Claims:1. A stable aqueous pharmaceutical formulation comprising a therapeutically effective amount of an anti-cd20 antibody, a buffer and a surfactant wherein the formulation is devoid of sodium/potassium chloride.
2. The formulation of claim 1 wherein the antibody is rDNA origin recombinant humanized IgG1 (kappa) monoclonal antibody.
3. The antibody as claimed in any preceding claims has SEQID 1.
4. The formulation of claim 1 wherein the antibody concentration in the formulation is up to 10 mg/mL.
5. The formulation of claim 1 wherein the surfactant is a polysorbate 80.
6. The surfactant of claim 5 is in the range of 0.01-0.02% w/v.
7. The formulation of claim 1 wherein the buffer is 10-20 mM L-Histidine at pH 6.5-7.0.
8. The formulation of claim 1 which is stable at a temperature of about 2-8°C for 24 months.
9. The formulation of claim 1 which is stable at about 25°C for at least one month.
, Description:FIELD OF INVENTION:
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 mg/mL in buffer and surfactant.

BACKGROUND OF INVENTION:
Non-hodgkin's lymphoma (NHL) is the most common clinical lymphoid malignancy. According to the characteristics of the disease, NHL can be divided into low, medium, and high degrees. Types of NHL vary significantly in their severity, from slow growing to very aggressive types. Many different subtypes of NHL exist. In recent years, clinical trials of monoclonal antibodies and treatment of NHL 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.
Proteins are larger and more complex than traditional organic/ inorganic drugs. The formulation of such proteins poses special problems. For a protein 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 proteins etc. are important.
BVX-20 is an anti-cd20 humanized monoclonal antibody (mAb) developed by Biocon Ltd in collaboration with Vaccinex Inc for non-hodgkin's lymphoma (NHL). The antibody binds specifically to the antigen CD20 a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD located on pre-mature B and mature B lymphocytes. The antigen is also expressed on >90% of B cell non-Hodgkin's lymphomas (NHL), but is not found on hematopoietic stem cells, pro B cells, normal plasma cells or other normal tissues.

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

SUMMARY OF INVENTION:
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 6.5±0.3 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 2-80C.

BRIEF DESCRIPTION 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 profiles (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 2-80C 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 containing different amounts of polysorbate 80 analysed by SEC.
Figure 9 shows the distribution of charge variants in formulations containing different amounts of polysorbate 80 analysed by IEX.
Figure 10 shows the CDC 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 checking at -200C and -800C by SEC.
Figure 16 shows the distribution of charge variants in formulated bulk product stored for stability checking at -200C and -800C by IEX.
Figure 17 shows the distribution of size variants in formulated bulk in repeated freeze-thaw for stability checking at -200C and -800C by SEC.
Figure 18 shows the distribution of charge variants in formulated bulk in repeated freeze-thaw for stability checking at -200C and -800C by IEX.

