 120 Da
[0171] As apparent from the data of Table 4, the molecular size is 77,244 Da for glycosylated IDS and 59,399 Da for deglycosylated IDS, which is similar to the molecular weight calculated on the basis of the amino acid sequence, which is 59,298 Da.
[0172] <1-7> Purity Measurement
[0173] The purity of IDS was measured using size exclusion chromatography. Size exclusion chromatography is a chromatographic method in which molecules in solution are separated by their relative molecular weight and shape. In size exclusion chromatography, proteins larger than the pore size of the column cannot penetrate the pore system and pass through the column at once. Subsequently, the analytes with smaller molecular weights or sizes elute later. For this chromatography, Alliance 2695 HPLC system (Waters, Wis., USA) coupled with 2487 UV/VIS detector (Waters, Wis., USA) was employed. Proteins were detected at 214 nm, and analyzed using Empower 2 Software. The analytes were loaded onto a TSK G3000SWXL column linked to a TSK SWXL guard column (Tosoh, Japan). IDS, after being diluted to a concentration of 1.0 mg/mL in a formulation buffer, was loaded in a volume of 10 L onto the column. They were allowed to flow with mobile phase (20 mM sodium phosphate buffer, 200 mM NaCl, pH 7.0) at a flow rate of 0.5 mL/min for 60 min.
[0174] Analysis results are shown in
[0175] <1-7a> Purity Measurement (2)
[0176] Reversed-phase high-performance liquid chromatography (RP-HPLC) involves the separation of molecules on the basis of hydrophobicity. The separation depends on the hydrophobic binding of the solute molecule from the mobile phase to the immobilized hydrophobic ligands attached to the stationary phase.
TABLE-US-00005 TABLE 5 RP-HPLC Operation Conditions Mobile A: Water + 0.1% (v/v) TFA Phase B: Acetonitrile + 0.1% (v/v) TFA Column Phenomenex Jupiter C4 (4.6 250 mm, 5 m) Flow Rate 0.8 mL/min Temperature Column: 30 C., Sampler: 4 C. Injection 10 L Volume Detector 214 nm Run Time 90 min Time Flow rate % A %B Gradient 0 0.8 70 30 10 0.8 70 30 70 0.8 30 70 75 0.8 10 90 80 0.8 70 30 90 0.8 70 30
[0177] <1-8> Activity Measurement Using Synthetic Substrate
[0178] The reaction of IDS with the synthetic substrate (4-methylumbelliferyl-L-idopyranosiduronic acid-2-sulfate sodium salt (4MU-IdoA-2S)) for 4 hours releases the sulfate moiety (primary reaction). After the primary reaction, the addition of recombinant human -L-iduronidase (rh IDUA) induces a secondary enzymatic reaction with the substrate 4-methylumbellifery-L-iduronide (reactant left after the release of the sulfate moiety in the primary reaction) to separate the 4-methylumbelliferyl moiety from the L-iduronide moiety. Because the remaining 4-methylumbelliferyl is fluorogenic, the activity of IDS was evaluated by measuring the intensity of fluorescence (Ex.355 nm/Em.460 nm). The IDS of the present invention was found to range in specific activity from 19 to 55 nmol/min/g. The IDS of the present invention was found to range in specific activity from 30.0 to 70.0 nmol/min/g. This activity indicates that formylglycine exists in the active site of the enzyme as a result of the post-translational modification of the cysteine residue at position 59 in IDS.
