M O NOC L O NA L A NTIBODY

FIE L D OF INV E NTION: The present invention relates to protein formulations suitable for therapeutic administration. In particular, the invention concerns a formulation comprising monoclonal antibody at a concentration of about 10 nrg/mL in buffer and surfactant.

BACKG ROU ND OF INV E NTION:

Non-hodgkin's lymphoma (NH L) is the most common clinical lymphoid malignancy. According to the characteristics of the disease, NH L can be divided into low, medium, and high degrees. Types of NH L vary significantly in their severity, from slow growing to very aggressive types. Many different subtypes of NH L exist In recent years, clinical trials of monoclonal antibodies and treatment of NH L has made significant progress, which has been widely used and an anti-cd20 monoclonal antibody preparations have been found to be effective.

It is known, that many protein preparations intended for human administration to require stabilizers to prevent denaturation due to aggregation or alterations during its shelf life. The instability is manifested in the formation of insoluble particles, known as ^" protein precipitation", is often increased when the protein preparation is stored at room temperature or higher. Therefore, the preparations are often refrigerated. Buffer and excipients may also impact the unfolding behaviour and consequently the stability of the monoclonal antibody, thus formulation requires use of stabilizers.

Protei ns are larger and more complex than traditional organic/ inorganic drugs. The formulation of such proteins poses special problems. For a protei n to remain biologically active, a formulation must preserve the intact conformational integrity of at least a core sequence of the protein's amino acids while at the same time protecting the protein's multiple functional groups from degradation. Reasons for degradation of proteins can involve chemical instability (i.e. any process which involves modification of the amino acid residues of a protein by bond formation or cleavage resulting in a new chemical entity) or physical instability (i.e. changes in the higher order structure or association status of the protein).

Various methods for stabilizing protein preparations have been used with varying degrees of success. However, such preparations may not always be acceptable for therapeutic purposes. Factors such as antigenicity, the chemical and biological activity of the specific protei ns etc. are i mportant

BVX-20 is an anti-cd20 humanized monoclonal antibody (mAb) developed by Biocon Ltd in collaboration with Vaccinex Inc for non-hodgkin's lymphoma (NH L). The antibody binds specifically to the antigen C D20 a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD located on pre-mature B and mature B lymphocytes. T he antigen is also expressed on >90% of B cell non-Hodgkin's lymphomas (NH L), but is not found on hematopoietic stem cells, pro B cells, normal plasma cells or other normal tissues. OBJ E CT OF INV E NTION:

The objective of invention is to develop antibody formulation which is biologically stable with shelf life up to two years.

SU M MA RY OF INV E NTION:

The present invention relates to a single-dose, sterile and clear buffered solution of rDNA origin monoclonal antibody as active ingredient formulated at approximately pH 6.5e0.3 in 10 ml USP type-1 sealed vial for intravenous (IV) administration. The formulation comprises antibody, buffer, surfactant and water wherein antibody is anti- cd20 monoclonal antibody, buffer is L-histidine and surfactant is polysorbate 80. The formulation of the present invention was found to be stable for up to 24 months at 2-8°C. B RIE F DESC RIPTION OF DRAWINGS:

Figure 1 is the amino acid sequence of heavy and light chain of recombinant humanized anti-cd20 antibody. Figure 2 shows the chromatography profi les (SEC and IEX ) of formulations with different pH values

Figures 3 to 4 show the monomer content of formulations 1-5 determined by SEC at various stress conditions Figure 5 is the CDC assay for formulations 5-10.

Figure 6 is the parallel line assay (PLA) of samples stored at 2-8°C for 9 months.

Figure 7 shows the physical stability of formulations containing different concentration of polysorbate 80 by A320 assay.

Figure 8 shows the monomer content of formulations contai ning different amounts of polysorbate 80 analysed by S E C .

Figure 9 shows the distribution of charge variants in formulations containing different amounts of polysorbate 80 analysed by IEX .

Figure 10 shows the C DC assay data for formulations containing different amount of polysorbate 80. Figure 11 shows the particle size distribution depicted by dynamic light scattering histograms of formulation.

Figure 12 shows the long term physical stability study by analysing monomer content by SEC.

