FORM-2
THE PATENT ACT,1970
(39 OF 1970)
AND
THE PATENT RULES, 2003
(As Amended)
COMPLETE SPECIFICATION (See section 10;rule 13)
"Improved feeding strategies and purification processes for monoclonal antibody production"
SERUM INSTITUTE OF INDIA PRIVATE LTD., a corporation organized and existing under the laws of India, of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411 028, Maharashtra, India.
The following specification particularly describes the invention and the manner in which it is to be performed:

Background of Invention:
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 accumulation, 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 effects on cell specific productivity(deZengotita VM, Schmelzer AE, Miller WM. 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 MM, Goyal A, Rank DL, Gupta SK, Boom TV, Lee SS. 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.

Summary of Invention:
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 degC,25 deg C,60degC, 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.
Brief Description of Drawings:
Figure 1(a- Amino acid concentrate, Acid Base Solution (ABS) followed by Feed A solution
Figure 1(b)- Feed A solution, Acid Base Solution (ABS)followed by Amino acid concentrate.
Figure 2- Batch Consistency in terms of high yield and
osmolality observed for feedings approach comprising addition of
Feed A solution, Acid Base Solution (ABS) followed by Amino acid
concentrate.

Figure 3- Rabies Monoclonal antibody (17C7) bulk indicates absence of aggregate formation after adjusting pH to 5.0 with neutralization Buffer having pH 6 comprising of 20mM citrate buffer in combination with 300mM sodium chloride and devoid of NaOH.
Figure 4- Rabies Monoclonal antibody (17C7) bulk indicates visible aggregate formation after adjusting pH to 5.0 with neutralization Buffer having 0.1 N NaOH.
Figure 5- Purified Rabies Monoclonal antibody (17C7)aggregates peak.
Figure 6- Rabies antibody aggregates spiked with Rabies antibody monomers
Figure 7- Overlay graph of Rabies antibody aggregates with Rabies antibody Monomers bulk.
Detailed Description of Invention:
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) adding “Feed solution A” containing vitamins, amino acids and glucose at log phase at a concentration between 0.2% to 0.8 % with a flow rate of about 5-15 ml/min wherein osmolality was maintained between 350 mOSm/kg and 400 mOSm/kg thereafter.

b) addition of concentrated amino acid feed solutions, Feed B and Feed C.
c) contacting the sample with a Protein A affinity chromatography column;
d) selecting a wash buffer containing salt to minimize aggregation during elution and low pH hold
e) eluting the monoclonal antibody from the Protein A affinity chromatography column with an elution buffer
f) neutralizing protein A eluate to pH 5 by using citrate buffer in combination with salt instead of base
g) subjecting sample to a second chromatography having strong cation exchange resin.
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 x 106 cells/ml and cell count thereafter reaches 5 to 7 x 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,L- 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 3™, 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
i) NaCl at a concentration between 100mM and 300mM, preferably between 200mM and 250mM to minimize aggregation during elution and low pH hold.
ii) Phosphate buffer between 5mM and 20mM, preferably between 10mM and 20mM
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
i) Citrate at a concentration between 10mM and 80mM,
preferably between 10mM and 30mM. ii) NaCl at a concentration between 100mM and 400mM, preferably between 250mM and 300mM
The protein A chromatographic resin of step (c)used may be any protein A or variant or a functional fragment thereof coupled to
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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 out 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 recombination 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
7

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 immunoglobulin (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 100IU/2.5ml to about 250IU/2.5ml.
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 atleast 85% of the potency before storage and ii) after 6 months storage at 25 deg C, 60 deg C was found to be atleast 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.
Examples:
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
8

calculated from the deduced amino acid sequence and is 145,280 Da.
Example 1
Cell-line Source and Development Summary
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 adjuvant 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 CHOK1SV 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
9

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 as reference).
Example 2
Cell Culture and Bioreactor Processes
Fermentation was carried out at temp 37°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 15ml /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 – 15ml/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.
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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.
Figure 1(a) & Figure 1(b) shows the effect of sequence of feed addition during the process, where Figure 1(a)shows higher osmolality and decrease in cell count and yield, on the other side Figure 1(b) shows lower Osmolality and increase in cell count and yield.
Table 1- Cell count ,Osmolality profiles and IgG productivity for i) AA, ABS, followed by FAS & ii) FAS, ABS followed by AA