[0179] <1-8a> Activity Measurement Using Synthetic Substrate (2)
[0180] The reaction of IDS with the synthetic substrate (4-methylumbelliferyl-L-idopyranosiduronic acid-2-sulfate sodium salt (4MU-IdoA-2S)) for 90 minutes releases the sulfate moiety (primary reaction). After the primary reaction, the addition of recombinant human -L-iduronidase (rh IDUA) induces a secondary enzymatic reaction with the substrate 4-methylumbellifery-L-iduronide (reactant left after the release of the sulfate moiety in the primary reaction) to separate the 4-methylumbelliferyl moiety from the L-iduronide moiety. Because the remaining 4-methylumbelliferyl is fluorogenic, the activity of IDS was evaluated by measuring the intensity of fluorescence (Ex.355 nm/Em.460 nm). The IDS was found to range in K.sub.m from 170 to 570 M and in k.sub.cat from 4,800 to 16,200 min.sup.1. This activity indicates that formylglycine exists in the active site of the enzyme as a result of the post-translational modification of the cysteine residue at position 59 in IDS.
[0181] <1-9> Activity Measurement Using Natural Substrate
[0182] In order to determine whether the reaction with the IDS and natural substrate, the sulfate ions released from the substrate (heparin disaccharide) by reaction with IDS were measured. The reaction mixture was loaded onto an ion column (Vydac 302IC) and allowed to flow with the mobile phase of 0.49 g/L phthalic acid at a flow rate of 2 ml/min, during which free sulfate ions were detected at 290 nm in negative mode.
[0183] As shown in
[0184] <1-10> In vivo Cellular Uptake Activity
[0185] Hunter syndrome (MPS II) is one type of lysosomal storage disorders (LSD); and in enzymatic replacement therapy for the treatment of LSD, IDS must be picked up by cells of a patient and enter into a lysosome to degrade glycosaminoglycans (dermatan sulfate and heparin sulfate).
[0186] Binding to M6P receptor on cells, mannose-6-phosphate (M6P) which are located on IDS allow IDS to be internalized into cells. In this Example, IDS was subjected to HPAEC-PAD (Bio-LC) to quantitatively analyze mannose-6-phosphate. As a result, it was confined that there is approximately 3.0 moles of M6P per mole of IDS. Also, cellular uptake activities were analyzed by assaying normal fibroblast cells and Hunter syndrome patient cells.
[0187] The cellular internalization activity of the IDS was measured using the normal fibroblast cells and Hunter syndrome patient cells. In this regard, normal fibroblast cells and Hunter syndrome patient cells (obtained from Samsung Medical Center, Seoul, Korea) were cultured and allowed to be internalized into cells while they were incubated with various concentrations of IDS at 37 C. for 20 hours in a 5% CO.sub.2 incubator. After being harvested, the cells were lyzed, and the level of the IDS internalized into the cells was determined in the lysate.
[0188] On the basis of the concentration ratio of internalized IDS to IDS added to the normal fibroblast cells, a Michaelis-Menten graph and a Lineweaver-Burk plot were constructed from which K.sub.uptake (IDS concentration at which the reaction rate is half of the maximum rate achieved at saturating substrate concentrations) was calculated. K.sub.uptake was calculated to be 18.0 nM or less, indicating that IDS is internalized into cells by the binding of the M6P of IDS to M6P receptors on the cell surface (
[0189] Also, the cellular uptake and activity of IDS in Hunter syndrome patient cells as well as normal human fibroblast cells were analyzed. The uptake and activity of the IDS were increased in both the cells, demonstrating that the IDS of the present invention is more efficiently internalized into cells (
[0190] <1-10a> In vivo Cellular Uptake Activity (2)
[0191] The cellular internalization activity of the IDS was measured using the normal fibroblast cells and Hunter syndrome patient cells. In this regard, normal fibroblast cells and Hunter syndrome patient cells (obtained from Samsung Medical Center, Seoul, Korea) were cultured and allowed to be internalized into cells while they were incubated with various concentrations of IDS at 37 C. for 6 hours in a 5% CO.sub.2 incubator. After being harvested, the cells were lyzed, and the level of the IDS internalized into the cells was determined in the lysate.