Figure 13-14 show the IEX chromatograms for long term chemical stability study. Figure 15 shows the distribution of size variants in formulated bulk product stored for stability checki ng at -20°C and -80°C by SEC.

Figure 16 shows the distribution of charge variants in formulated bulk product stored for stabi I ity checki ng at -20°C and -80°C by IEX . Figure 17 shows the distribution of size variants in formulated bulk in repeated freeze- thaw for stabi I ity checki ng at -20°C and -80°C by S E C .

Figure 18 shows the distribution of charge variants in formulated bulk in repeated freeze- thaw for stability checking at -20°C and -80°C by IEX . DESC RIPTION OF INV E NTION:

BVX-20 is a recombinant humanized IgG1 (kappa) monoclonal antibody directed against antigen C D20. Antigen C D20 is a transmembrane glycoprotein expressed exclusively on normal and malignant B cells. Monoclonal antibody BVX-20 causes B cell depletion by a variety of mechanisms including apoptosis, complement dependent cytotoxicity (CDC) and antibody dependent cell mediated cytotoxicity (A DCC). It may be targeted for treatment of non-Hodgkin s lymphoma, rheumatoid arthritis and B cell chronic lymphocytic leukaemia. BVX -20 was produced in CHO cells and purified by affinity and IEX chromatography.

Present invention discloses a single-dose, sterile and preservative free buffered solution of anti-cd20 monoclonal antibody (BVX -20) which is pharmaceutically acceptable for i ntravenous admi ni strati on.

Defi nitions:

The term "pharmaceutical formulation" refers to a sterile preparation which is in a form as to permit the biological activity of the active ingredient (for example monoclonal antibody) to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are uncontaminated by other immunoglobuli ns. Specific examples of monoclonal antibodies herein i nclude chimeric antibodies, human antibodies and humanized antibodies. A "therapeutic monoclonal antibody" is a monoclonal antibody used for therapy of a human subject

In one embodiment therapeutic monoclonal antibody is anti cd-20 monoclonal antibody (BVX -20) of approximate molecular weight of 147 kDa and the approximate pi of 8.6.

" Pharmaceutically acceptable" is with respect to an excipient in a pharmaceutical formulation means that the excipient is suitable for administration to a human patient.

A "sterile" formulation is aseptic or free from all living microorganisms and their spores.

A "stable" formulation is the one in which the active agent (e.g. monoclonal antibody) therein essentially retains its physical stability, chemical stability and biological activity upon storage. The storage period is generally selected based on the i ntended shelf-life of the formulation.

A protein is said to retain its ^" physical stability" in a formulation if it shows l ittle or no change in aggregation, precipitation and/or denaturation as observed by visual examination of colour and/or clarity, or as measured by UV light scattering (measures visible aggregates) or size exclusion chromatography (SEC). SEC measures soluble aggregates that are not necessari ly a precursor for vi si bl e aggregates.

A protein is said to retain its ^" chemical stability" in a formulation, if the chemical stability at a given time is such that the protein is considered to retain its biological activity. Chemically changed species may be biological ly active and chemically unstable. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Chemical alteration may involve size modification (e.g. clipping) which can be evaluated using SEC, SDS-PAG E and/or matrix-assisted laser desorption ionizati on/time- of -flight mass spectrometry (MA L DI/TOF MS), for example. Other types of chemical alteration include charge alteration (e.g. occurring as a result of de- ami dati on) which can be evaluated by ion-exchange chromatography.

An antibody is said to retain its ^" biological activity" in a formulation, if the change in the biological activity of the antibody at a given time is within about 10% (within the errors of the assay) of the biological activity exhibited at the time the formulation was prepared as determined in an antigen binding assay.

In one embodiment stability can be evaluated when the formulation is held at a selected temperature for a sel ected ti me peri od.

In one embodiment, stability is assessed by evaluating suspension physical stabil ity, e.g. visual i nspecti on of settl i ng and/or parti cl e sedi mentati on rate.

" Buffer" refers to a buffered solution that resists changes in pH by the action of its acid- base conjugate components. The buffer of this invention generally has a pH from about 5.0 to about 8.0. Examples of buffers that will control the pH in this range include acetate, succinate, citrate, phosphate, L-histidi ne, and other organic acid buffers. In one embodiment herein, the buffer is L-histidine buffer.

