WO2022154762A1 - Method of producing adalimumab - Google Patents
Method of producing adalimumab Download PDFInfo
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- WO2022154762A1 WO2022154762A1 PCT/TR2021/050034 TR2021050034W WO2022154762A1 WO 2022154762 A1 WO2022154762 A1 WO 2022154762A1 TR 2021050034 W TR2021050034 W TR 2021050034W WO 2022154762 A1 WO2022154762 A1 WO 2022154762A1
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- citric acid
- naoh
- nacl
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- sodium phosphate
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- 238000000034 method Methods 0.000 title claims abstract description 42
- 229960002964 adalimumab Drugs 0.000 title claims abstract description 12
- 238000004519 manufacturing process Methods 0.000 claims abstract description 30
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims 2
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/241—Tumor Necrosis Factors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
Definitions
- the present invention relates to manufacturing of a anti-TNFa monoclonal antibody (adalimumab), produced in high density cell culture, purified with flow-through chromotography unit operations.
- adalimumab anti-TNFa monoclonal antibody
- Process of the improved method provides to handle with high cell density culture in stirred tank reactor (STR) while chromotography steps can handle more than 99.5% purity of the desired mAb to be deliverable to the patients.
- Dysregulation in TNFa activity can mediate pathogenic effects and can play a role in autoimmune disease initiation and maintenance.
- the overexpression of this soluble factor may result in an additional increase of pro-inflammatory cytokines, increased T cell survival and suppression of regulatory macrophages, endothelial cell dysfunction, and tissue destruction.
- Systemic TNFa overexpression causes TNFa-induced pro-inflammatory activities observed during disease states, especially in all autoimmune diseases.
- Fab domain of adalimumab was shown to bind to the antigen TNFa with high affinity and specificity.
- Monoclonal antibodies are structurally complex molecules due to the post-translational modifications (PTMs) and state-of-art analytical instruments are currently used in order to define this complexity.
- CQA critical quality attribute
- mAbs Monoclonal antibodies
- PTMs post-translational modifications
- CQA critical quality attribute
- These CQAs are mainly based on intact/reduced mass measurement, PTM (e.g oxidation, deamidation) and glycosylation analysis, aggregate/monomer amount and acidic/basic variant analysis.
- Cell culture parameters such as pH, temperature, and dissolved oxygen (DO) have impact on glycosylation.
- additional parameters affecting glycan compositions arise. Besides pH, temperature and DO; agitation, shear stress, sparging, dissolved carbondioxyde come into account. These parameters may alter charge variants or glycosylation patterns; thus, can impact product quality.
- Microheterogeneity is significantly affected by culture conditions. Glycoform heterogeneity might increase, batch-to-batch variations can occur and therapeutic efficacy can be reduced accordingly.
- Downstream processing refers to the recovery or purification of biological products such as monoclonal antibodies (mAbs). Following cell culture expression in bioreactors and subsequent harvesting actions, the target product is subjected to downstream stages of manufacture. Downstream processing of a monoclonal antibody consists of various stages. Firstly, a capture step (Protein A chromatography) is followed, in which the mAb in the culture supernatant is separated from other components.
- a capture step Protein A chromatography
- an intermediate step [Cation Exchange Chromatography (CEX), Anion Exchange Chromatography (AEX), Hydrophobic Intreaction Chromatography (HIC)] is carried out, in which the mAb is decontaminated from impurities similar in size or other biochemical specifications; and lastly, a polishing step (Virus filtration, Ultrafiltration/Diafiltration, sterile filtration) provides complete removal of dash contaminations.
- CEX Cion Exchange Chromatography
- AEX Anion Exchange Chromatography
- HIC Hydrophobic Intreaction Chromatography
- the present invention provides a method for producing adalimumab by Cation Exchange Chromatography (CEX) chromotography technique with flow-through mode. Since the CEX chromatography technique is used with the flowthrough method, it allows the purification of a much higher amount of product with a much lower resin volume. Therefore, due to its high loading capacity, low resin volume can be used and thus the buffer solution volumes used are reduced. Less resin volume requirement of CEX chromotography provided cost improvement per pure protein quantity. Flow-through mode also provided some advantages for scale up of purification steps.