DESCRIPTION OF INVENTION:
BVX-20 is a recombinant humanized IgG1 (kappa) monoclonal antibody directed against antigen CD20. Antigen CD20 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 (ADCC). 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 intravenous administration.
Definitions:
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 immunoglobulins. Specific examples of monoclonal antibodies herein include 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 intended shelf-life of the formulation.
A protein is said to retain its “physical stability" in a formulation if it shows little 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 necessarily a precursor for visible 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 biologically 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-PAGE and/or matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS), for example. Other types of chemical alteration include charge alteration (e.g. occurring as a result of de-amidation) 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 selected time period.
In one embodiment, stability is assessed by evaluating suspension physical stability, e.g. visual inspection of settling and/or particle sedimentation 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-histidine, 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 adjust osmolality. Examples of excipients include, but are not limited to, stabilizers, sugars, polyols, amino acids, surfactants, chelating agents, and polymers.
A "stabilizer" 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 sulphate (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 milli-osmoles per kilogram.
"Stress condition" refers to an environment which is chemically and physically unfavourable for a protein and may render unacceptable protein stability (e.g. thermal, shear, chemical stress).
The antibody BVX-20 has heavy chain and the light chain amino acid sequences defined by SEQ ID NOS: 1 and 2 respectively. The amino acid sequences are depicted in figure 1.
Formulation development achieved through the procedure that included stability check of antibody at various check-points. Pre-formulation studies were conducted that included screening of pH, buffers and surfactant individually for optimizing stability of BVX-20 in a solution.
The invention is further illustrated by way of the following 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 within the scope of the invention.
The examples below are carried out using standard techniques as per table 1. Such standard techniques are well known and routine to those are skilled in the art.
Parameter Technique
General Visual Appearance
pH pH meter
Protein Content/Concentration A280 (UV Spectroscopy)
Particulate Matter A320,
Particle Count by Light obscuration
Physical Stability (SEC) Size Exclusion Chromatography,
SDS PAGE,
(DLS) Dynamic Light Scattering
Chemical Stability (IEX) Ion Exchange Chromatography
Conformational Stability (DSC) Differential Scanning Calorimetry,
(IFS) Intrinsic Fluorescence Spectroscopy
Biological Activity (CDC) Complement Dependent Cytotoxicity Assay
Table 1: the parameters analysed and methodologies used
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 stability of formulations
Tests Specification
Description (visual observation) Colourless and transparent solution
Particulate matter = 10 µm : not more than 6000 particles/ container
=25 µm: not more than 600 particles/ container
pH 6.5±0.3
Purity by SEC Not less than 95% of monomer
Protein Concentration (A280) 9.0 mg/ml to 11.0 mg/ml
Identity by gel electrophoresis (SDS-PAGE under reducing conditions) 2 Major bands at molecular weight of 45 kDa and 25 kDa
Residual HCDNA Less than 10 ng/dose
Residual Host Proteins Less than 100 ppm
Residual Protein A Less than 10 ppm
Bacterial Endotoxin Less than 5 EU/mg
Sterility by Membrane Filtration Preparation for injections should meet the USP <71> requirements of sterility
Determination of vol. of injection in containers USP Between 10.0 ml to 10.5 ml
Biological Activity,
Potency using CDC assay EC50 of Reference Standard between 0.05 nM and 0.5 nM
Test sample should be 70%-130% of the EC50 of the internal reference standard value
Table 2: the specifications

A number of experimental test were conducted for screening of formulations according to various criteria such as different pH values, buffers and excipients

Example 1: Preparation of the formulation: screening 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 all the formulations to maintain the ionic strength.
Formulation pH Composition
1 5.5 25mM Citrate, 150 mM NaCl
2 6.0 25mM Histidine, 150 mM NaCl
3 6.5 25mM Histidine, 150 mM NaCl
4 7.0 25mM Phosphate, 150 mM NaCl

Table 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 molecule at pH 6.5 and pH 7.0 (table 4) indicating 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 formulation.

pH Tm1(°C) Tm2(°C) Tm3(°C)
6 52.9 71.2 83.4
6.5 70.5 72.8 84
7 70.3 72.1 84.2
Table 4: Melting temperatures of antibody formulated at different pH
Pre-formulation study further continued to in two parts as per example 2 and 3.

Example 2: Preparation of the formulation – screening of buffers and pH
Based on the results of the pH screening study, first pre-formulation study was initiated 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. Solutions 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, A320 (clarity), reducing SDS-PAGE. The techniques were selected to have an overall understanding of the stability of the molecule.
The stress conditions mainly included various storage conditions viz. 2-80C, 250C, 370C and Freeze-Thaw at short term using time-points of 1 week and 2 week (table 6).