A " L-histidine buffer" is a buffer comprising histidine ions. Examples of histidine buffers include histidine chloride, histidine acetate, histidine phosphate, histidine sulphate.

The term "excipient" refers to an agent that may be added to a preparation or formulation, for example: as a stabilizer, to achieve a desired consistency (e.g., altering the bulk properties), and/or to adj ust osmolality. Examples of excipients include, but are not limited to, stabilizers, sugars, polyols, amino acids, surfactants, chelating agents, and polymers.

A "stabi lizer" herein is an excipient, which stabilizes a pharmaceutical formulation. Typical stabilizers herein include saccharides, surfactants, and amino acids. A "surfactant" herein refers to a surface-active agent, preferably a non-ionic surfactant. Examples of surfactants herein include polysorbate (for example, polysorbate 20 and, polysorbate 80); poloxamer (e.g. poloxamer 188); triton; sodium dodecyl sul phate (SDS); sodium laurel sulphate etc. The surfactant may be included to prevent or reduce aggregation or denaturation of the monoclonal antibody in the preparation and/or formulation. In one embodiment the surfactant is polysorbate 20 or polysorbate 80.

"Isotonic" means that the formulation of interest has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 to 350 mi 11 i -osmol es per ki I ogram

"Stress condition" refers to an environment which is chemical ly and physically unfavourable for a protein and may render unacceptable protein stability (e.g. thermal, shear, chemical stress).

The antibody BVX -20 has heavy chai n and the light chain amino acid sequences defined by SE Q ID NOS: 1 and 2 respectively. The amino acid sequences are depicted in figure 1.

Formulation development achieved through the procedure that i ncluded stability check of antibody at various check-poi nts. Pre-formulation studies were conducted that included screening of pH, buffers and surfactant individually for optimizing stability of BVX -20 in a sol ution.

The invention is further ill ustrated by way of the followi ng examples which are intended to elucidate the invention. These examples are not intended to, nor are they to be construed, as limiting the scope of the invention. Many modifications and variations of the present invention are possible in view of the teachings herein and, therefore, are withi n the scope of the invention.

The examples below are carried out using standard techniques as per table 1. Such standard techni ques are wel I known and routi ne to those are ski 11 ed i n the art.

The observations of the studies were conducted as per parameters and methodologies from table 1 and further cross-checked with a standard set of values to determine the stabi I ity of formulati ons Table 2: the specifications

A number of experi mental test were conducted for screeni ng of f ormul ati ons accordi ng to various criteria such as different pH values, buffers and excipients E xampl e 1 : Preparati on of the f ormulati on: screeni ng of pH

In one embodiment of the invention, the choice of a particular buffer for a particular pH was determined by the buffering capacity of a buffer at the chosen pH.

Screening of pH is first step of pre-formulation process. Antibody was formulated at 10 mg/ml at the different pH (viz. 5.5, 6.0, 6.5, and 7.0) as listed in Table 3. Sodium chloride was added to al I the f ormul ati ons to mai ntai n the i oni c strength.

T able 3: Details of evaluation of different formulation pHs

Size exclusion chromatography and ion-exchange chromatography were used to check physical stability and chemical stability, respectively. As shown in figure 2 no significant difference was observed in physical stability (SEC) while the presence of greater number of basic variants were seen at pH 5.5 (IEX ). Comparison of the conformational stability at pH 6.0 and 6.5 showed that the melting temperatures were higher for the mol ecul e at pH 6.5 and pH 7.0 (tabl e 4) i ndi cati ng better stability at pH 6.5-7.0 that at pH 6.0.

In one embodiment of the invention, pH 6.5 and pH 7.0 were found to be most suitable for the f ormul ati on.

T able 4: M elting temperatures of antibody formulated at different pH

Pre-formulation study further continued to in two parts as per example 2 and 3.

E xample 2: Preparation of the formulation ^" screening of buffers and pH B ased on the resul ts of the pH screeni ng study, fi rst pre-f ormul ati on study was i ni ti ated to evaluate formulations with different buffers and excipients at pH 6.5-7.0.