[0192] On the basis of the concentration ratio of internalized IDS to IDS added to the normal fibroblast cells, a Michaelis-Menten graph and a Hanes-Woolf plot were constructed from which K.sub.uptake (IDS concentration at which the reaction rate is half of the maximum rate achieved at saturating substrate concentrations) was calculated. K.sub.uptake was calculated between 3.0 nM and 23.0 nM, indicating that IDS is internalized into cells by the binding of the M6P of IDS to M6P receptors on the cell surface (
[0193] Also, the cellular uptake and activity of IDS in Hunter syndrome patient cells as well as normal human fibroblast cells were analyzed. The uptake and activity of the IDS were increased in both the cells, demonstrating that the IDS of the present invention is more efficiently internalized into cells (
[0194] <1-11> Determination of Host Cell-Derived DNA Contents
[0195] According to the recommendation from the World Health Organization (WHO), Guidelines on the Quality, Safety, and Efficacy of Biotherapeutic Protein Products Prepared by Recombinant DNA Technology, adopted by the 64.sup.th meeting of the WHO Expert Committee on Biological Standardization, 21-25 Oct. 2013, the level of cell-derived and plasmid-derived DNA should be not more than 10 ng per purified dose.
[0196] The contents of host cell-derived DNA contents were measured on the IDS composition obtained in Example 1 <1-5>, using a Threshold system (Threshold total DNA assay kit, Molecular Devices Corp). Threshold system is equipment for the determination of total DNA quantity. It is intended for use in screening for total DNA contamination of recombinant DNA. In the first step, DNA was isolated from the proteins in the sample. In the second step, the sample is heat denatured to convert all DNA to the single stranded form. The denatured DNA samples are incubated with the DNA labeling reagent, which contains a conjugated enzyme. In the third step, the labeled DNA is captured onto a membrane by filtration. In the last step, enzyme-catalyzed pH response is measured on captured membranes.
[0197] A standard curve was obtained using standard solutions of concentrations of 6.25, 12.5, 25, 50, 100, 200, and 400 pg/ml.
[0198] Aliquots of the purified IDS composition obtained in Example <1-5-J>and the zero calibrator were dispensed to a pair of 2 mL sterile Sarstedt microcentrifuge tube with cap, and 50 uL of spike solution (1 ng/mL) was added to one of the tube. 20 uL of Sodium N-Lauroyl Sarcosinate solution to the tube and mix, following by adding 500 uL of NaI solution containing glycogen to the mixture, vortex and then incubate at about 40 C. for about 15 minutes. 900 uL of isopropanol is added to the mixture, vortex and then let stand at room temperature for about 15 minutes, followed by centrifugation to obtain a pellet containing DNA and glycogen.
[0199] The pellet is reconstituted using a calibrator buffer (500 uL), and subject the resulting sample to denaturation and labeling. The labeled DNA was captured onto a membrane by and the enzyme-catalyzed pH response was measured on the captured membranes. The host-cell derived DNA was measured in a range of 0-0.03 ng/mg, which is far lower than the limit 1.6 ng/mg set by the FDA.
[0200] <1-12> Determination of Host Cell-Derived Protein Contents
[0201] The level of host-cell proteins should be not more than 10 parts per million, for biological medicines used chronically over a lifetime (e.g. human insulin, erythropoietin or factor VIII). E.g., TGA Guidance 18. Australian Government, Version 1.0, August 2013).
[0202] The contents of host cell-derived protein were measured on the purified IDS composition obtained in Example 1 <1-5>, using two-site immunoenzyme assay (ELISA). Aliquots of the composition obtained in Example 1 were reacted with an affinity purified capture antibody (anti-CHO HCP antibody, Rabbit 3). An IDSspecific HCP assay kit (Young In Frontier, Korea) was used for this purpose, which allows a test performed in microtiter wells coated with an anti-CHO HCP capture antibody. The complex was reacted with anti-CHO HCP antibody (Rabbit 7)-biotin labeled antibody and then reacted with Avidine linked Horse Radish Peroxidase. The sandwich complex was reacted with TMB substrate after the microtiter strips were washed to remove and unbound reactants.