Formulation Composition
1 150 mM Sodium Chloride, 0.02% (w/v) Polysorbate 80 25 mM Histidine, pH 6.5
2 150 mM Sodium Chloride, 0.02% (w/v) Polysorbate 80 15 mM Sodium Phosphate, pH 7
3 150 mM Sodium Chloride, 0.02% (w/v) Polysorbate 80 25 mM Sodium Phosphate, pH 7
4 150 mM Sodium Chloride, 0.02% (w/v) Polysorbate 80 15 mM Potassium Phosphate, pH 7
5 150 mM Sodium Chloride, 0.02% (w/v) Polysorbate 80 15 mM Histidine, pH 6.5
Table 5: Details of the formulations evaluated

Stress Conditions
2-80C
370C
300C Agitation (220 RPM)
Freeze Thaw
(Storage of sample at -200C for 1 week followed by thawing at RT (22±20C))
Table 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 melting temperatures and hence the better stability compared to other formulations.
Formulation Tm1 (0C) Tm2 (0C) Tm3 (0C)
1 70.1 75.4 83.5
2 70.3 72.6 84.2
3 67.2 73.2 80.5
4 69.5 76.4 82.7
5 70.7 76.4 84.2
Table 7: Melting Temperatures 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 2-80C.
2. The molecule was found to degrade with increase in fragmentation and a corresponding decrease in monomer content upon storage at 370C. 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 300C 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 first pre-formulation study of formulations 1-5 subjected to different stress conditions are summarized in table 8.
Stress/Storage Condition Ranking of Formulations
DSC (thermal denaturation)
(Based on average of Tm1, Tm2, Tm3) 3 < 2 <1 < 4< 5

agitation 1 < 5< 2< 3, 4
freezing and thawing 2 < 3 < 1 < 4,5
37°C 5, 1 < 4 < 3, 2
2-8°C 1 < 2, 3, 4, 5
Table 8: Results summary of first pre-formulation study

Example 2: Preparation of the formulation – screening 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 filled 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. During 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 included various storage conditions namely 2-80C, 370C and 300C agitation at various time-points up to 4 weeks (table 10). The samples were analysed by the techniques and parameters outlined in table 1 and 2 such as SEC, reducing SDS-PAGE, IEX, DSC, CDC assay. The techniques were selected to have an overall understanding of the stability of the molecule.

Formulation Composition
6 150 mM Sodium Chloride, 0.05% (w/v) Polysorbate 80 15 mM Sodium Phosphate, pH 7.0
7 150 mM Potassium Chloride, 0.05% (w/v) Polysorbate 80, 15 mM Potassium Phosphate, pH 7.0
8 150 mM Potassium Chloride, 0.05% (w/v) Polysorbate 80 15 mM Histidine, pH 6.5
9 0.05% (w/v) Polysorbate 80 15 mM Histidine, pH 6.5
10 0.05% (w/v) Polysorbate 80 15 mM Sodium Phosphate, pH 7.0
Table 9: Details of the formulations evaluated

Stress Conditions
2-80C
370C
300C agitation (220 RPM)
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.

Formulation Tm1 (0C) Tm2 (0C) Tm3 (0C)
6 70.1 76.1 83.7
7 59.9 70.1 81.1
8 60.2 70.8 78.6
9 70.7 75.4 83.4
10 69.9 75.2 83.9
Table 11: Evaluation of melting Temperatures of the formulation 6-10 by DSC

Formulation % Degradant (%HMWP+%LMWP)
T0 T-4W (2-8°C) T-2W (30°C, Agitation) T-2W 37°C
6 2.54 2.44 11.16 8
7 2.63 2.52 13.95 29.63
8 2.25 1.97 39.95 5.49
9 2.12 1.87 3.07 5.37
10 2.47 2.4 33.22 11.68
Table 12: SEC results of formulations subjected 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. SEC, SDS-PAGE and biological activity analysis indicated that formulation 9 is consistently marginally better than the other formulations.
Storage Condition A320 SEC Reducing SDS Potency (done at high conc.) Potency (done at low conc.)
2-8°C 9, 8 >6 >>10 > 7 9 > 8 >10 > 6 > 7 6=7=8=9=10 6=7=8=9=10 6=7=8=9=10
37°C 7, 6 > 8, 10 > 9 9 > 8 >6 >> 10 >>> 7 9 >6 >8 >10 > 7 6=7=9>8=10 8=9>6>10>7
30°C Agitation 7, 6 > 8 > 10 > 9 9 >> 6, 7 > >10 > 8 8, 9# > 6, 7, 10 6=7=9>8=10 9>6>7=8=10
Table 13: Summarized result of the formulations 6-10 based on analysis of samples subjected to different stress conditions
Based on the results of pre-formulation studies, the formulation 9 was chosen for further studies.