AA, ABS, FAS, ABS AA, ABS, FAS, ABS AA, ABS, FAS, ABS followed by followed by followed by followed followed followed by FAS AA FAS by AA by FAS AA
Day Cell Count (Milli./ml) Cell Count (Milli./ml) IgG Conc. (gm/L) IgG
Conc.
(gm/L) OSM (mOsm/Kg) OSM (mOsm/Kg)
0 0.46 0.45 0.00 0.00 310 310
1 0.9 0.82 0.00 0.00 317 318
2 1.53 1.65 0.00 0.00 329 322
3 2.8 3.02 0.00 0.00 350 333
4 3.6 4.00 0.00 0.00 359 342
5 4.2 5.10 0.25 0.24 378 348
6 4.8 5.80 0.29 0.33 390 341
7 5.2 6.00 0.35 0.42 400 388
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8 5.3 5.90
9 4.9 5.90
10 4.6 5.70
11 4.5 5.60
12 4.2 4.50
0.37 0.63 405 401
0.42 0.72 412 403
0.50 0.81 419 405
0.58 0.87 423 411
0.64 0.93 430 420
AA: Amino Acid concentrate ABS: Acid Base Solution FAS: Feed A Solution OSM:Osmolality
Table 2 - Batch Consistency for Feed A solution, Acid Base Solution (ABS)followed by Amino acid concentrate(Refer figure 2)

Cell Count (Milli./ml) B1 Cell Count (Milli./m l) B2 Cell Count (Milli./m l) B3 OSM
(mOsm/K g) B1 OSM
(mOsm/K g) B2 OSM
(mOsm/K g) B3 IgG Conc. (gm/L ) B1 IgG Conc. (gm/L ) B2 IgG Conc. (gm/L ) B3
0.45 0.50 0.54 310 306 305 ND ND ND
0.82 0.97 1.00 318 314 314 ND ND ND
1.65 1.75 1.96 322 319 315 ND ND ND
3.02 3.40 2.70 333 325 320 ND ND ND
4.00 4.40 4.40 342 339 339 ND 0.15 ND
5.10 5.20 4.50 348 342 345 0.25 0.25 0.28
5.80 5.80 4.90 341 340 340 0.33 0.35 0.36
6.00 6.20 5.50 388 356 354 0.42 0.44 0.41
5.90 6.10 5.50 401 400 405 0.63 0.58 0.54
5.90 6.40 5.40 403 401 409 0.72 0.69 0.66
5.70 5.90 5.30 405 406 410 0.81 0.77 0.73
5.60 5.00 5.20 411 410 412 0.87 0.84 0.78
4.50 4.50 4.50 420 410 414 0.93 0.86 0.85
OSM:Osmolality
Example 3
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Purification of Rabies Human Monoclonal Antibodies (17C7) at 350L Scale
Table 3

Steps Sub-Step
Step 1- Prosep vA Ultra protein A Chromatography Packed Bed Height - 11.3 cm

Binding capacity~column loaded >25 mg/mL

HETP Testing

Initial Sanitization

Equilibration (5CV, ≤300 cm/hr)

Loading (≤300 cm/hr)

Post Load Wash I (≤300 cm/hr)

Post Load Wash II
(≤300 cm/hr, 10 mM Phosphate buffer, 250 mM NaCl, pH 6.0)

Elution(5CV, ≤150 cm/hr)

CIP(5CV, ≤300 cm/hr)
Storage(3CV, ≤300 cm/hr)
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Step 2- Viral Inactivation Neutralization of low pH treated Protein A Eluate carried out with 20mM citrate buffer, 300 mM NaCl, pH 6.0
Step 3- Fractogel EMD SE HiCap Chromatography Packed Bed Height(11.3 cm)

Binding capacity(~column loaded >25 mg/mL)

Sanitization (0.5M NaOH, 5 CV, ≤300 cm/hr)

Static Hold

Charge

Equilibration (5CV, ≤300 cm/hr)

Loading (≤300 cm/hr)

Post Load Wash (3CV, ≤300 cm/hr)

Elution(10-15 CV, ≤150 cm/hr(0-60% Buffer B (20 mM Citrate buffer, pH 6.0, 300 mM NaCl))

CIP / Sanitization (0.5M NaOH, 5 CV, ≤300 cm/hr)

Storage (0.1 M NaOH, 3 CV, ≤300 cm/hr)
Example 4
Protein A Wash 2 buffer, Neutralization buffer (With or Without Sodium chloride): Effect on Aggregation Profile
Sodium chloride in Wash 2 buffer
Table 4: pH and conductivity of 10 mM Sodium phosphate buffer
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(Wash 2 Buffer) at different NaCl concentration.