- CEX Cation Exchange Chromatography
- Temperature shifting is the general strategy used in modulating secretion system. However temperature shift day and temperature changes from process to process. In this invention, both cell growth and quality of the product were controlled using down temperature shift to 32 ⁇ 1°C on production day 6.
- Galactose is generally used as a glycosylation modulating agent in the industrial processes.
- specific galactose addition strategy was developed to provide product quality improvement.
- Culture media was supplemented with galactose solution with a concentration of 0.2% (v/v) on the production day (PD) of PD2, PD4, PD6, PD8, PD10.
- CEX flow-through mode provided to remove aggregates, host cell protein (HCP), residual protein A, residual DNA, impuritie. Less resin volume requirement of CEX chromotography provided cost improvement per pure protein quantity. Flow-through mode also provided some advantages for scale up of purification steps.
- Hydrophobic Intreaction Chromatography can be used since it can provide to work in low pH (4.0-5.0) condition for further impurity clearance instead of anion exchange. This step is also used as a potential viral clearance step in this invention.
- FIG. 1 Flow Chart of Downstream Process Flow A) Pre- Viral (DSP1) B) Post- Viral (DSP2)
- the present invention relates to a method of producing an anti-TNFa antibody (adalimumab), wherein the method comprises; thawing of Chinese Hamster Ovary (CHO) Master Cell Bank (MCB) or Working Cell Bank (WCB) vial, cell expansion in shake flask, cell expansion in wave bag, production in bioreactor, bioreactor harvest clarification, Protein A affinity chromatography, viral inactivation, cation exchange chromatography, viral filtration, hydrophobic interaction chromatography and tangential flow filtration.
- CHO Chinese Hamster Ovary
- WCB Working Cell Bank
- a method of producing an anti-TNFa monoclonal antibody comprises following steps: a. Culturing mammalian cells comprising a nucleic acid encoding anti-TNFa monoclonal antibody in a high density cell culture production medium, b. Production of anti-TNFa monoclonal antibody in bioreactor at temperature 37 ⁇ 1°C during equilibration until Day 6 and shifting temprerature on Day 6 to 32 ⁇ 1°C until to the end of culture, c. Supplementing of culture medium with a concentration of 0.2% (v/v) galactose solution on the production day (PD) of PD2, PD4, PD6, PD8, PD10, d. Bioreactor harvest clarification, e.
- Cells from the MCB or WCB are thawed and used for inoculum preparation. These cells are serially expanded in shake flasks and wave-bag. The inoculum is then transferred to a 200 L production bioreactor where the cells are cultivated for 14 days. At the end of the fed- batch fermentation, the crude harvest is clarified by depth and membrane filtration. The clarified harvest is then transferred to the downstream process (DSP). Each step is carefully controlled by in-process testing and controls to ensure process and product consistency. A general overview of the expansion and production phases is presented in Figure 1 and Table 1. The in-process controls (IPC) are further discussed in Controls of Critical Steps and Intermediates section.
- IPC in-process controls
- Table 1 Overview of the Upstream Process Steps IVVT: in vitro virology test, HCP: Host cell protein, PA: Protein A, TEM: Transmission electron microscopy, MVM: MinuteVirus of Mice, HPLC: High performance liquid chromatography
- One vial of MCB or WCB is thawed and expanded using a series of subculture steps (5 passages), comprising shake flasks and wave bags.
- 50 L wave bag is used as seeding bioreactor for the 200 L production.
- the expansion medium and supplements used are chemically defined and free from components of human or animal origin.
- the 200 L production bioreactor is inoculated with cells coming from the 50 L wave bags, and culture is run for 14 days.
- the crude harvest is clarified by D0HC/X0HC depth filtration.
- the filters should be equilibrated with at least 40 L/m 2 of purified water.
- the working Apressure should not exceed 1.5 bars.
- D0HC/X0HC filters are rinsed with at least 15 L/m 2 of purified water. The wash is then collected with the filtered material.
- the clarified harvest is filtered by SHC filters (post clarification filtration).