Example 4: Long term stability study
The formulation number 9, (0.05% (w/v) Polysorbate 80, 15 mM Histidine, pH 6.5) was further studied for long 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 fill volume of 10 ml. The vials were stoppered with cream coloured bromo butyl closures from WEST pharma and crimped with 13 mm aluminium seals.
The stress conditions included various storage conditions viz. 2-80C (example 4.1), 250C (example 4.2), 370C (example 4.3) and Freeze-Thaw up to 4 thaw cycles (example 4.4) at various time-points 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.

Example 4.1 Stability at 2-8ºC (Tables 14-18)
An increase in fragmentation was observed after 9 months and fragmentation continued to increase till 24 months. Acidic variants were found to gradually increase over a period 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.
Time point
(months) Protein Concentration (mg/ml)
(Limits: 9-11 mg/ml) pH
(Limits: 6.5 ± 0.3) Appearance
1 M 9.7 6.70 Clear/Colorless
2 M 9.7 6.78 Clear/Colorless
6 M 9.6 6.75 Clear/Colorless
12 M 9.7 6.76 Clear/Colorless
16 M 9.5 6.78 Clear/Colorless
22 M 9.6 6.65 Clear/Colorless
24M 9.7 6.67 Clear/Colorless
Table 14: A280, pH and visual appearance of samples stored at 2-8ºC

Condition 2-8ºC
Time Point (Months) % Aggregate %Monomer %Fragments
0 1.89 98.11 0.00
1 M 1.70 98.27 0.03
2 M 1.80 98.19 0.02
3 M 1.60 98.30 0.10
4 M 1.55 98.35 0.10
5 M 1.58 98.31 0.11
6 M 1.46 98.43 0.11
7 M 1.53 98.33 0.11
8 M 1.44 98.43 0.12
9 M 1.58 98.28 0.12
11 M 1.57 96.46 1.84
12 M 1.50 95.99 2.11
16 M 1.71 96.92 1.36
22 M 1.81 96.50 1.69
24 M 1.55 95.39 3.05
Table 15: Physical stability by SEC for samples stored at 2-8ºC

Condition 2-8ºC
Time point (months) Acidic % Main Peak % Basic %
0 (initial) 22.1 64.8 13.1
1 M 19.7 58.2 22.1
2 M 22.1 60.4 17.4
3 M 19.3 64.0 16.3
4 M 22.3 61.3 16.4
5 M 26.2 61.0 12.8
6 M 22.1 59.5 18.5
7 M 30.3 54.9 14.7
8 M 31.0 54.1 14.8
9 M 31.1 54.5 14.4
12 M 33.9 52.2 13.9
16 M 51.0 37.0 11.0
22 M 52.0 37.0 9.7
24 M 53.2 36.9 9.9
Table 16: Chemical stability by IEX for samples stored at 2-8ºC

The % cytotoxicity over control is calculated by dividing the cytotoxicity of the sample by that of the standard, and multiplying by 100 to obtain the result as a percentage.
Time (months) % Cytotoxicity over Control
0 (initial) 100
0.5 M 117
1 M 79
2 M 94
3 M 98
4 M 128
5 M 119
6M 80
Table 17: Biological activity (cytotoxicity) by CDC assay of samples stored at 2-8ºC (carried out at 0.015µg/ml)

The CDC assay was carried out at 0.015 µg/ml till the 6th month. The samples after 9th (Figure 6), 12th month, 16th 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.