[0203] A dilution buffer (10 mg/ml BSA in TBS) was used to dilute the samples. The following reagents were used:
[0204] (a) 1 wash buffer
[0205] Mix 10 wash buffer 100 ml with distilled water 900 ml and make it to 1 washing solution
[0206] Store at 4 C. for 1 month.
[0207] (b) Working secondary antibody solution (Dilution fold may be changed, if necessary)
[0208] Add secondary antibody/AV-HRP dilution buffer 150 pi to a vial containing freeze-dried secondary antibody and mix well to obtain 100x diluted secondary antibody solution.
[0209] Add secondary antibody solution (100) 40 uL to secondary antibody/AV-HRP dilution buffer 3,960 uL and mix well.
[0210] (c) Working AV-HRP solution
[0211] Add AV-HRP concentrated solution (100) 40 uL to Secondary antibody/AV-HRP dilution buffer 3,960 uL and mix well.
[0212] As standard solutions, solutions containing standard CHO HCP in an amount of 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, and 50 ng/mL were prepared.
[0213] The results show that the host cell derived proteins in the samples were in a range of 0-13.7 ng/mg(=1-13.7 ppm), which is far lower than the limit of 100 ppm set by the FDA.
[0214] <1-13> Determination of Sialic Acid Contents
[0215] Sialic acid is a generic term for derivatives of neuraminic acid having a nine-carbon backbone, which is a monosaccharide with a complex structure including carboxylate, ketone and acetamide. The presence of carboxylates in sialic acid is of great importance because they are widely distributed in non-reducing terminus of glycoprotein and give acidic characteristics.
[0216] The contents of sialic acid of the IDS in the composition obtained in Example 1 <1-5>, were measured. Aliquots of the test composition were diluted with distilled water to a final concentration of 1.0 mg/ml. Standard solutions were prepared by dissolving N-acetylneruaminic acid in distilled water to make 10 mg/ml, and diluting it with distilled water to final concentrations of 0, 20, 40, 60, 80, 100, 1000, and 10,000 ug/ml.
[0217] In the first analysis, the amount of sialic acid in IDS was quantified based on the fact that sialic acid causes color formation in the resorcinol method (Seliwanoff's test,
[0218] Seliwanoff reaction: 100 ul of standard solutions and test solutions, respectively, were loaded to glass cab tubes, and 1 ml of resorcinol reagent (prepare by mixing hydrochloride acid R1 80 ml, 0.1M cupric sulfate 0.25 ml and 2% resorcinol solution 10 ml, and filling up to 100 ml with distilled water) was added and mixed. The resulting mixtures were incubated at 100-105 C. heating block for about 30 min. and cooled for about 10 min immediately after heat processing.
[0219] Extraction: 2 ml of extraction solution (butanol 24 ml and butyl acetate 96 ml) was added to each tube. When layers were completely separated by oxidizing at room temperature for about 30 min, transfer 1.5 ml of the supernatant to 1.5 ml tube and centrifuged for 3 min (12,000 rpm, room temperature). Adjust zero point with standard H and the absorbance at 580 nm was measured.
[0220] A standard curve for the absorbance values of standard solutions and sialic acid's concentration (ug/ml) in the test solutions from the standard curve.
[0221] 309 g/mol: Molecular weight of sialic acid
[0222] 78,000 g/mol: Molecular weight of IDS
[0223] The results showed that the sialic acid contents in the samples were in a range of 13.5-17.8 mol/mol, falling within the acceptance criteria of 11-20 mol/mol.