- the pCPPs are presented in Table 5.
- DSP Downstream Process
- the first TUR01 purification process step consists of several chromatographic and filtration steps, summarized in Table 7. Each unit operation is carefully controlled by means of in-process testing and controls to ensure process and product consistency.
- a process flow diagram for the downstream manufacturing process is presented in Figure 2.
- HCP Host cell protein
- rPA residual Protein A
- rDNA residual DNA
- HMW High molecular weight
- Protein A (PA) capture is performed using a ProSep Ultra Plus capture resin.
- the capture step allows concentration of the product, removal of the main cell culture components, and also removal of aggregates and virus-like particles that could potentially be derived from the production cell bank.
- the pCPPs during capture are presented in Table 8. Table 8: pCPPs - Protein A Chromatography
- PA HPLC Protein A High Performance Liquid Chromatography No storage will be performed after capture step and before viral inactivation step.
- resin will be just regenerated but not sanitised or stored between them.
- the pCPPs for storage of the product after the viral inactivation step are described in the Table 10 below.
- the post virus inactivation step storage is either at 2-8°C or 15-25°C.
- the CEX step is performed in flow through mode.
- the pCPPs are presented in Table 11.
- Table 11 pCPPs - CEX and Filtration post CEX
- the pCPPs for storage of the product after the CEX step are described in Table 12 below.
- the post CEX step storage is either at 2-8°C or 15-25°C.
- Table 12 pCPPs for Storage post CEX Step
- Each CEX intermediate is subjected to a nanofiltration step for virus removal.
- the pCPPs for the viral filtration step are presented in Table 13. Table 13: pCPPs - Viral Filtration
- the viral filtration intermediate is subject to further purification by HIC.
- this step allows HCP and rPA, but also provide a slight decrease of rDNA, LMW, and HMW forms removal.
- resin will be just regenerated but not sanitised or stored between them.
- the pCPPs for storage of the product after the HIC step are described in the Table below.
- the post HIC step storage is either at 2-8°C or 15-25°C.
- TMP Transmembrane pressure
- DV Dia-volume Drug Substance (DS) Storage and Filling
- the pCPPs for DS storage and filling are described in the Table below.
- the DS storage is either at - 70°C, 2-8°C or 15-25°C.
- Table 17 pCPPs for DS Storage Table 18: Acceptance Criteria for Filling
- Buffers used for purification are listed in Table 21.
- the wild-type CHO-K1 cell line was derived as a subclone from the parental CHO cell line. After purchasing one vial of CHO-K1 cells, Merrimack Pharmaceuticals, Inc., established a serum free cultivated cell bank, this adapted cell bank was named CHO-M.
- Cell lines were generated by transfecting the gene of interest in the CHO cell line by using Microporation, followed by a second transfection (SuperTransfection) using another selection marker (e.g. monocistronic vectors including hygromycin resistance cassette, ratio Hc:Lc 1:1.3)
- Two additional single cell dilution rounds (ClonePix) were conducted to provide high productive supertransfected clone cell lines. ClonePix FL was used for cell line subcloning. To ensure monoclonality only good sized and well isolated single colonies were selected.
- IVVT in vitro virology test
- TFF process was studied to determine the optimal transmembrane pressure (TMP) and recirculation flow rate for the concentration and diafiltration process. It was also assessed the number of diavolumes necessary to put the molecule in formulation conditions. The process was fixed with 1 bar TMP and 6 LMM flux.
- the TUR01 drug product is an injectable liquid intended for subcutaneous administration. It is presented as a sterile solution with adalimumab at a concentration of 50 mg/mL in a 1 mL type I glass pre-filled syringe containing 0.8 mL of DP.
- the formulation also includes: monobasic sodium phosphate dihydrate, dibasic sodium phosphate dihydrate, sodium chloride, sodium citrate dihydrate, citric acid monohydrate, polysorbate 80, mannitol and water for injection.
- Adalimumab production was provided in high producing, stable cell lines. Gal+ addition, pH adjustment, temperature shift and flow-through chromotography were used as a strategical approach to provide the high biosimilarity. Process conditions provided highly similar charge variants and glycosylation profiles which falls in CQA limits. Successful scale-up process was accomplished providing consistent and high biosimilarity.