Sample name EC50 (nM) 95% confidence interval (CI)
Reference standard 0.071 6.86 to 7.33 x 10-11
Time-12M 0.103 9.95 x 10-11 to 1.08 x 10-10

Sample name EC50 (nM) 95% CI
Reference standard 0.156 0.147 - 0.166 x10 -9
Time-16M 0.132 0.122 - 0.143 x10 -9

Sample name EC50 (nM) 95% CI
Reference standard 0.098 0.092 – 0.104 x10 -9
Time-22M 0.101 0.095 – 0.109 x10 -9

Sample name EC50 (nM) 95% CI 70 – 130% EC50 range
Reference standard 0.128 0.12 – 0.138 0.089 – 0.166
Time-24M 0.137 0.126 – 0.148 Passes
Table 18: Cytotoxicity of sample stored at 2-8ºC at end of 12, 16, 22 and 24 months by parallel line assay

Example 4.2 Stability at 25ºC (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 yellow at end of 5 months at 250C.

Time point Protein concentration (mg/ml) pH Appearance
1 M 9.7 6.7 Clear/Colorless
2 M 9.74 6.71 Clear/Colorless
6 M 9.87 6.75 Clear/Yellow discoloration
Table 19: pH, visual appearance and protein concentration of samples stored at 25ºC

Condition 25°C
Time (months) % Aggregate %Monomer %Fragments
0 1.89 98.11 0.00
0. 25 M 1.70 98.24 0.06
0.5 M 1.73 98.17 0.10
1 M 1.57 98.26 0.16
2 M 1.59 98.09 0.32
3 M 1.91 95.11 2.92
4 M 2.46 93.68 3.87
5 M 2.55 93.22 4.23
6M 1.93 90.90 7.16
Table 20: Physical Stability by SEC for samples stored at 25ºC

Condition 25°C
Time Points Acidic Variant Zero Variant Basic Variant
0 22.13 64.80 13.07
0.5 M 23.61 59.28 17.12
1 M 27.74 51.05 21.22
2 M 48.74 37.90 13.35
3 M 61.96 27.75 10.29
4 M 67.31 23.27 9.43
5 M 74.23 18.21 7.56
6M 73.94 15.49 10.58
Table 21: Chemical stability by IEX for samples stored at 25ºC

Time (weeks) % Cytotoxicity over Control
0 100
0.25 M 97
0.5 M 107
1 M 80
2 M 53
3 M 61
4 M 30
6 M 10
Table 22: Biological activity (cytotoxicity) by CDC assay of samples stored at 25ºC (carried out at 0.015µg/ml)

Example 4.3 Stability at 37ºC (Table 23-26)
Upon physical, chemical stability and biological activity check at various time-points as per tables 23-26, antibody was found to increase in aggregation 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 followed by a complete loss of activity at the end of 2 months. The formulation exhibited yellow discolouration at the end of 2 months.

Time point
(months) Protein concentration (mg/ml)
(Limits: 9-11 mg/ml) pH
(Limits: 6.5±0.3) Appearance
T 1M 9.74 6.7 Clear/Colorless
T 2M 9.63 6.62 Clear/Yellow discoloration
Table 23: pH, visual appearance and protein concentration of samples stored at 37ºC

Condition 370C
Time (months) % Aggregate %Monomer %Fragments
0 1.89 98.11 0.00
0.25 M 1.53 97.37 1.10
0.5 M 1.59 96.62 1.79
0.75 M 1.56 96.16 2.28
1 M 1.67 95.69 2.63
2 M 2.34 92.77 4.89
3 M 2.30 91.24 6.47
Table 24: Physical Stability by SEC for samples stored at 37ºC

Condition 370C
Time Points
(months) Acidic Variant Zero Variant Basic Variant
0 (initial) 22.13 64.80 13.07
0.25 M 27.38 57.08 15.54
0.5 M 35.43 48.19 16.39
0.75 M 48.28 36.80 14.92
1 M 55.72 27.97 16.31
2 M 75.64 14.62 9.75
Table 25: Chemical stability by IEX for samples stored at 37ºC