Sr.No. NaCl Concentration pH Conductivity (mS/cm)
1 10 mM sodium phosphate Buffer + 150 Mm NaCl 6.00±0.2 15.8
2 10 mM sodium phosphate Buffer + 200 mM NaCl 6.00±0.2 20.81
3 10 mM sodium phosphate Buffer + 250 mM NaCl 6.00±0.2 25.12
4 10 mM sodium phosphate Buffer + 300 mM NaCl 6.00±0.2 29.33
Table 5: pH and conductivity of Wash 2 buffer of protein A chromatography with and without NaCl.

Sr.No. Parameters Wash 2 Buffer with NaCl Wash 2 Buffer without NaCl
1 pH 6.00±0.2 6.00±0.2
2 Conductivity 25.12 mS/cm 1.36 mS/cm
3 Chemical Composition 10 mM Sodium
phosphate buffer +
250 mM NaCl 10 mM Sodium phosphate buffer
Sodium chloride in Neutralization Buffer
Table 6 - pH and conductivity of 20 mM Citrate buffer (Neutralization Buffer)at different NaCl concentration.

Sr.No. NaCl Concentration pH Conductivity (mS/cm)
1 20 mM Citrate Buffer + 150 mM NaCl 6.00±0.2 17.5
2 20 mM Citrate Buffer + 200 mM NaCl 6.00±0.2 22.1
3 20 mM Citrate Buffer + 250 mM NaCl 6.00±0.2 26.67
4 20 mM Citrate Buffer + 300 mM NaCl 6.00±0.2 31.3
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Example 5
Neutralization Buffer (With and Without NaOH)of Protein A chromatography:
Earlier 0.1M NaOH was used to neutralize the antibody i.e to raise the pH from 3.5 to 5.0.
Table 7-

Sr.No. Parameters Sodium Hydroxide Citrate Buffer
1 pH 13.40 6.00±0.2
2 Conductivity 15.97 mS/cm 26.67 mS/cm
3 Chemical Composition 0.1M Sodium hydroxide 20 mM citrate buffer + 250 mMNaCl
Example 6
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 Figure 4.
Whereas adjusting pH with neutralization buffer containing citrate and sodium chloride, particles are not observed and solution appears clear, probably indicating minimum aggregation. Refer Figure 3.
Refer figures 5,6 and 7 for aggregate peak analysis.
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
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i)wash buffer 2 having pH 6.0 containing 250mM sodium chloride and 10mM 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 20mM citrate buffer in combination with 300mM sodium chloride. Refer Figure 4.
Example 7
Formulation of HuMab 17C7
Each mL contains: Rabies Human Monoclonal Antibody-100 IU/40IU, 20 mM Citrate Buffer(Sodium citrate and citric acid),150mM Sodium Chloride and 0.025% (w/v) Polysorbate 80.
Example 8
RFFIT protocol for potency estimation of rabies human monoclonal antibody (17C7)
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.
Dilutions :
Sample Dilutions :
1) Once chamber slide was taken and marked as sample slide. 75µl of media (MEM+10% FBS) was added to well 1.
2) 100µl of media (MEM+10% FBS) was added to wells 2-8 of the
17

chamber slide.
3) 50µl of test sample was added to well 1 and mixed thoroughly (1:5).
4) 25µl of well 1 was transferred to well 2 and mixed thoroughly (1:25).
5) Similarly dilution upto 1: 390625 (Well 8) was prepared and then 25µl of well 8 was discarded.
Table 8

4 5
3 6
2 7
1 8
Test Sample
Typically this assay was performed in duplicate. So above mentioned procedure was repeated.
Control Dilutions :
1) One chamber slide was taken and marked as the control slide for the assay.
2) 75µl of media (MEM+10% FBS) was added to well 1
3) 100µl of media (MEM+10% FBS) was added to wells 2 through 7 of the control slide.
4) 200µl of media (MEM+10% FBS) was added to well 8 of the control slide.
5) 50µl of 2 IU/ml reference standard was added to well 1 and mixed thoroughly (1:5).
6) 25µl of well 1 was transferred to well 2 and mixed thoroughly (1:25).
7) 25µl of well 2 was transferred to well 3 and mixed thoroughly (1:125).
8) 25µl of well 3 was transferred to well 4 and mixed thoroughly (1:625).
9) 25µl of well 4 was discarded.
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Table 9