Abstract
The present invention relates to a method of producing an anti-TNFα monoclonal antibody (adalimumab), wherein the method comprises; thawing of CHO MCB/WCB vial, cell expansion in shake flask, cell expansion in wave bag, production in bioreactor, bioreactor harvest clarification, Protein A affinity chromatography, viral inactivation, cation exchange chromatography, viral filtration, hydrophobic interaction chromatography and tangential flow filtration.
Description
METHOD OF PRODUCING ADALIMUMAB
Technical Field
The present invention relates to manufacturing of a anti-TNFa monoclonal antibody (adalimumab), produced in high density cell culture, purified with flow-through chromotography unit operations. Process of the improved method provides to handle with high cell density culture in stirred tank reactor (STR) while chromotography steps can handle more than 99.5% purity of the desired mAb to be deliverable to the patients.
Prior Art
Dysregulation in TNFa activity can mediate pathogenic effects and can play a role in autoimmune disease initiation and maintenance. The overexpression of this soluble factor may result in an additional increase of pro-inflammatory cytokines, increased T cell survival and suppression of regulatory macrophages, endothelial cell dysfunction, and tissue destruction. Systemic TNFa overexpression causes TNFa-induced pro-inflammatory activities observed during disease states, especially in all autoimmune diseases. Fab domain of adalimumab was shown to bind to the antigen TNFa with high affinity and specificity.
Monoclonal antibodies (mAbs) are structurally complex molecules due to the post-translational modifications (PTMs) and state-of-art analytical instruments are currently used in order to define this complexity. The critical quality attribute (CQA) of originator molecule must be determined implicitly due to their impacts on efficacy, safety and immunogenicity of drugs. These CQAs are mainly based on intact/reduced mass measurement, PTM (e.g oxidation, deamidation) and glycosylation analysis, aggregate/monomer amount and acidic/basic variant analysis. By analyzing different originators, CQAs of the originators are identified and the upstream and downstream processes are developed due to these CQAs in the small scale. Process development studies in labscale are pivotal regarding process scale up. The purpose of scale up is to transfer the conditions optimized in laboratory and/or pilot scale to that of production scale, providing that the alterations will not affect efficiency/productivity or product quality.
Cell culture parameters such as pH, temperature, and dissolved oxygen (DO) have impact on
glycosylation. When cell culture processes are carried to bioreactor systems, additional parameters affecting glycan compositions arise. Besides pH, temperature and DO; agitation, shear stress, sparging, dissolved carbondioxyde come into account. These parameters may alter charge variants or glycosylation patterns; thus, can impact product quality. Microheterogeneity is significantly affected by culture conditions. Glycoform heterogeneity might increase, batch-to-batch variations can occur and therapeutic efficacy can be reduced accordingly.
Downstream processing refers to the recovery or purification of biological products such as monoclonal antibodies (mAbs). Following cell culture expression in bioreactors and subsequent harvesting actions, the target product is subjected to downstream stages of manufacture. Downstream processing of a monoclonal antibody consists of various stages. Firstly, a capture step (Protein A chromatography) is followed, in which the mAb in the culture supernatant is separated from other components. Then, an intermediate step [Cation Exchange Chromatography (CEX), Anion Exchange Chromatography (AEX), Hydrophobic Intreaction Chromatography (HIC)] is carried out, in which the mAb is decontaminated from impurities similar in size or other biochemical specifications; and lastly, a polishing step (Virus filtration, Ultrafiltration/Diafiltration, sterile filtration) provides complete removal of dash contaminations.
In conclusion, in this study a new process was developed specifically for anti-TNFa monoclonal antibody (adalimumab) by using novel technologies.
Aims of the Invention and Brief Description
The present invention provides a method for producing adalimumab by Cation Exchange Chromatography (CEX) chromotography technique with flow-through mode. Since the CEX chromatography technique is used with the flowthrough method, it allows the purification of a much higher amount of product with a much lower resin volume. Therefore, due to its high loading capacity, low resin volume can be used and thus the buffer solution volumes used are reduced. Less resin volume requirement of CEX chromotography provided cost improvement per pure protein quantity. Flow-through mode also provided some advantages for scale up of purification steps.