TABLE-US-00006 TABLE 6 Analysis results for sialic acid (Resorcinol method) Average Batch (mol/ No. 20R 707R9001 707R9002 707R9003 707R9004 mol) Neu5Ac 15.0 17.2 16.7 16.1 17.6 16.5 (mol/ mol) (R20: 200 L scale batch for laboratory use, 707R9001~707R9004: GMP 500 L scale batch)
[0224] <1-13a> Determination of Sialic Acid Contents (Bio-LC) (2)
[0225] In the second analysis, IDS was hydrolyzed in 0.1 N hydrochloric acid (HCl), and then the hydrolysate was analyzed using liquid chromatography (High Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection; HPAEC-PAD). The assay was carried out by Protagen AG. A reference sialic acid concentration of which was already known was analyzed under the same conditions, and molar ratios of sialic acid to glycoprotein were obtained by comparison of the areas. Analysis was conducted in triplicate. Sialic acid reference and sialic acid composition chromatograms of the IDS are shown in
[0226] IDS was hydrolyzed with 0.5M HCl and the hydrolysate was analyzed using liquid chromatography (High Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection; HPAEC-PAD). Sialic acid of known concentrations was analyzed under the same condition, and molar ratios of Sialic acid to glycoprotein were obtained by comparison of the areas.
TABLE-US-00007 TABLE 7 Analysis results for sialic acid (Bio-LC method) Rel. Retention Rel. Stand. Amount Stand. Molar ratio Sample time [min] Dev. [pmol/10 l] Dev. (mol/mol) Neu5Ac 5.82 1.03% 439.16 2.95% 14.68 Neu5Gc 10.72 0.18% 17.84 1.36% 0.60
[0227] <1-15> Determination of Oligosaccharide Pattern
[0228] The oligosaccharide pattern of the sample was determined using IE-HPLC(Ion Exchange-High Performance Liquid Chromatography). In this test, samples are treated with PNGase F to deglycosylate the proteins in the sample, and then the released glycans are labeled 2-AB(2-aminobenzamide). And 2-AB labeled glycans are analyzed by ion exchange HPLC with a fluorescence detector. A glycoprofiling scheme for antibody and chemistry of 2-AB labeling are shown in
[0229] The sample of the IDS composition obtained in Example 1 <1-5> (after affinity chromatography) was diluted to 1 mg/ml using water. 45 ul of 1 mg/ml samples and 5 ul of 10 denaturing buffer were mixed and allowed to stand at 50 C. for about 10 minutes, and 1 uL of PNGase F was added and incubate the mixture at 37 C. for about 6 hours. Glycans are isolated through solid phase extraction and label the isolated glycans with 2-AB dye. Oligosaccharide pattern was determined using GlycoSep C HPLC column (mobile phase A: 200 ml of 100% acetonitrile and 800 ml of filtered water; mobile phase B: 40% acetonitrile (500 ml) and 500 mM ammonium formate (500 ml) were mixed and adjusted to pH 4.5 using formic acid).
[0230] The results are shown in
[0231] <1-14> Determination of IDS Charge Variance
[0232] Proteins migrate to the negative pole when the pH is higher than the isoelectric point (pH and pI at which the total electric charge becomes 0) and to the positive pole when the pH is lower than the isoelectric point. There are two types of ion exchanger used for ion exchange chromatography: cation exchangers and anion exchangers, to each of which counter ions (Na.sup.+, Cl.sup., etc.) are electrostatically bound. Therefore, when the target protein is a basic protein that migrates to the positive pole, it is bound to a cation exchanger with a negative electric charge. When it is an acidic protein that migrates to the negative pole, it is bound to an anion exchanger with a positive electric charge. Bond strength increases according to the size of the total electric charge of the protein. When the ion concentration (salt concentration) of the elution buffer is gradually increased, the bound proteins are eluted in order of weakest to strongest bonds.
[0233] In this test, a purity of the IDS obtained in Example 1 <1-5> (after affinity chromatography) and ELAPRASE, a commercially available therapeutic agent for Hunter syndrome, were measured using Ion Exchange High performance Liquid Chromatography (IE-HPLC). A formulation buffer (as a blank formulation) was prepared, which contains 950 mL of ultrapure distilled water, 0.22 g of polysorbate 20, 2.25 g of sodium phosphate monobasic monohydrate, 0.99 g of sodium phosphate dibasic heptahydrate, and 8 g of sodium chloride, pH 6.0).