Ref Std CVS
1;625 50
FFD50
1:125 10-1
1:25 10-2
1:5 Cells Only
Control Slide
One more chamber slide was taken and procedure was repeated for control dilutions.
Challenge Virus Standard (CVS) dilution:
1) CVS i.e. CVS-11 was thawed and diluted to 50FFD50 in media (MEM+10% FBS).
2) 100 µl of diluted CVS Rabies virus was added to wells 1-8 of all sample slides and to wells 1-5 of the control slide and mixed thoroughly.
3) Two sterile glass vials labeled -1 and -2 were taken.
4) 1.80 ml of media (MEM+10% FBS) was added to both vials.
5) 200 µl of diluted virus was added to the -1 vial and mixed thoroughly.
6) 200 µl from the -1 vial was transferred to the -2 vial and mixed thoroughly.
7) 100 µl of -1 virus was added to well 6 of the control slide.
8) 100 µl of -2 virus was added to well 7 of the control slide.
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9) Well 8 of the control slide was “cells control only”.
10) Slides were incubated for 90 minutes at 36±10C in
humidified CO2 incubator.
MNA cells:
1) MNA cells suspension was prepared and cell count adjusted to 5–6 x 105 cells per ml.
2) 200 µl of the cell suspension was added to each well of the chamber slides and mixed thoroughly.
3) chamber slides were incubated at 36±10C for 20-24 hours in humidified CO2 incubator (CO2 -2.0 to 2.5%).
Fluorescence staining:
After completion of incubation period , slides were removed from CO2 incubator and fluorescence staining was done as follows :
Fixation:
1) Two glass/plastic beakers filled with 80% chilled acetone were taken, slide cover was removed and the media was decanted in the SS bowl.
2) slide was submerged immediately in the first 80% chilled acetone beaker , rinced once and then wells were filled with 80% chilled acetone from second beaker.
3) Slides were kept for 10-15 minutes at room temperature.
4) Acetone was discarded in the SS bowl.
5) Slides were allowed to dry at room temperature or in the incubator.
Staining:
1) Antirabies antibody conjugated to fluorescein Isothiocyanate (FITC) dye was diluted with PBS to a predetermined dilution (1:40).
2) 100 µl –150 µl of conjugate was added to each well so that entire cell monolayer was covered.
3) Slides were incubated at 36±10C in humidified incubator 30-45 minutes.
Washing:
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After completion of incubation period , slides were removed from CO2 incubator and washed as follows :
1) Top chamber was removed and discarded.
2) slide was dip rinsed twice in two beakers containing PBS.
3) Then slide was dip rinsed in WFI.
Slides Observation
1) Slides were observed on an inverted fluorescence microscope at a magnification of 160X to 200X. Observation of bright Green intracellular granules indicated a positive result i.e. the MNA cell was infected with rabies virus.
2) 20 distinct fields of the chamber were counted.
3) Number of fields containing infected cells were noted.
4) The assay was considered valid if the control slide had the following range of infected fields out of twenty.
Table 10

Ref Std CVS (FFD50)
18-20/20 20/20
0-10/20 10-20/20
0/20 <10/20
0/20 0/20
Control Slide
Calculation:
ND50 of the test and standards sample was calculated as per the Reed and Muench method and potency was calculated as follows:
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P.D.= [(Infectivity next above 50%-50)/ ( infectivity next above 50% - infectivity next below 50%)] x log of dil.factor
Neutralizing titer (IU/ml) of the test sample was determined as follows:
(ND50 of the test sample/ ND50 of the reference standard)x Potency of the reference standard
Example 9
Phase II/III Clinical Trial results (HRIG Vs HuMab 17C7):
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 atleast 4 fold enhanced potency measured by RFFIT
relative to human rabies immunoglobulin (hRIG)
Table 11