In this process; high cell density (>35xl06 cells/ml) was used with high oxygenation level which provides cell survival and high production level in STR while not causing any effect on PTMs of adalimumab.
Temperature shifting is the general strategy used in modulating secretion system. However temperature shift day and temperature changes from process to process. In this invention, both cell
growth and quality of the product were controlled using down temperature shift to 32±1°C on production day 6.
Galactose is generally used as a glycosylation modulating agent in the industrial processes. In this invention specific galactose addition strategy was developed to provide product quality improvement. Culture media was supplemented with galactose solution with a concentration of 0.2% (v/v) on the production day (PD) of PD2, PD4, PD6, PD8, PD10.
CEX flow-through mode provided to remove aggregates, host cell protein (HCP), residual protein A, residual DNA, impuritie. Less resin volume requirement of CEX chromotography provided cost improvement per pure protein quantity. Flow-through mode also provided some advantages for scale up of purification steps.
In the manufacturing of mAbs; Hydrophobic Intreaction Chromatography (HIC) can be used since it can provide to work in low pH (4.0-5.0) condition for further impurity clearance instead of anion exchange. This step is also used as a potential viral clearance step in this invention.
Definition of the Figures of the Invention
Figure l:Flow Chart of Upstream Process Flow (USP)
Figure 2: Flow Chart of Downstream Process Flow A) Pre- Viral (DSP1) B) Post- Viral (DSP2)
Detailed Description of the Invention
The present invention relates to a method of producing an anti-TNFa antibody (adalimumab), wherein the method comprises; thawing of Chinese Hamster Ovary (CHO) Master Cell Bank (MCB) or Working Cell Bank (WCB) vial, cell expansion in shake flask, cell expansion in wave bag, production in bioreactor, bioreactor harvest clarification, Protein A affinity chromatography, viral inactivation, cation exchange chromatography, viral filtration, hydrophobic interaction chromatography and tangential flow filtration.
In general, a method of producing an anti-TNFa monoclonal antibody comprises following steps: a. Culturing mammalian cells comprising a nucleic acid encoding anti-TNFa monoclonal antibody in a high density cell culture production medium, b. Production of anti-TNFa monoclonal antibody in bioreactor at temperature 37±1°C during equilibration until Day 6 and shifting temprerature on Day 6 to 32±1°C until to the end of culture,
c. Supplementing of culture medium with a concentration of 0.2% (v/v) galactose solution on the production day (PD) of PD2, PD4, PD6, PD8, PD10, d. Bioreactor harvest clarification, e. Purification by Protein A affinity chromatography, f. Viral inactivation step in pH 3.5-4.5, g. Purification by cation exchange chromotography in an overloading flow-through mode, h. Viral filtration, i. Purification by hydrophobic interaction chromotography performed in flow-through mode, j . Tangential flow filtration.
Description of Manufacturing Process and Process Controls
1. Description of the Upstream Process (USP)
Cells from the MCB or WCB are thawed and used for inoculum preparation. These cells are serially expanded in shake flasks and wave-bag. The inoculum is then transferred to a 200 L production bioreactor where the cells are cultivated for 14 days. At the end of the fed- batch fermentation, the crude harvest is clarified by depth and membrane filtration. The clarified harvest is then transferred to the downstream process (DSP). Each step is carefully controlled by in-process testing and controls to ensure process and product consistency. A general overview of the expansion and production phases is presented in Figure 1 and Table 1. The in-process controls (IPC) are further discussed in Controls of Critical Steps and Intermediates section.
Table 1: Overview of the Upstream Process Steps
IVVT: in vitro virology test, HCP: Host cell protein, PA: Protein A, TEM: Transmission electron microscopy, MVM: MinuteVirus of Mice, HPLC: High performance liquid chromatography
Cell Culture
Cell Expansion
One vial of MCB or WCB is thawed and expanded using a series of subculture steps (5 passages), comprising shake flasks and wave bags. 50 L wave bag is used as seeding bioreactor for the 200 L production. The expansion medium and supplements used are chemically defined and free from components of human or animal origin.