[0234] The IE-HPCL operation conditions are shown in Table 8 below:
TABLE-US-00008 TABLE 8 IE-HPLC Operation Conditions Mobile A: 20 mM Bis-Tris, pH 7.0 Phase B: 20 mM Bis-Tris + 0.5M sodium chloride, pH 7.0 Column TOSOH SuperQ-5PW (7.5 75 mm, 10 um) Flow Rate 0.5 mL/min Temperature Column: 30 C., Sampler: 4 C. Injection 100 L Volume Detector 280 nm Run Time 70 min Time Flow rate %A %B Gradient 0 0.5 70 30 10 0.5 70 30 45 0.5 0 100 50 0.5 0 100 55 0.5 70 30 70 0.5 70 30
[0235] The results are shown in
[0236] <1-14A> Isoelectric Point Analysis
[0237] The purpose of this test is to confirm isoelectric point of IDS by using vertical isoelectric focusing technique.
[0238] Various amounts of electrical charge carried by proteins can be used to separate proteins based on the charge that they carry when an electrical field is applied. Due to its amphotropic nature, a protein has a net negative charge when the pH is greater than pI, and a protein has a net positive charge when the pH is smaller than pI. Thus, when a strong electrical force is applied to an established pH gradient increasing from anode to cathode, and a protein migrate according to the gradient until the protein reaches the pH region that corresponds to its pI. This assay was conducted to analyze isoelectric point by using a 2D concentration gradient.
[0239] As a result, the IDS showed a band within the pH range of 3.5 or lower (
EXPERIMENTAL EXAMPLE 2
Clinical Analysis for Effect of IDS
[0240] Thirty one patients with Hunter syndrome were divided into three groups, administered with the IDS of the present invention and analyzed for parameters associated with Hunter syndrome. ELAPRASE, a commercially available therapeutic agent for Hunter syndrome, was used as a positive control.
[0241] <2-1> Change in Urine GAG Level (Primary Check Parameter for Validity Test)
[0242] The three groups of Hunter syndrome patients were administered for 24 weeks with ELAPRASE (0.5 mg/kg) and the IDS of the present invention (0.5 mg/kg and 1.0 mg/kg), and urine GAG (Glycosaminoglycan) levels were measured as reported previously (Conn. Tissue Res. Vol. 28, pp 317-324, 1990.; Ann. Clin. Biochem. Vol 31, pp 147-152, 1994). Measurements are summarized in Table 9, below.
TABLE-US-00009 TABLE 9 Change in Urine GAG Level with IDS Administration ELAPRASE Inventive IDS Inventive IDS Group (0.5 mg/kg) (0.5 mg/kg) (1.0 mg/kg) Change in urine 18.7 29.5 41.1 GAG level (%)
[0243] In Hunter syndrome patients, as shown in Table 9, urine GAG levels were decreased by 18.7% upon the injection of ELAPRASE, but by 29.5% upon the injection of the IDS of the present invention at the same dose. In addition, when injected at a dose of 1.0 mg/kg, the IDS of the present invention reduced the urine GAG level by as much as 41.1%. These results demonstrate that the IDS of the present invention is effectively therapeutic for Hunter syndrome, a disease caused as a result of the accumulation of GAG.
[0244] <2-2> 6-MWT(6 Minute Walking Test) Change (Secondary Checking Parameter for Validity Test)
[0245] After Hunter syndrome patients were administered with ELAPRASE and the IDS of the present invention for 24 weeks, the distances which they walked for 6 minutes were measured according to the method described in AM. J. Respir. Crit. Care. Med., Vol 166, pp 111-117, 2002. The results are given in Table 10, below.