The antibody was buffer exchanged into the respective formulations and filled at 10 mg/ml in 3ml 2R USP type-1 glass vials stoppered with 13mm cream coloured un-coated bromo- butyl closures and crimped with aluminium flip-off seals. Sodium chloride and polysorbate were added to all the formulations. Sol utions formulated at different buffer species such as sodium phosphate, potassium phosphate and histidine at different concentration and pH (table 5), were subjected to different stress conditions and were analysed by the techniques and parameters outlined in table 1 and 2 such as DSC, SEC, A ₃₂₀ (clarity), reducing SDS-PAG E. The techniques were selected to have an overal I understand! ng of the stabi I ity of the mol ecul e. The stress conditions mainly included various storage conditions viz. 2-8°C, 25°C, 37°C and Freeze-Thaw at short term using time-points of 1 week and 2 week (table 6).

T able 6: Stress conditions to evaluate stability for first pre-formulation study

Results of differential scanning calorimetric (DSC) analysis for conformational stability are summarized in table 7. This analysis indicated that formulations 5 had slightly higher melti ng temperatures and hence the better stabi I ity compared to other f ormul ati ons.

Table 7: M elting T emperatures of the formulations evaluated by DSC

Results of SEC analysis are summarized as below (figures 3-4).

1. No significant degradation was observed in formulation 1-5 upon storage at 2-8°C.

2. The molecule was found to degrade with increase in fragmentation and a corresponding decrease in monomer content upon storage at 37°C. Degradation was found to be highest in formulation 1 while formulation 2 appeared to be most stable.

3. Similar degradation pattern was observed upon storage at 30°C with agitation.

Formulation 1 showed highest degradation.

4. During freeze- thaw, formulations 1, 4, 5 appeared stable. Formulations 2, 3 showed aggregation as monomer content reduced significantly.

Results of fi rst pre-formulation study of formulations 1-5 subjected to different stress condi ti ons are summari zed i n tabl e 8.

T able 8: R esults summary of first pre-formulation study

E xampl e 2: Preparati on of the f ormulati on screeni ng of buffers and pH

In order to further evaluate different excipients second pre-formulation study was performed.

The antibody was buffer exchanged into the respective formulations and fil led at 10 mg/ml in 3 ml 2R USP type-1 glass vials stoppered with 13 mm cream coloured un- coated bromo-butyl closures and crimped with an aluminium flip-off seals.

During first study, all formulations contained sodium chloride, however, a clear leader was not apparent among the different formulations tested. Duri ng this round of study, in order to evaluate if sodium chloride did in fact have a beneficial effect it was present in one formulation only. The buffer systems were otherwise similar to the ones tested in first study. Here polysorbate 80 was added in all the formulations.

Buffer species including sodium phosphate, potassium phosphate and histidine at different concentration and pH are described in table 9. These were subjected to stress conditions i ncluded various storage conditions namely 2-8°C, 37°C and 30°C agitation at various time-points up to 4 weeks (table 10). T he samples were analysed by the techniques and parameters outlined in table 1 and 2 such as SEC, reducing SDS-PAG E, IEX , DSC, C DC assay. The techniques were selected to have an overall understanding of the stabi I ity of the mol ecul e.

Table 9: Details of the formulations evaluated

Table 10: Stress conditions to evaluate stability of the different formulation for pre- formulation study 2 DSC analysis (table 11) showed that formulations 6, 9 and 10 had higher melting temperatures as compared to formulation 7 & 8.

Table 11 : Evaluation of melting T emperatures of the formulation 6-10 by DSC

Table 12: SE C results of formulations subj ected to different stress condition.

Additional technique such as CDC assay (figure 5) for biological activity of formulation was added during this part of pre-formulation study. Results of first pre-formulation study of formulations 6-10 subjected to different stress conditions are summarized in table 13. When subjected to different stress conditions, formulation 9 showed better physical stability than the other formulations. SE C, SDS-PAG E and biological activity analysis indicated that formulation 9 is consistently marginally better than the other formulations.

Table 13: Summarized result of the formulations 6-10 based on analysis of samples subj ected to different stress conditions

Based on the results of pre-formulation studies, the formulation 9 was chosen for further studies.

E xample 4: Long term stability study

The formulation number 9, (0.05% (w/v) Polvsorbate 80, 15 mM H istidine, pH 6.5) was further studi ed for I ong term stability up to 24 months.

