Patent Application
20170088873
Monoclonal antibody stability represents a current challenge in the purification and formulation of these proteins. MAb instability leads to high levels of aggregated mAb in protein formulations, which can have several disadvantages including changing protein activity and potentially leading to undesirable immunological responses in patients. Protein A affinity chromatography is a powerful and widely-used tool for purifying antibodies. In order to elute a protein or antibody from the Protein A resin, acidic conditions are required due to the high affinity of the monoclonal antibodies to the resin. Exposure to these acidic conditions can result in the formation of protein aggregates. Some strategies to address aggregation during Protein A chromatography have been previously described in the literature. Furthermore, a low pH hold step following elution is required for viral inactivation and can also result in the formation of protein aggregates.
Furthermore, association and aggregation tend to occur during frequently. Extensive research into changes in antibody structure caused by acidic pH has been conducted. However, resolution of the issues regarding structural change and the association and aggregation reactions has yet to be proposed.
The performance of the cell culture process can have significant effects on product quality and potency, especially with respect to glycosylation, post-transcriptional modifications and impurity profiles. Since CHO cell and other continuously cultured cells have low efficiency in completely oxidizing glucose to CO2 and H2O, one by-product of cell culture process is lactate accumlation, which can cause acidification of culture medium and lead to high osmolality and low viability due to the alkali added to control the medium pH. Thus, when lactate accumulation exceeds the buffering capacity of the culture medium, pH drifts downward, which could trigger base addition leading to increased osmolality of the culture medium. This could be risky in cell lines that synthesize excessive amounts of lactate since high pH, high lactate and high osmolality cascade often causes delayed cell growth and accelerated cell death.
The impact of osmolality has been reported on growth inhibition with increasing osmolality and effect on cell specific productivity (deZengotita V M, Schmelzer A E, Miller W M. Characterization of hybridoma cell responses to elevated pCO2 and osmolality: intracellular pH, cell size, apoptosis and metabolism. Biotechnol Bioeng. 2002; 77:369-380). These deleterious effects could be exacerbated when combined with high dissolved CO2 levels that could occur in high cell density cultures, and hence it is vital to ensure during process development that the osmolality profile is acceptably low, especially towards the latter stages of the cell culture process. (Zhu M M, Goyal A, Rank D L, Gupta S K, Boom T V, Lee S S. Effects of elevated pCO2 and osmolality on growth of CHO cells and production of antibody-fusion protein B1: A case study. Biotechnol Prog. 2005; 21:70-77). Also it has been reported earlier that when high feeding rates are utilized both lactate and ammonium start accumulating at higher concentrations in the cultures resulting in an osmolality as high as 500 mOsm/kg to 700 mOsm/kg.
The applicant has surprisingly found that it is possible to minimize the amount of aggregates produced during the cell culture process as well as to improve yield of monoclonal antibody, ultimately resulting in improved potency that is retained over longer duration storage at 2-8 deg C., 25 deg C., 60 deg C., attributed to i) carefully selection of the optimal cell line and optimizing cell culture conditions such as media components that will impact media osmolality and conductivity, feed strategy, temperature, and pH ii) Most importantly, applicant's purification process a) includes sodium chloride as one of the components of solutions used across entire purification, b) is devoid of strong bases (such as sodium hydroxide) as despite the advantage of low volume addition use of base can be associated with risk of product denaturation in the localized region where the solution is added.
The instant invention describes an improved process for manufacturing a monoclonal antibody that maintains low osmolality during logarithmic phase, minimum secondary metabolites like ammonia and lactate, enhanced cell growth and productivity minimum aggregation or degradation of monoclonal antibody during purification, thereby improving potency of monoclonal antibody comprising:
A preferred embodiment of invention is that said wherein Feed A addition can be done during log phase, particularly when cell count is between 2-4×106 cells/ml and cell count thereafter reaches 5 to 7×106 cells/ml.
One of the preferred embodiment of instant invention is that said flow rate of Feed solution A was found to maintain low osmolality, thereby providing improved growth and productivity.
Accordingly feed solution A can comprise of a mixture of water soluble amino acids, vitamins and glucose, wherein amino acids selected from but not limited to L-Aspartic acid, L-Glutamic, Aspargine, L-Serine, L-Histidine Hydrochloride, Monohydrate, L-Glycine, Threonine, L-Alanine, L-Arginine, L-Tyrosine, L-Cystine-SS-CysL-Valine, L-Methionine, L-Phenylalanine, L-Isoleucine, L-Leucine, L-Lysine Hydrocloride and L-Proline.
Most preferably the feed solution A can be selected from commercially available feeds like Cell Boost 1™, Cell Boost 2™, Cell Boost ™, Cell Boost 4™, IS CHO-CD XP™, CHO CD EfficientFeed™ A, CHO CD EfficientFeed™ B, preferably Cell Boost 2™ or Cell Boost 4™, most preferably Cell Boost 2™ (R15.4) such that the feed solution A does not contain growth factors, lipids or Cholesterol.