Time (months) % Cytotoxicity over Control
0 100.48
0.25 M 101.47
0.5 M 80.24
0.75 M 32.95
1 M 6.97
2 M -22.33
3 M -11.06
Table 26: Biological activity (cytotoxicity) by CDC assay of samples stored at 370C (carried out at 0.015µg/ml)

Example 4.4 Stability upon repeated freezing and thawing (Table 27-29)
Freeze thaw stability of the samples was evaluated by freezing the samples at –20ºC for 1 week followed by thawing at room temperature. Four such cycles were performed.

Condition Freeze Thaw
Freeze Thaw Cycle % Aggregate %Monomer %Fragments
0 1.89 98.11 0
Cycle 1 1.94 98.06 0
Cycle 2 1.90 98.10 0
Cycle 3 1.78 98.22 0
Cycle 4 1.83 98.17 0
Table 27: Physical stability by SEC for samples subjected to freezing and thawing

Condition Freezing and Thawing
Time Points Acidic Variant Zero Variant Basic Variant
0 22.13 64.80 13.07
Cycle 1 17.07 59.02 23.91
Cycle 2 19.70 63.27 17.03
Cycle 3 20.16 59.89 19.95
Cycle 4 19.00 59.02 21.98

Table 28: Chemical stability by IEX for samples subjected to repeated freezing and thawing
Freeze thaw cycle % Cytotoxicity over control
0 100
Cycle 1 105
Cycle 2 112
Cycle 3 125
Cycle 4 106
Table 291: Biological activity (cytotoxicity) by CDC assay of samples subjected to repeated freezing and thawing (carried out at 0.015µg/ml)

The formulation was found to be physically stable over a period of 9 months at 2-80C. A significant increase in fragmentation observed at the end of 12 months at 2-80C. 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 250C, 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 370C, 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 370C. (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. Variations observed in the % basic variants are due to differences in resolution of the zero and basic variants.
Example 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 fill 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 WEST pharma and crimped with 13 mm aluminium seals.
The stress conditions mainly included various storage conditions viz. 2-80C, 260C with agitation and 370C using different time-points till 12 weeks.

Samples Composition Conditions
1 10 mg/ml BVX-20, 15 mM Histidine 0 % (w/v) Polysorbate 80 2-80C,
260C Agitation,
370C
2 10 mg/ml BVX-20, 15 mM Histidine 0.005 % (w/v) Polysorbate 80
3 10 mg/ml BVX-20, 15 mM Histidine 0.02 % (w/v) Polysorbate 80
4 10 mg/ml BVX-20, 15 mM Histidine 0.05 % (w/v) Polysorbate 80
5 10 mg/ml BVX-20, 15 mM Histidine 0.08 % (w/v) Polysorbate 80
Table 30: Details of the formulations evaluated

No significant difference was observed for formulations containing different quantities of polysorbate 80 upon storage at 2-8°C. 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).

Storage Condition Time Point (Weeks) 0% 0.005% 0.02% 0.05% 0.08%
Emission maxima (nm)
2-8 ºC 0 347 347 347 347 347
1 347 347 347 348 347
4 347 348 347 347 347
7 347 347 348 348 348
10 347 347 347 347 347
Table 31: Intrinsic fluorescence data of samples stored at 2-8ºC

Antibody was found to degrade upon agitation at 260C. 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 RPM) at 260C. No difference was observed in the CDC assay (figure 10) and in intrinsic fluorescence data (table 32) for all samples.