The antibody was formulated at 10 mg/ml and filled in USP type 1 glass vials (10 R) at a fil l volume of 10 ml. The vials were stoppered with cream coloured bromo butyl closures from WE ST pharma and crimped with 13 mm aluminium seals.

The stress conditions included various storage conditions viz. 2-8°C (example 4.1), 25°C (example 4.2), 37°C (example 4.3) and Freeze-Thaw up to 4 thaw cycles (example 4.4) at vari ous ti me-poi nts between 0 to 24 months. Formulations were analysed to evaluate physical and chemical stability and biological activities using techniques as per table 1 and 2.

E xample 4.1 Stability at 2-81C (Tables 14-18)

An increase in fragmentation was observed after 9 months and fragmentation continued to i ncrease ti 11 24 months. Acidic vari ants were found to gradual ly i ncrease over a peri od of 9 months and a steep increase was observed at 16 months. This increase in acidic variants continued over the 24 month time period. Biological activity was found to be comparable to the standard over period of 24 months.

Table 14: A ₂ so, pH and visual appearance of samples stored at 2-81C

T able 16: C hemical stability by I EX for samples stored at 2-81C

The % cytotoxicity over control is calculated by dividing the cytotoxicity of the sample by that of the standard, and multi plyi ng by 100 to obtai n the result as a percentage.

Table 17: Biological activity (cytotoxicity) by C DC assay of samples stored at 2-81C

(carried out at 0.015i g/ml)

The CDC assay was carried out at 0.015 ι g/ml till the 6 ^th month. The samples after 9 ^th (Figure 6), 12 ^th month, 16 ^th and 22 month incubation were analysed by a parallel line assay at 7 different concentrations (Table 18).

Parallel work performed for optimizing the assay to obtain the complete dose response curves using several concentrations. This allowed to evaluate the results in PLA software (parallel line analysis software: from Stegmann Systems), hence the samples of later stages (6 months onwards) were evaluated using the PLA software. The ratio of EC50 values obtained from PLA analysis for standard and test was considered as pass, if the ratio was between 70 ^" 130% of the reference standard.

The reference standard EC50 was between 0.05 and 0.5 nM, and cytotoxicity was between 70-130% of that of the standard run.

Table 18: Cytotoxicity of sample stored at 2-81C at end of 12, 16, 22 and 24 months by parallel l ine assay

E xample 4.2 Stability at 251C (Table 19-22) Upon physical, chemical stability and biological activity check at various time-points as per table 19-22, antibody was found to be physically stable and biologically active over a period of 2 months. After 2 months, fragmentation of antibody was observed and the activity gradually dropped. A significant drop in biological activity was observed at the end of 4 and 6 months. The acidic variants also increased markedly at end of 2 months. The discoloration of the formulation was observed and the solution had turned yel low at end of 5 months at 25°C.

Table 19: pH, visual appearance and protein concentration of samples stored at

251C

Table 22: Biological activity (cytotoxicity) by C DC assay of samples stored at 25IC

(carried out at 0.015i g/ml)

E xample 4.3 Stability at 371C (Table 23-26)

Upon physical, chemical stability and biological activity check at various time-points as per tabl es 23-26, anti body was found to i ncrease i n aggregati on at the end of 2 months. A marked increase in the percentage of acidic variants was observed over the period of months. A large increase in the percentage of acidic variants at the end of 3 months rendered the chromatogram un-analysable. The biological activity was found to decrease markedly over the first month fol lowed by a complete loss of activity at the end of 2 months. T he f ormulati on exhi bited yel I ow discol ourati on at the end of 2 months.

Table 23: pH, visual appearance and protein concentration of samples stored at

371C

Table 24: Physical Stability by SE C for samples stored at 371C Table 26: Biological activity (cytotoxicity) by C DC assay of samples stored at 37°C

(carried out at 0.015i g/ml)

E xample 4.4 Stability upon repeated freezing and thawing (Table 27-29)

Freeze thaw stability of the samples was evaluated by freezing the samples at ^" 201C for 1 week fol I owed by thawi ng at room temperature. Four such cycl es were performed.