TABLE-US-00010 TABLE 10 6-MWT Test Results ELAPRASE Inventive IDS Inventive IDS Group (0.5 mg/kg) (0.5 mg/kg) (1.0 mg/kg) 6-MWT 5.9 67.6 52.8 Distance (m) 6-MWT Change 1.3 18.2 13.4 (%)
[0246] As shown in Table 10, the 6-WMT change was merely 1.3% for the patients administered with ELAPRASE, but increased to 18.2% for the patients administered with the same dose of the IDS of the present invention. Hunter syndrome patients have trouble walking due to contracture. However, the IDS of the present invention improves the symptoms and thus is effective for the treatment of Hunter syndrome.
[0247] <2-3> Efficacy and Toxicity Tests in Mouse Model
[0248] Efficacy and toxicity tests of GC1111 of the present invention were conducted using mouse model and the results are summarized in
TABLE-US-00011 TABLE 11 Terminal Test IV C.sub.max AUC.sub.last T.sub.1/2 MRT.sub.last Item (n = 3) (ng/ml) (ng * h/ml) (h) (h) GC1111 1.5 mg/kg 2478.03 1043.73 2.47 0.78 Elaprase 3184.67 1743.8 1.68 1.07 GC1111 uptake into the cell takes place at a faster rate than that of Elaprase based on in vitro test results Toxicity test GC1111 Elaprase* Single-dose 0/5/10/20 mg/kg Same as left toxicity in No abnormal clinical rats signs were observed Single-dose Same as left toxicity in monkeys and safety pharmacology 4 wk repeated Not conducted toxicity test in monkeys 26 wk 0/0.5/2.5/12.5 mg/kg 0/0.5/2.5/12.5 mg/kg repeated NOEL: 12.5 mg/kg Histopathologic toxicity Histopathologic diagnosis test in diagnosis One subject from monkeys No abnormal clinical high-dose group: liver signs were observed granuloma observed Immunogenicity One subject from diagnosis intermediate-dose Some (2 subjects) were group: Some test positive after 13 wk histiocytosis was seen 26 All subjects were in mesenteric lymph test negative after 26 wk nodes No other abnormal clinical signs were observed Immunogenicity diagnosis Antibodies were formed in 2 out of 4 subjects from 2.5 mg/kg group, and 4 out of 6 subjects from 12.5 mg/kg group, but no effect when drug was exposed in blood Reproductive 0/0.5/1.5/5 mg/kg Same as left toxicity No abnoiival clinical test in rats signs were observed *Reference for Elaprase non-clinical studies: FDA BLA (Biologics License Applications)
[0249] <2-4> Change in Liver Volume (Secondary Check Parameter for Validity Test): Liver Volume
[0250] Hunter syndrome patients were administered for 24 weeks with Elaprase and GC1111, and their liver volume was measured by liver ultrasonography, and the results were evaluated by comparing with baseline values.
[0251] Hepatomegaly was observed in Hunter syndrome patients due to accumulation of GAG. The results are shown in Table 12.
TABLE-US-00012 TABLE 12 Elprase_0.5 GC1111_1.0 Group mg/kg GC1111_0.5 mg/kg mg/kg Change in liver 258 110 195.5 volume (vol, cc) Change in liver 14.6 6.2 8.5 volume (rate, %)
[0252] <2-5> Change in Urine GAG Level (Secondary Check Parameter for Validity Test)
[0253] Hunter syndrome patients were administered for 24 weeks with Elaprase and GC111, and their urine GAG levels were measured, and evaluated by comparing with baseline values.
[0254] Urinary GAG was detected from Hunter syndrome patients because degradation of GAG did not take place due to iduronate-2-sulfatase deficiency. The results are shown in Table 13.
TABLE-US-00013 TABLE 13 Elprase_0.5 GC1111_1.0 Group mg/kg GC1111_0.5 mg/kg mg/kg Change in urine 23.8 49.6 50.2 GAG (mg GAG/g Creatinine)
[0255] <2-6> Change in LV End Diastolic, Systolic Volume (Secondary Check Parameter for Validity Test): Contraction and Relaxation of Heart
[0256] Hunter syndrome patients were administered with Elaprase and GC 1111 for 24 weeks, and LV end diastolic and systolic volumes were measured by using echocardiography, and the results were evaluated by comparing with baseline values.