Condition Time Point
(Weeks) 0.005% 0.02% 0.05% 0.08%
Emission maxima (nm)
260C Agitation 0 347 347 347 347
1 347 348 347 347
4 348 348 347 346
7 348 348 348 347
10 348 348 346 347
Table 32: Intrinsic Fluorescence data of samples subjected to agitation at 26ºC
Antibody was found to degrade upon storage at 370C. All formulations showed an increase in fragmentation, absorbance at 320nm (figure 7) and acidic variants over a period of 10 weeks when stored at 370C. 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.
Storage Condition Time Point (Weeks) 0% 0.005% 0.02% 0.05% 0.08%
Emission Maxima (nm)
37 ºC

0 347 347 347 347 347
1 347 347 347 348 348
4 346 347 347 347 347
7 346 347 347 347 347
10 346 347 346 346 346
Table 33: Intrinsic fluorescence data of samples stored at 37ºC
Hence, formulation containing 0.02% polysorbate 80 showed a marginally 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 Histidine 0.02% (w/v) Polysorbate 80 pH 6.5.

Example 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 2-80C, 250C and 370C 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. The antibody 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 aluminium flip-off seals. The vials were stored at various conditions as shown in table 34 and analysed by methodologies and parameters outlined in Table 1 and table 2.

Stress Condition
2-80C
250C
370C
Table 34: stress conditions for long-term stability check

Example 6.1 stability check at 2-80C
Time (months) Protein Concentration (mg/ml) pH Description
Initial 10.65 6.41 Complies
3 10.65 6.43 Complies
4 10.71 6.44 Complies
5 10.56 6.42 Complies
6 10.54 6.38 Complies
7 10.71 6.35 Complies
9 10.67 6.43 Complies
12 10.61 6.41 Complies
18 10.71 6.42 Complies
24 10.75 6.41 Complies
Table 35: pH, appearance and protein concentration of samples stored at 2-8ºC
Time (months) Particulate matter Sterility Endotoxin
= 10 µm = 25 µm
Initial 785 29 Complies Complies
3 151 4 -- --
4 438 12 -- --
5 229 12 -- --
6 239 9 -- --
7 71 8 -- --
9 814 61 -- --
12 872 61 -- --
18 225 86 -- --
24 329 23 Complies Complies
Limits NMT 6000 particles per container NMT 600 particles per container Preparation for injections should meet the requirements of sterility Less than
5 EU/mg
Table 36: Particulate matter, sterility and endotoxin limits of samples stored at 2-8ºC
Time Point (Months) % Aggregate %Monomer %Fragments
Initial 1.98 97.97 0.05
3 2.34 97.58 0.09
4 1.85 97.33 0.82
5 2.28 97.03 0.69
6 2.26 96.60 1.13
7 2.00 97.15* 0.85
9 2.02 96.95 1.03
12 1.92 97.06 1.02
18 1.91 96.47 1.61
24 1.97 96.27 1.76
Table 37: Distribution of High molecular Weight protein (HMWP), Monomer and Low Molecular Weight Protein (LMWP) in BVX20 samples stored at 2-8ºC (SEC)
Time point (months) Acidic % Zero % Basic %
Initial 29.9 39.7 30.4
3 34.9 37.4 27.7
4 33.9 40.0 26.1
5 33.5 40.8 25.8
6 32.4 41.3 26.3
7 32.4 39.1 28.5
9 34.4 38.2 27.5
12 34.5 38.5 27.0
18 35.7 35.6 28.7
24 40.0 34.6 25.5
Table 38: Distribution of charge variants in BVX-20 samples stored at 2-8ºC (IEX)