Another preferred embodiment of instant invention is that said Wash buffer 2 having pH between 5.8 and 6.2, used during protein A chromatography can comprise of
Yet another important embodiment of the invention is that said neutralization of Protein A eluate to pH 5.0 can be carried using a neutralization solution devoid of NaOH, having pH between 5.8 and 6.2 comprising of
The protein A chromatographic resin of step (c) used may be any protein A or variant or a functional fragment thereof coupled to any chromatographic support. Preferably, the protein A resin is Prosep vA Ultra (from Millipore), wherein animal-free protein A is immobilized on porous glass.
Cation exchange chromatographic step (g) mentioned in the embodiments may be carried our using any weak or strong cation exchange chromatographic resin or a membrane which could function as a weak or a strong cation exchanger. Commercially available cation exchange resins include, but are not limited to, those having a sulfonate based group e.g., MonoS, MiniS, Source 15S and 30S, SP Sepharose Fast Flow, SP Sepharose High Performance from GE Healthcare, Toyopearl SP-650S and SP-650M from Tosoh, S-Ceramic Hyper D, from Pall Corporation or a carboxymethyl based group e.g., CM Sepharose Fast Flow from GE Healthcare, Macro-Prep CM from BioRad, CM-Ceramic Hyper D, from Pall Corporation, Toyopearl CM-650S, CM-650M and CM-650C from Tosoh. Preferably, the cation exchange resin in step (g) can be a strong cation exchange resin, preferably Fractogel EMD SE Hicap (M) resin.
The antibody of instant invention can be selected from the group consisting of a natural human antibody, a humanized antibody, a human-type antibody, an antibody prepared by genetic recombinantion and a monoclonal antibody. Preferably, said antibody is a human monoclonal antibody that binds to rabies virus selected from the group consisting of 17C7, 6Gl 1 5G5, 2B10, or 1E5. More preferably, said antibody is HuMab 17C7 (WO2006084006-incorporated by reference) that neutralizes rabies virus by interacting with a discontinuous epitope on the rabies virus glycoprotein which includes amino acids 336-342 of the glycoprotein (antigenic site III).
According to one of the preferred embodiment, HuMab 17C7 potency measured by RFFIT of was found to be 4-6 fold better as compared to human rabies immmoglobulin (hRIG), wherein such significant improvement in potency can be attributed to i) use of “Feed solution A” followed by “Amino acid concentrate” and optimal feeding rate that ensures rapid growth, high expression and low osmolality iii) purification utilizing salt at a particular concentration as part of wash II buffer iv) using neutralization solution comprising of salt and citrate buffer, devoid of NaOH.
Further, Rabies virus neutralization potency for 17C7 was found to be ranging from about 100 IU/2.5 ml to about 250 IU/2.5 ml.
An important embodiment of the instant invention is that said rabies virus neutralization potency of 17C7 monoclonal antibody i) after 1 year storage at 2-8 deg C. was found to be at least 85% of the potency before storage and ii) after 6 months storage at 25 deg C., 60 deg C. was found to be at least 85% of the potency before storage.
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
17C7 is a fully human IgG1 monoclonal antibody specific to the rabies virus surface G glycoprotein used for local and intramuscular administration. The molecular weight of 17C7 was calculated from the deduced amino acid sequence and is 145,280 Da.
The 17C7 monoclonal antibody has been shown to neutralize multiple isolates of the rabies virus in both in vitro and in vivo assays. The 17C7-expressing hybridoma was isolated from transgenic HuMAb mice (Medarex) containing human immunoglobulin genes and inactivated mouse heavy chain genes and kappa light chain genes and thus is a fully human IgG1 antibody containing human kappa light chains.
HuMAb mice were injected with 1/10 of a human dose of RabAvert™ (Chiron) or Imovax® (Aventis) rabies vaccines using complete Freund's adjutant in the first week, and RIBI adjuvant in subsequent weeks for a total of 6-8 weeks. Hybridomas were generated by fusion of splenocytes and partner cells (P3X63Ag8.653 mouse myeloma cells). Hybridoma supernatants were screened for reactivity in a rabies virus glycoprotein ELISA and RFFIT assays and reactive antibodies were purified from hybridoma cultures by protein A sepharose chromatography.
The antibody genes from the 17C7 hybridoma were isolated and cloned into an expression vector (pConKappa/Gamma, Lonza) designed to promote the production of high levels of antibody. The expression vector containing the 17C7 antibody genes (designated 17C7) was transfected into CHOKISV cells and tranfectants were selected for the glutamine synthetase gene (contained in the vector) using methionine sulfoximine as a selection agent. High-expressing cells were isolated, subcloned and ultimately banked. The most favorable cell line, based on expression levels, stability, gene copy number, production in small scale bioreactors and growth properties, was selected for manufacturing of the 17C7 antibody for use in clinical trials.