The potential critical process parameters (pCPPs) during cell expansion are presented in Table 2.
Table 2: pCPPs during Cell Expansion
Production in Bioreactor
The 200 L production bioreactor is inoculated with cells coming from the 50 L wave bags, and culture is run for 14 days.
The pCPPs in the production bioreactor are presented in Table 3.
Table 3: pCPPs during Production in Bioreactor
PD: Production day
Harvest Clarification
The crude harvest is clarified by D0HC/X0HC depth filtration. The filters should be equilibrated with at least 40 L/m2 of purified water. The working Apressure should not exceed 1.5 bars.
Post clarification, D0HC/X0HC filters are rinsed with at least 15 L/m2 of purified water. The wash is then collected with the filtered material.
The pCPPs in the clarification of the harvest are presented in Table 4.
Table 4: pCPPs for the Clarification Step
LMH: Liter per square meter per hour
The clarified harvest is filtered by SHC filters (post clarification filtration). The pCPPs are presented in Table 5.
Table 5: pCPPs for the Post-Clarification Filtration Step
Storage Conditions for the Filtered Clarified Harvest
The pCPPs for the storage of the filtered clarified harvest are described in Table 6.
Table 6: pCPPs for the Storage of the Filtered Clarified Harvest
2. Description of the Downstream Process (DSP)
The first TUR01 purification process step consists of several chromatographic and filtration steps, summarized in Table 7. Each unit operation is carefully controlled by means of in-process testing and controls to ensure process and product consistency. A process flow diagram for the downstream manufacturing process is presented in Figure 2.
Table 7: Overview of the Downstream Process Steps
HCP: Host cell protein, rPA: residual Protein A, rDNA: residual DNA, HMW: High molecular weight
Capture Step (Protein A Affinity Chromatography) Protein A (PA) capture is performed using a ProSep Ultra Plus capture resin. The capture step allows concentration of the product, removal of the main cell culture components, and also removal of aggregates and virus-like particles that could potentially be derived from the production cell bank. The pCPPs during capture are presented in Table 8.
Table 8: pCPPs - Protein A Chromatography
PA HPLC: Protein A High Performance Liquid Chromatography No storage will be performed after capture step and before viral inactivation step.
If several runs are performed the same day, resin will be just regenerated but not sanitised or stored between them.
Viral Inactivation (VI)
Each Protein A eluate is acidified for viral inactivation and then neutralized. The pCPPs are described in Table 9.
Table 9: pCPPs - Viral Inactivation and Filtration post VI Step
Storage post Viral Inactivation Step
The pCPPs for storage of the product after the viral inactivation step are described in the Table 10 below. The post virus inactivation step storage is either at 2-8°C or 15-25°C.
Table 10: pCPPs for Storage post VI Step
Cation Exchange Chromatography (CEX)
The CEX step is performed in flow through mode. The pCPPs are presented in Table 11.
Table 11: pCPPs - CEX and Filtration post CEX
OD: Optical Density
Storage post CEX Step
The pCPPs for storage of the product after the CEX step are described in Table 12 below. The post CEX step storage is either at 2-8°C or 15-25°C. Table 12: pCPPs for Storage post CEX Step
Viral Filtration
Each CEX intermediate is subjected to a nanofiltration step for virus removal. The pCPPs for the viral filtration step are presented in Table 13.
Table 13: pCPPs - Viral Filtration
Hydrophobic Interaction Chromatography (HIC)
The viral filtration intermediate is subject to further purification by HIC. For our process, this step allows HCP and rPA, but also provide a slight decrease of rDNA, LMW, and HMW forms removal.
The pCPPs are presented in Table 14.
Table 14: pCPPs - HIC
If several runs are performed the same day, resin will be just regenerated but not sanitised or stored between them.
Storage post HIC Step
The pCPPs for storage of the product after the HIC step are described in the Table below. The post HIC step storage is either at 2-8°C or 15-25°C.