17C7 HRIG GMCs Ratio of GMCs
Mab
GMCs
12.9 4.4 2.9
Table 11 indicates clinical 17C7 material that was prepared using
1. Protein A wash buffer 2 containing 10 mM Phosphate buffer,
pH 6.0.
2. Neutralization solution containing 0.1 M NaOH
Table 12

17C7 HRIG GMCs Ratio of GMCs
Mab
GMCs
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24.9 5.5 4.5
Table 12 indicates clinical 17C7 material that was prepared using
1. Protein A wash buffer 2 containing 10 mM Phosphate buffer,250mM NaCl, pH 6.0.
2. Neutralization solution containing 20mM citrate buffer, 300 mM NaCl, pH 6.0
Example 10
Stability testing of 17C7 at 2-8,25,60 deg C
Results of Stability Studies: Table 13
a) Long term condition (2-8ºC):

TESTS Initial 3rd m 6th m 9th m 12th m 18th m 24th m
Appearance
(Clear,
colorless
liquid
free from
any
visible
particles) C C C C C C C
pH (5.50-6.50) 6.01 6.02 5.90 5.98 6.00 6.04 6.09
Osmolality (250-350mOsm/kg 270 269 260 267 263 268 261
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)
Protein conc(4.00-5.00) 4.78 4.69 4.83 4.73 4.80 4.85 4.85
Purity-SEC HPLC (monomer should be >90%.Reten tion time of monomer should be comparable to ref std.) Monomer =99.25% Monomer =98.59% Monomer =99.21% Monomer= 99.90% Monomer= 100.00% Monomer= 100.00% Monomer= 100.00%
Purity-IEF(pI value must be within + 10 % of pI value of ref std) Spl=8.9 9
RS=8.99 Spl=8.9 6
RS=8.96 Spl=9.1 1
RS=9.08 Spl=8.97 RS=9.01 Spl=8.74 RS=8.86 Spl=9.09 RS=9.06 Spl=9.01 RS=9.03
Purity-SDS
page(R)
(Molecular
weights of
heavy and
light
chains
must be Spl HC=46KD
LC=25KD
RS HC=46KD
LC=26KD Spl HC=46KD
LC=25KD
RS HC=47KD
LC=25KD Spl HC=46KD
LC=26KD
RS HC=46KD
LC=26KD Spl HC=46KD
LC=25KD
RS HC=47KD
LC=26KD Spl HC=47KD
LC=25KD
RS HC=49KD
LC=25KD Spl HC=48KD
LC=26KD
RS HC=48KD
LC=26KD Spl HC=48KD
LC=26KD
RS HC=47KD
LC=25D
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within Total% Total% Total% Total%Sp Total%Sp Total%Sp Total%Sp
+10% of Spl;=10 Spl;=10 Spl;=10 l;=100% l;=100% l;=100% l;=100%
mol wt of 0% 0% 0%
Ref
Std.Total;
% of heavy
and light
chains
must be
>95%)
Mol Mol Mol
Purity-SDS weight weight weight Mol Mol Mol Mol
page (NR) of of of weight weight weight weight
(Molecular major major major of major of major of major of major
weight of band of band of band of band of band of band of band of
major must
be within Spl=149 Spl=150 Spl=150 Spl=151K Spl=149K Spl=159K Spl=155K
+10% of KD KD KD D D D D
mol wt of
Ref Std. RS=149K RS=152K RS=152K RS=149KD RS=150KD RS=161KD RS=154KD

D D D
Bacterial
Endotoxin(
<2.50EU/mg <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 <0.10
of
protein)
Sterility
(Shall be Passes - - - - - Passes
sterile)
Potency-
RFFIT 591 586 577 542 520 495 422
(Should
25

not be less than 300IU/ml)
C- Complies
Table 14
b) Accelerated Data (25+2°C; 60+5% RH):