[0257] Cardiomegalia was observed in Hunter syndrome patients. Increased diastolic volume and decreased systolic volume indicate an improved myocardial contractility. The results are shown in Table 14.
TABLE-US-00014 TABLE 14 Elprase_0.5 GC1111_1.0 Group mg/kg GC1111_0.5 mg/kg mg/kg Change in LV end 0.6 5.8 5 diastolic volume (vol, cc) Change in LV end 1.7 9.1 6.9 diastolic volume (rate, %) Change in LV end 3.3 1.2 3.2 systolic volume (vol, cc) Change in LV end 17.2 5.7 10.8 systolic volume (rate, %)
[0258] <2-7> Change in LV Mass Index (Secondary Check Parameter for Validity Test): Hypertrophic Myocytes
[0259] Hunter syndrome patients were administered for 24 weeks with Elaprase and GC1111, and LV mass indices were measured by using echocardiography, and the results were evaluated by comparing with baseline values.
[0260] The degree of hypertrophic myocytes was analyzed by using LV mass index. The results are shown in Table 15.
TABLE-US-00015 TABLE 15 Elprase_0.5 Group mg/kg GC1111_0.5 mg/kg GC1111_1.0 mg/kg Change in LV 0.9 1.9 0.6 mass index (g/m.sup.2.7) Change in LV 1.7 2.9 0.6 mass index (%)
[0261] <2-8> Change in LV Ejection Fraction (Secondary Check Parameter for Validity Test): Cardiac Function
[0262] Hunter syndrome patients were administered for 24 weeks with Elaprase and GC1111, and their LV ejection fractions were measured by echocardiography, and the results were evaluated by comparing with baseline values. The results are shown in Table 16.
TABLE-US-00016 TABLE 16 Elprase_0.5 GC1111_1.0 Group mg/kg GC1111_0.5 mg/kg mg/kg Change in LV 1.5 3.6 0.6 ejection fraction (%) Change in LV 2 5 1 ejection fraction (%)
[0263] <2-9> Change in Absolute FVC (Secondary Check Parameter for Validity Test): Pulmonary Function During Respiration
[0264] After Hunter syndrome patients were administered with Elaprase and GC1111 for 24 weeks, absolute FVC was measured by using pulmonary function test, and the results were evaluated by comparing with base line values.
[0265] Hunter syndrome patients experience narrowed respiratory tract due to GAG accumulation in respiratory tract; in severe cases, respiratory obstruction occurs and tracheotomy must be performed. The results are shown in Table 17.
TABLE-US-00017 TABLE 17 Elprase_0.5 GC1111_1.0 Group mg/kg GC1111_0.5 mg/kg mg/kg Change in 0 0.1 0.2 absolute FVC (L) Change in 0 12.6 17 absolute FVC (%)
[0266] <2-10> Safety Evaluation
[0267] 1) Adverse drug reaction (ADR) [0268] In Elaprase_0.5 mg/kg group, 2 of 11 subjects experienced a total of 19 adverse drug reactions. [0269] In GC1111_0.5mg/kg group, 1 of 10 subjects experienced a total of 4 adverse drug reactions. [0270] In GC1111_1.0mg/kg group, 2 of 10 subjects experienced a total of 3 adverse drug reactions.
[0271] All the adverse drug reactions observed were mild symptoms as expected, and these ADRs were controllable by discontinuation of drug at lower and less frequent dosing regimens.
[0272] 2) Immunogenicity
[0273] Three groups of Hunter syndrome patients were administered with GCIII for 24 weeks and immunogenicity test was performed. As a result, no changes were observed in the status on prevalence of GC1111 antibodies and neutralizing antibodies in Hunter syndrome patients before and after the administration.