Table 15: pCPPs for Storage post HIC Step
Tangential Flow Filtration (TFF)
Tangential flow filtration is performed for each HIC intermediate. The pCPPs are presented in
Table 16.
Table 16: pCPPs - TFF and Filtration post TFF
TMP: Transmembrane pressure; DV: Dia-volume Drug Substance (DS) Storage and Filling
The pCPPs for DS storage and filling are described in the Table below. The DS storage is either at - 70°C, 2-8°C or 15-25°C.
Table 17: pCPPs for DS Storage
Table 18: Acceptance Criteria for Filling
Raw and Starting materials used in Cell Culture (USP)
Raw materials used for cell culture are provided in Table 19. The composition of the cell culture media and solutions are presented in Table 20.
Table 19: Raw Materials
Table 20: Media and Solutions used for USP
Raw and Starting Materials used in Purification (DSP)
Buffers used for purification are listed in Table 21.
Table 21:Buffers Used in the Purification
3. Host Cell Line
The wild-type CHO-K1 cell line (ATCC) was derived as a subclone from the parental CHO cell line. After purchasing one vial of CHO-K1 cells, Merrimack Pharmaceuticals, Inc., established a serum free cultivated cell bank, this adapted cell bank was named CHO-M. Cell lines were generated by transfecting the gene of interest in the CHO cell line by using Microporation, followed by a second transfection (SuperTransfection) using another selection marker (e.g. monocistronic vectors including hygromycin resistance cassette, ratio Hc:Lc 1:1.3) Two additional single cell dilution rounds (ClonePix) were conducted to provide high productive supertransfected clone cell lines. ClonePix FL was used for cell line subcloning. To ensure monoclonality only good sized and well isolated single colonies were selected.
2.1.S.2.4 Controls of Critical Steps and Intermediates
Potential CPPs are listed in Description of Manufacturing Process and Process Controls Section (Table 2 to Table 6 and Table 8 to Table 17). An in- process sampling program has been developed to monitor the manufacturing consistency and quality of TUR01 (please refer to Table 1 and Table 7 in Description of Manufacturing Process and Process Controls
Section). The IPCs performed along each run and their limits are detailed in the following tables (Table 22 and Table 23). A systematic process characterization and validation program will follow the recommendations given in ICH Q8/Q9/Q10. Table 22:In-Process Testing for the Upstream Process
IVVT: in vitro virology test
Table 23:In-Process Testing for the Downstream Process
*in case of intermediate storage; SE-UPLC: Size Exclusion Ultra Performance Liquid Chromatography
4. FORMULATION (COMPOSITION) MANUFACTURING METHOD
TFF process was studied to determine the optimal transmembrane pressure (TMP) and recirculation flow rate for the concentration and diafiltration process. It was also assessed the number of diavolumes necessary to put the molecule in formulation conditions. The process was fixed with 1 bar TMP and 6 LMM flux.
Tangential flow filtration is performed for each HIC intermediate. The pCPPs are presented in Table 24.
Table 24: pCPPs - TFF and Filtration post TFF
TMP: Transmembrane pressure; DV: Dia-volume
5. DOSE REGIME (APPLICATION METHOD AND DOSE)
The TUR01 drug product (DP) is an injectable liquid intended for subcutaneous administration. It is presented as a sterile solution with adalimumab at a concentration of 50 mg/mL in a 1 mL type I glass pre-filled syringe containing 0.8 mL of DP. The formulation also includes: monobasic sodium phosphate dihydrate, dibasic sodium phosphate dihydrate, sodium chloride, sodium citrate dihydrate, citric acid monohydrate, polysorbate 80, mannitol and water for injection.
6. CONCLUSION
Success of scalable highly biosimilar Adalimumab production was provided in high producing, stable cell lines. Gal+ addition, pH adjustment, temperature shift and flow-through chromotography were used as a strategical approach to provide the high biosimilarity. Process conditions provided highly similar charge variants and glycosylation profiles which falls in CQA limits. Successful scale-up process was accomplished providing consistent and high biosimilarity.