TESTS Initial 1m 2m 3m 6m
Appearance(Cle ar, colorless liquid free from any visible particles) C C C C C
pH (5.50-6.50) 6.01 6.00 6.00 6.01 5.79
Osmolality (250-350mOsm/kg) 270 276 271 269 261
Protein
concentration
(4.00-5.00) 4.78 4.73 4.75 4.67 4.81
Purity-SEC HPLC (monomer should be >90%.Retention time of monomer should Monomer=
99.25%
Spl RT comparabl e to Ref Monomer=
99.17%
Spl RT comparable to Ref std Monomer=
97.73%
Spl RT comparable to Ref std Monomer=
98.58%
Spl RT comparable to Ref std Monomer=
98.79%
Spl RT comparable to Ref std
26

be comparable to ref std.) std RT RT RT RT RT
Purity-IEF(pI value must be within + 10 % of pI value of ref std) Spl=8.99 RS=8.99 Spl=8.97 RS=9.01 Spl=8.96 RS=8.99 Spl=8.98 RS=8.96 Spl=9.10 RS=9.08
Purity-SDS page(R) (Molecular weights of heavy and light chains must be within +10% of mol wt of Ref Std.Total; % of heavy and light chains must be >95%) Spl HC=46KD
LC=25KD
RS HC=46KD
LC=26KD
Total%Spl ;=100% Spl HC=46KD
LC=25KD
RS HC=47KD
LC=26KD
Total%Spl;= 100% Spl HC=49KD
LC=26KD
RS HC=46KD
LC=25KD
Total%Spl;= 100% Spl HC=46KD
LC=25KD
RS HC=47KD
LC=25KD
Total%Spl;= 100% Spl HC=47KD
LC=26KD
RS HC=46KD
LC=26KD
Total%Spl;= 100%
Purity-SDS page (NR) (Molecular weight of major must be within +10% of mol wt of Ref Std. Mol weight of major band of
Spl=149KD
RS=149KD Mol weight of major band of
Spl=148KD
RS=148KD Mol weight of major band of
Spl=149KD
RS=153KD Mol weight of major band of
Spl=154KD
RS=152KD Mol weight of major band of
Spl=152KD
RS=152KD
Bacterial Endotoxin(<2.5 0 EU/mg of <0.10 <0.10 <0.10 <0.10 <0.10
27

protein)
Sterility (Shall be sterile) Passes NA NA NA NA
Potency-RFFIT (Should not be less than 300IU/ml) 591 529 507 520 510
Based on Stability observations disclosed in Table 13 & Table 14, following 17C7 was found to possess following stability attributes:
1) Final Bulk for Rabies Human Monoclonal Antibody(17C7) was found to have a shelf life of 36 months at 2-8 deg C.
2) Rabies virus neutralization potency of 17C7 monoclonal antibody i) after 1 year storage at 2-8 deg C was found to be atleast 85% of the potency before storage and ii) after 6 months storage at 25 deg C, 60 deg C was found to be atleast 85% of the potency before storage
3) The monomer content of all batches at all time intervals showed above 98%. The aggregation levels of rabies antibody remained negligible and there was no increase in dimer content during longer storage periods.
4) pI of 17C7 antibody remained stable.
28

5) The pH at all time points was stable, which indicates that even during longer storage periods, there was no alteration or biophysical modification.
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.

We claim,
1. A method for producing a monoclonal antibody with enhanced potency comprising:
a) adding “Feed solution A” containing vitamins, amino acids and glucose at log phase at a concentration between 0.2% to 0.8% with a flow rate of about 5-15 ml/min at an initial osmolality of about 350 mOSm/kg;
b) addition of concentrated amino acid feed solutions, Feed B and Feed C;
c) contacting the sample with a Protein A affinity chromatography column;
d) selecting atleast one wash buffer having pH between 5.8 and 6.2 containing salt and phosphate buffer to minimize aggregation during elution and low pH hold;
e) eluting the monoclonal antibody from the Protein A affinity chromatography column with an elution buffer;
f) neutralizing protein A eluate to pH 5 by using a neutralization solution having pH between 5.8 and 6.2 devoid of NaOH, instead comprising of citrate buffer in combination with salt;
g) subjecting sample to a second chromatography having cation exchange resin; and
wherein the said fermentation method maintains low osmolality during logarithmic phase, minimum accumulation of secondary metabolites like ammonia and lactate, provides higher cell growth, high yield followed by a purification

method that provides an antibody solution lacking aggregation and devoid of significant turbidity, thereby providing a monomeric antibody with a yield greater than 98% and purity of greater than > 99.0 %, having an endotoxin content of <0.10 EU/mg.
2. The method according to Claim 1, wherein the feed solution A is a mixture of water soluble amino acids, vitamins and glucose, wherein amino acids can be selected from L-Aspartic acid, L-Glutamic,Aspargine ,L-Serine,L-Histidine Hydrochloride, Monohydrate,L- Glycine,L- 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.
3. The method according to Claim 2, wherein the feed solution A is selected from Cell Boost 1™, Cell Boost 2™, Cell Boost 3™, Cell Boost 4™, IS CHO-CD XP™, CHO CD Efficient Feed™ A, CHO CD EfficientFeed™ B.
4. The method according to Claim 3, wherein the feed solution A is Cell Boost 2™ (R15.4) such that does not contain growth factors, lipids or Cholesterol.
5. The method according to Claim 1,wherein the osmolality during logarithmic phase is maintained between 350 mOSm/kg and 400 mOSm/kg.