T able 27: Physical stability by SE C for samples subj ected to freezing and thawing

Table 28: C hemical stabil ity by I EX for samples subj ected to repeated freezing and thawing

T able 291 : Biological activity (cytotoxicity) by C DC assay of samples subj ected to repeated freezing and thawing (carried out at 0.015i g/ml)

The formulation was found to be physically stable over a period of 9 months at 2-8°C. A significant increase in fragmentation observed at the end of 12 months at 2-8°C. Furthermore, the acidic variants were found to increase over the period of 22 months (Tables 14-18).

At the accelerated conditions evaluated, the antibody was found to degrade gradually at 25°C, with a sharp decline in activity and increase in fragmentation at the end of 3 months. Furthermore, the acidic variants also increased significantly at end of 6 months. (Tables 19-22).

At 37°C, an increase in fragmentation and decrease in activity was observed at the end of 1 month. A complete loss of activity was observed at the end of 2 months at 37°C. (Tables 23-26). Antibody was found to be physically and chemically stable upon freezing and thawing (up to 4 cycles) (Tables 27-29). The biological activity was not compromised upon repeated freezing and thawing. V ariations observed in the % basic variants are due to differences in resolution of the zero and basic variants.

E xample 5: Preparation of the formulation ^" optimization of concentration of polysorbate 80

The antibody was formulated at 10 mg/ml and filled in USP type 1 glass vials (10 R) at a fil l volume of 3 ml and had different concentration of polysorbate 80 in the range of 0- 0.08% w/v (table 30). The vials were stoppered with cream coloured bromo butyl closures from WE ST pharma and crimped with 13 mm aluminium seals. The stress conditions mainly included various storage conditions viz. 2-8°C, 26°C with agi tati on and 37°C usi ng di ff erent ti me- poi nts ti 11 12 weeks.

No significant difference was observed for formulations containing different quantities of polysorbate 80 upon storage at 2-8eC. This was evident upon evaluation of physical stability by A320 (figure 7) and SEC (figure 8), chemical stability by IEX (figure 9), biological activity (figure 10) and conformational stability by intrinsic fluorescent analysis (table 31).

T able 31: Intrinsic fluorescence data of samples stored at 2-81C

Antibody was found to degrade upon agitation at 26°C. All formulations showed an increase in fragmentation, absorbance at 320nm (figure 7) and acidic variants over a period of 10 weeks when subjected to agitation (220 R PM) at 26°C. No difference was observed in the CDC assay (figure 10) and in intrinsic fluorescence data (table 32) for all samples.

T able 32: Intrinsic Fluorescence data of samples subj ected to agitation at 26IC

Antibody was found to degrade upon storage at 37°C. All formulations showed an increase in fragmentation, absorbance at 320nm (figure 7) and acidic variants over a period of 10 weeks when stored at 37°C. DLS analysis showed the presence of higher order aggregates in samples with 0 and 0.005% polysorbate 80 (figure 11). These species were not detected in the formulation containing 0.02% polysorbate 80.

T able 33: Intrinsic fluorescence data of samples stored at 371C

Hence, formulation contai ning 0.02% polysorbate 80 showed a margi nally higher stability. Based on the results of this study, the polysorbate 80 concentration in the formulation of monoclonal antibody was fixed at 0.02% (w/v) and optimized as below: 15 mM H istidine 0.02% (w/v) Polysorbate 80 pH 6.5. E xample 6: Long term stability evaluation of formulation

A long term stability study of product in the chosen formulation, 15 mM histidine 0.02% (w/v) polysorbate 80, pH 6.5 was initiated to evaluate stability of the product.

The formulation was evaluated at 2-8°C, 25°C and 37°C to evaluate long term stability, stability under accelerated and stress condition (table 34). The analysis was done at various time-points from 0 to 24 months. The stability of the molecule was monitored by evaluating physical, chemical and biological stability.

The stability of BVX -20 was evaluated in the optimized solution comprising of 15 mM histidine and 0.02% (w/v) polysorbate 80 formulated at pH 6.5. T he anti body was formulated at 10 mg/ml and filled in 10 ml USP type 1 glass vials. The vials were stoppered with cream coloured bromo-butyl closures from West Pharma and sealed with 13 mm al uminium flip-off seals. The vials were stored at various conditions as shown in table 34 and analysed by methodologies and parameters outlined i n Table 1 and table 2.