Claims
1. A method of producing an anti-TNFa. monoclonal antibody, wherein the method comprises; a. Culturing mammalian cells comprising a nucleic acid encoding anti-TNFa monoclonal antibody in a high density cell culture production medium, b. Production of anti-TNFa monoclonal antibody in bioreactor at temperature 37±1°C during equilibration until Day 6 and shifting temprerature on Day 6 to 32±1°C until to the end of culture, c. Supplementing of culture medium with a concentration of 0.2% (v/v) galactose solution on the production day (PD) of PD2, PD4, PD6, PD8, PD10, d. Bioreactor harvest clarification, e. Purification by Protein A affinity chromatography, f. Viral inactivation step in pH 3.5-4.5, g. Purification by cation Exchange chromotography in an overloading flow- through mode, h. Viral Filtration, i. Purification by hydrophobic interaction chromotography performed in flow- through mode, j . Tangential flow filtration.
2. The method of claim 1, wherein the pH of the formation is 6.9± 0.2.
3. The method of claim 1 or 2, wherein the concentration of the cells is over 35 x 106 cells/ml.
4. The method of claim 1, wherein step (f) is performed at pH 4.5 ± 0.1 and/or conductivity is between 1.5 to 1.9 mS/cm.
5. The method of claim 1, wherein step (h) is performed at pH 4.5 ± 0.1 and/or conductivity is between 60 to 80 mS/cm.
6. The method of claim 1, wherein the Protein A affinity chromatography comprises one or more buffers selected from the group consisting of: a. 30 mM citrate + 150 mM NaCl pH 7.0
27
b. 30 mM citrate + IM NaCl pH 7.0 c. 10 mM citric acid pH 7.0 d. 10 mM citric acid pH 3.5 e. 5 M NaOH
7. The method of claim 1, wherein viral inactivation step comprises using one or more buffers selected from the group consisting of: a. 200 mmol/kg citric acid b. 0.5 M NaOH c 5 M NaOH
8. The method of claim 1, wherein cation exchange step comprises using one or more buffers selected from the group consisting of: a. 15 mM citric acid, pH 4.5 b. 200 mmol/kg citric acid c. 0.5 M NaOH d. 5 M NaOH e. 2 M NaCl
9. The method of claim 1, wherein viral filtration step comprises using one or more buffers selected from the group consisting of: a. 15 mM citric acid, pH 4.5 b. 200 mmol/kg citric acid c. 0.5 M NaOH d. 5 M NaOH
10. The method of claim 1, wherein hydrophobic interaction chromotography comprises one or more buffers selected from the group consisting of: a. 15 mM citric acid, pH 4.5 b. 200 mmol/kg citric acid c. 15.6 mM citric acid, 0.52 M ammonium sulfate, pH 4.5 d. 0.5 M NaOH e. 5 M NaOH
The method of claim 1, wherein tangential flow filtration step comprises using one or more buffers selected from the group consisting of: a. 15 mM citric acid, pH 4.5 b. 200 mmol/kg citric acid c. 15.6 mM citric acid, 0.52 M ammonium sulfate, pH 4.5 d. 0.5 M NaOH e. 6.16 mg/mL NaCl, 0.86 mg/mL monobasic sodium phosphate dihydrate, 1.53 mg/mL dibasic sodium phosphate dihydrate, 0.342 mg/mL sodium citrate dihydrate, 1.30 mg/mL citric acid monohydrate, pH 5.2 f. 6.16 mg/mL NaCl, 0.86 mg/mL monobasic sodium phosphate dihydrate, 1.53 mg/mL dibasic sodium phosphate dihydrate, 0.342 mg/mL sodium citrate dihydrate, 1.30 mg/mL citric acid monohydrate, 10 mg/mL polysorbate 80, pH 5.2 g. 6.16 mg/mL NaCl, 0.86 mg/mL monobasic sodium phosphate dihydrate, 1.53 mg/mL dibasic sodium phosphate dihydrate, 0.342 mg/mL sodium citrate dihydrate, 1.30 mg/mL citric acid monohydrate, 12.0 mg/mL mannitol, 1 mg/mL polysorbate 80, pH 5.2 The method of any of the preceding claims, wherein anti-TNFa monoclonal antibody is Adalimumab.
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