Further details of methods for generation of Anti-Rabies Monoclonal Antibody—HuMab 17C7 have also been disclosed in William D. Thomas, et al EP1851315 and S. E. Sloan et al, Vaccine 25 (2007), 2800-2810 (incorporated an reference).
Fermentation was carried out at temp 3720 C. (±) 0.3 for a duration of 12 days (±) 1 day. Feed A solution i.e. Cell Boost 2™ (R15.4) comprising of Glucose, vitamins and amino acids was prepared as 5 to 10% solution, in medium component or WFI and was fed at a flow rate of 5 to 15 ml/min or more, and final concentration in the reactor was made as 0.2 to 0.5%. Feed A was added when the cell count was between 2-4 mill Cells/mL generally at day 2 or 3, and at a flow rate of 5-15 ml/min such that the final concentration of Feed A in the fermenter was around 0.2% to 0.5%, thereafter during day 4th to 7th Feed B & C were added which were basically Amino acid concentrates.
A chemically defined Medium (CD-CHO) was used as fermentation medium.
Table 1 indicates that when Amino acid solution was fed first followed by Feed A, high Osmolality was observed especially during 4th to 9th day which finally affected the cell growth and also the yield. Whereas applicant found that when Feed ‘A’ solution was fed first and followed by Aminoacid Concentrate, osmolality was maintained on lower side during log phase wherein cell count was on higher side and consequently a rise in yield was observed.
Earlier 0.1M NaOH was used to neutralize the antibody i.e to raise the pH from 3.5 to 5.0.
Physical Appearance of Protein A Eluate after adjusting pH with neutralization buffer (With and Without NaOH).
Protein A eluate is clear before adjusting pH with neutralization buffer. However after adjusting pH with neutralization buffer containing NaOH, thread like particles are observed and solution becomes hazy, probably indicating aggregation. Refer
Whereas adjusting pH with neutralization buffer containing citrate and sodium chloride, particles are not observed and solution appears clear, probably indicating minimum aggregation. Refer
Refer
Applicant has found that minimum aggregation, preservation of antibody integrity and minimum unfolding of 17C7 Monoclonal antibody was observed during elution and low pH hold, when i) wash buffer 2 having pH 6.0 containing 250 mM sodium chloride and 10 mM phosphate buffer was utilized ii) protein A eluate was neutralized to pH 5 by using a neutralization solution having pH 6 devoid of NaOH, instead comprising of 20 mM citrate buffer in combination with 300 mM sodium chloride. Refer
Each mL contains: Rabies Human Monoclonal Antibody-100 IU/40 IU, 20 mM Citrate Buffer (Sodium citrate and citric acid), 150 mM Sodium Chloride and 0.025% (w/v) Polysorbate 80.
The RFFIT (rapid fluorescent focus inhibition test) assay is based on the principle that un-neutralized Rabies virus does not produce any cytopathic effect in MNA cells. But when antibodies labelled with fluorescent dye are added, they bind to the rabies virus infected MNA cells. When observed under fluorescence microscope, these cells fluoresce due to the dye and indirectly confirm the presence of rabies virus.
Typically this assay was performed in duplicate. So above mentioned procedure was repeated.
2) 75 μl of media (MEM+10% FBS) was added to well 1
3) 100 μl of media (MEM+10% FBS) was added to wells 2 through 7 of the control slide.
4) 200 μl of media (MEM+10% FBS) was added to well 8 of the control slide.
5) 50 μl of 2 IU/ml reference standard was added to well 1 and mixed thoroughly (1:5).
(6) 25 μl of well 1 was transferred to well 2 and mixed thoroughly (1:25).
7) 25 μl of well 2 was transferred to well 3 and mixed thoroughly (1:125).
8) 25 μl of will 3 was transferred to well 4 and mixed thoroughly (1:625).
9) 25 μl of well 4 was discarded.
One more chamber slide was taken and procedure was repeated for control dilutions.
After completion of incubation period, slides were removed from CO2 incubator and fluorescence staining was done as follows:
After completion of incubation period, slides were removed from CO2 incubator and washed as follows:
<10/20
ND50 of the test and standards sample was calculated as per the Reed and Muench method and potency was calculated as follows:
P.D.=[(Infectivity next above 50%−50)/(infectivity next above 50%−infectivity next below 50%)]×log of dil.factor
Neutralizing titer (IU/ml) of the test sample was determined as follows:
(ND50 of the test sample/ND50 of reference standard)×Potency of the inference standard
Clinical results discussed below indicate that huMab 17C7 anti-rabies monoclonal antibody prepared by above mentioned fermentation and purification processes surprisingly was found to have at least 4 fold enhanced potency measured by RFFIT relative to human rabies immunoglobulin (hRIG)
Table 11 indicates clinical 17C7 material that was prepared using
Table 12 indicates clinical 17C7 material that was prepared using
In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 3654/MUM/2015 | Sep 2015 | IN | national |