T able 34: stress conditions for long-term stability check E xample 6.1 stability check at 2-8°C

Table 35: pH, appearance and protein concentration of samples stored at2-81C Table 36: Particulate matter, sterility and endotoxin limits of samples stored at 2-

T able 39: Potency of samples stored at 2-8eC, at various time points by C DC assay

No significant change was observed in the protein concentration on storage for 24 months. The particle count was within limits at end of 24 months (table 36). The pH and appearance of samples stored at 2-8°C complied with the specifications, at the end of 24 months (table 35).

By size exclusion chromatography, an increase in fragmentation was observed over the period of 24 months at 2-8°C (Table 37/ Figure 12). A corresponding decrease in monomer content was observed (1.7% decrease over 24 months). There was no increase in aggregation at the end of 24 months at 2-8°C. The overall purity was greater than 95% and within the specified limits at the end of 24 months at 2-8°C (Table 38/ Figure 13). The percentage of acidic variants increased (by approximately 10%) at the end of 24 months. The main peak percentage dropped by approximately 4-5%. There appeared to be a slight decrease in the basic content The biological activity of the sample remained withi n specifications at the end of 24 months storage at 2-8°C (table 39). E xample 6.2 stability check at 25°C

T able 40: pH, appearance and concentration of samples stored at 251C

Table41: Particulate matter, sterility and endotoxin limits of samples stored at251C

Table 43: Distribution of charge variants in samples stored at 25IC (IEX)

The pH, appearance and protein concentration of samples stored at 25°C complied with the specifications, at end of 6 months (table 41). The particle count was within limits at end of 6 months (table 42). A decrease in percentage of monomer and increase in fragmentation was observed over the period of 6 months at 25°C with the product going out of specification at the end of 6 months (figure 12). There was also considerable increase in acidic and reduction in zero variants over six months (Table 43/ Figure 13). The biological activity of samples was intact and within specifications at end of two months. However, there was a considerable drop in activity at end of three months and the data was not within specification, although the monomer content was > 95% (95.03%).

Under accelerated storage condition (25°C), the molecule was found to degrade gradually with increase in fragmentation and decrease in monomer at the end of 6 months. The acidic variants also increased significantly at end of 6 months. A lthough the monomer content was found to be within the specification (> 95%) at the end of 3 months, the biological activity decreased on storage at 25°C and was outside the specification I imits.

E xample 6.3 stability check at 37°C

44: pH, appearance and concentration of samples stored at 371C

T able 452: Particulate matter, sterility and endotoxin limits of samples stored at 371C

T able 48: Percentage cytotoxicity of samples stored at 371C by C DC assay

Following storage of the product at 37°C, the pH, appearance and protein concentration of samples complied with the specifications at end of 7 weeks (table 44-47). The particle count was also within limits up to 7 weeks. A decrease in percentage of monomer and increase in fragmentation was observed over the period of 7 weeks at 37°C with the product going out of specification at the end of 5 weeks (table 47). There was also considerable increase in acidic and reduction in zero variants over 7 weeks (figure 14). The biological activity of sample was found to decrease considerably at the end of 3 weeks (tabl e 48), although the monomer content was >95% (98.18). E xample 7: Hold time study

Earlier, freezi ng was considered the best option to maintain the stability of the antibody. Therefore, the study was initiated to evaluate the stability of the formulated bulk.

This study involved evaluation of the physical, chemical and biological stability of the formulated BVX -20 antibody, filled into bags and frozen by parameters and methodologies from table 1 and 2. The cytotoxicity of the sample and standard was calculated individually. The potency ratio calculated by dividing the cytotoxicity of the sample by that of the standard. The results were compared agai nst the limits set for the assay which should be within 70-130% of the standard. Low molecular weight protei ns (L MWP) and high molecular weight proteins (H MWP) were calculated. The study conducted at 2 different freezing temperatures: -20°C and -80°C. Thawing temperature in both cases was room temperature (RT) 22e2°C. E xample 7.1 Stability foil owing storage at -20°C and -80°C

Frozen state stability involved testing the ability of the monoclonal antibody to remain frozen for long periods of time. In this study, the monoclonal anti body in bags was left frozen at -20°C and -80°C for a total period of 12 months, and stabil ity was evaluated once every 3 months. A different bag was used for every time point, such that once a bag is thawed, it was not ref rozen.