6. The method according to Claim 1, wherein the concentration of NaCl in the wash buffer is from about 200mM to about 250mM.
7. The method according to Claim 1, wherein the concentration of phosphate buffer in the wash buffer is from about 10mM to about 20mM.
8. The method according to Claim 1, wherein the concentration of citrate in the neutralization solution is from about 10mM to about 30mM.
9. The method according to Claim 1, wherein the concentration of NaCl in the neutralization solution is from about 250mM to about 300mM.

10. The method according to Claim 1, wherein the cation exchange resin in step (g) is a weak or strong cation exchange resin .
11. The method according to Claim 10, wherein said cation exchange resin is having a sulfonate based group or a carboxymethyl based group.
12. The method according to Claim 10, wherein said cation exchange resin is a strong cation exchange resin.

13. The method according to Claim 12, wherein said strong cation exchange resin is a Fractogel EMD SE Hicap (M) resin.
14. The method according to Claim 1, wherein said antibody can be a natural human antibody, a humanized antibody, a human-type antibody, an antibody prepared by genetic recombination and a monoclonal antibody.
15. The method according to Claim 14, wherein said antibody is an IgG monoclonal antibody.
16. The method according to Claim 15, wherein said IgG monoclonal antibody is an IgGl antibody.
17. The method according to Claim 16, wherein the IgG1 antibody is a human monoclonal antibody that binds to rabies virus.
18. The method according to Claim 17, wherein the anti-rabies IgG1 antibody is a human monoclonal antibody selected from the group consisting of HuMab 17C7, 6Gl 1 5G5, 2B10 and 1E5.
19. The method according to Claim 18, wherein the said anti-rabies IgG1 human monoclonal antibody is 17C7.

20. A method according to claim 1, wherein the yield of monomeric monoclonal antibody is greater than 98% and the monoclonal antibody is purified to a purity of greater than > 99.0 % as assessed by high performance size exclusion chromatography (HP-SEC).
21. A method for producing a HuMab 17C7,an anti-rabies monoclonal antibody with atleast 4 fold enhanced potency measured by RFFIT relative to human rabies immunoglobulin (hRIG) comprising:

a) adding Cell Boost 2™ (R15.4) containing vitamins, amino acids and glucose at log phase at a concentration between 0.2% to 0.5 % with a flow rate of 5 to 15ml /min at an initial osmolality of less than 350 mOSm/kg;
b) addition of concentrated amino acid feed solutions, Feed B and Feed C;
c) contacting the sample with a Protein A affinity chromatography column;
d) selecting wash buffer 2 having pH 6.0 containing 250mM sodium chloride and 10mM phosphate buffer to minimize aggregation during elution and low pH hold;
e) eluting the monoclonal antibody from the Protein A affinity chromatography column with an elution buffer;
f) neutralizing protein A eluate to pH 5 by using a neutralization solution having pH 6 devoid of NaOH, instead

comprising of 20mM citrate buffer in combination with 300mM sodium chloride;
g) subjecting sample to Fractogel EMD SE HiCap Chromatography; and
wherein the said fermentation method maintains low osmolality during logarithmic phase, minimum accumulation of secondary metabolites like ammonia and lactate, higher cell growth and antibody yield, followed by a purification method that provides an antibody solution substantially lacking significant turbidity and/or aggregation thereby providing a monomeric antibody with a yield greater than 98% and purity of greater than > 99.0 %, having an endotoxin content of <0.10 EU/mg.
22. A method according to claim 1 or claim 21, wherein said 17C7 antibody is stable i) for 36 months at 2-8 deg C with retention of atleast 85% potency ,ii) for atleast 6 months at 25 deg C with retention of atleast 85% potency and iii)for atleast 6 months at 60 deg C with retention of atleast 85% potency.