49: pH , UV 280 data of samples stored at -201C and -80eC

T able 50: C DC Assay results of formulated bul k stored at -20IC and -80eC

The pH of the samples was maintained and no visible particulates under all conditions tested. By UV280, the concentration of the samples varies by less than 5% compared to TO.

There was a marginal increase in H MWP% and L MWP% from TO at 4 Months at -20eC. After 4 months, the H MWP and L MWP % remains relatively stable. The distribution of H MWP, Monomer and L MWP in the frozen state stability -80eC samples is comparable to the TO val ues (figure 15).

The distri bution of acidic variant main peak and basic variant in the frozen state stabil ity (9 months) samples is comparable to the TO values (figure 16) All samples that were tested had a Potency ratio between 0.7 and 1.3 of the standard, i ndi cati ng that the samples and the standard had comparable activity (table 50)

Although the results of this study indicated that BVX-20 formulation was amenable to storage as formulated bulk for long term storage, when the BVX -20 bulk was stored at - 20eC in the production facil ity, it was found to aggregate. This may be due to the fact that there was no cryo- protectant used in the formulation that might protect the monoclonal antibody when frozen.

E xampl e 7.2 Stabi I ity f ol I owi ng repeated f reezi ng and thawi ng

Repeated freeze-thaw stability evaluates the ability of the monoclonal antibody to withstand repeated freezing and thawing cycles. The formulated monoclonal antibody was frozen at -20°C or -80°C, left in the frozen state for 1 week and then thawed. Three such repeated freezing and thawing cycles were conducted. After each freezing and thawing cycle, the monoclonal antibody was evaluated for stability, and the profiles were compared to the initial (TO) profile.

Additionally, in this experiment after the completion of 3 freeze-thaw cycles, the bags containing formulation were left at room temperature for a period of 72 hours, to understand if any stress conditions that the molecule may have undergone during the freezing and thawing cycles have had detrimental after-effects that may make the molecule less stable at room temperature.

Table 52: C DC Assay results of formulated bulk upon FT (-801C/ RT )

There was no significant difference in the distribution of monomer, H MWP and L MWP in the repeated freeze thaw samples (3 freeze thaw cycles at -20eC and -80eC) compared to the TO samples (figure 17). The samples incubated at room temperature for 72 hours after undergoing 3 cycles of freeze-thaw at -20eC and -80eC showed comparable distri bution of H MWP, monomer and L MWP to other samples.

There was no significant difference found in the charge variant distribution in the repeated freeze thaw samples (3 freeze thaw cycles at -20eC and -80eC) compared to the TO samples (figure 18). The samples incubated at RT for 72 hours after undergoing 3 cycles of freeze-thaw at -20eC and -80eC showed comparable distribution of acidic variant, main peak and basic variant to other samples.

All samples that were tested had a potency ratio between 0.7 and 1.3 of the standard, indicating that the samples and the standard had comparable activity (table 52).

E xample 9: Final Formulation The formulation was manufactured of BVX 20 mAb finished product involves the buffer exchange of the anti body i nto buffer contai ni ng15 mM histi di ne at pH 6.5. T he sol uti on is then diluted in formulation buffer without polysorbate 80 to a concentration of - 12 mg/ml. This solution is further diluted to 10 mg/ml using formulation buffer containing polysorbate 80 required to make the final concentration of polysorbate 80 in the formulated bulk 0.02% (w/v). In case of a delay in the filling of the bulk for a period greater than 72 hours, the bulk is frozen in bioprocessing bags at -20tC/-80tC. In this case, the bulk is thawed at RT (22 e 21C) prior to filling. The formulated bulk is then steri le filtered through 0.22i filter and filled into 10 ml USP type-1 glass vials at a fill volume of 10.5 ml, stoppered with 13 mm Fluoro coated bromo butyl closures and crimped with aluminum flip-off seals. T he vials are labeled and stored at 2-81C.

Although the results of this study indicated that BVX-20 formulation was amenable to storage as formulated bulk for long term storage, when the BVX -20 bulk was stored at - 20eC in the production facil ity, it was found to aggregate. This may be due to the fact that there is no cryo- protectant in the formulation that may help protect the mAb when frozen. T herefore the bul k of BVX -20 conti nues to be stored at 2-8eC.