Testing Interval (in months) EC50 of reference Standard (nM) 95% CI (nM) of reference standard EC50 of the Test sample 95% CI (nM) of Test sample 70% - 130% Range (Standard, nM) Result
3 0.075 0.071-0.078 0.076 0.072-0.081 0.052 - 0.097 Complies
4 0.082 0.073-0.093 0.085 0.078-0.093 0.057 - 0.107 Complies
5 0.075 0.071-0.078 0.087 0.078-0.096 0.052 - 0.097 Complies
6 0.080 0.068-0.094 0.092 0.086-0.097 0.056 - 0.104 Complies
7 0.118 0.112-0.125 0.141 0.134-0.149 0.082 - 0.153 Complies
9 0.164 0.154-0.175 0.171 0.161-0.181 0.115 - 0.213 Complies
12 0.22 0.217-0.232 0.194 0.171-0.219 0.154 - 0.286 Complies
18 0.156 0.123-0.143 0.133 0.147-0.166 0.10 - 0.20 Complies
24 0.146 0.135-0.157 0.149 0.141-0.157 0.102-0.189 Complies
Limits 70 -130% of the EC50 of the reference standard which should lie within 0.05 – 0.5 nM
Table 39: Potency of samples stored at 2-8°C, at various time points by CDC 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 2-80C 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 2-80C (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 2-80C. The overall purity was greater than 95% and within the specified limits at the end of 24 months at 2-80C (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 within specifications at the end of 24 months storage at 2-80C (table 39).

Example 6.2 stability check at 250C
Sample Protein Concentration (mg/ml) pH Description
Initial 10.65 6.41 Complies
1 10.65 6.46 Complies
2 10.69 6.46 Complies
3 10.58 6.42 Complies
6 10.63 6.39 Complies
Table 40: pH, appearance and concentration of samples stored at 25ºC

Time (months) Particulate matter Sterility Endotoxin
= 10 µm = 25 µm
Initial 785 29 Complies Complies
1 280 13 -- --
2 302 6 -- --
3 107 3 -- --
6 113 1 -- --
Table 41: Particulate matter, sterility and endotoxin limits of samples stored at 25ºC
Time Points (Months) % HMWP % Monomer % LMWP
Initial 1.98 97.97 0.05
1 1.98 96.50 1.52
2 1.83 96.63 1.55
3 2.13 95.07 2.80
6 2.21 94.68 3.11
Table 42: Distribution of High molecular Weight protein (HMWP), Monomer and Low Molecular Weight Protein (LMWP) in BVX-20 samples stored at 25ºC (SEC)

Time Point (Months) % Acidic % Main Peak % Basic
Initial 29.9 39.7 30.4
1 37.3 36.2 26.5
2 41.0 33.1 25.9
3 46.9 29.4 23.7
6 54.3 25.4 20.3

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

The pH, appearance and protein concentration of samples stored at 250C 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 250C 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 (250C), 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. Although the monomer content was found to be within the specification (> 95%) at the end of 3 months, the biological activity decreased on storage at 250C and was outside the specification limits.

Example 6.3 stability check at 370C

Time
(Weeks) Protein Concentration (mg/ml) pH Description
Initial 10.65 6.41 Complies
1 10.56 6.42 Complies
3 10.56 6.43 Complies
5 10.75 6.41 Complies
7 10.58 6.40 Complies
Table 44: pH, appearance and concentration of samples stored at 37ºC

Time (Weeks) Particulate matter Sterility Endotoxin
= 10 µm = 25 µm
Initial 785 29 Complies Complies
1 150 5 -- --
3 173 14 -- --
5 87 5 -- --
7 552 14 -- --
Table 452: Particulate matter, sterility and endotoxin limits of samples stored at 37ºC
Time
(Weeks) % HMWP % Monomer % LMWP
Initial 1.98 97.97 0.05
1 1.87 96.27 1.87
3 2.12 95.18 2.70
5 2.26 93.87 3.86
7 2.26 93.44 4.32
Table 46: Distribution of High molecular Weight protein (HMWP), Monomer and Low Molecular Weight Protein (LMWP) in BVX-20 samples stored at 37ºC (SEC)
Time
(Weeks) % Acidic % Zero % Basic
Initial 29.9 39.7 30.4
1 41.6 32.3 26.1
3 52.3 25.2 22.6
5 58.5 23.0 18.6
7 60.6 21.7 17.8
Table 47: Distribution of charge variants in BVX-20 samples stored at 37ºC (IEX)

Concentration (ng/ml) 62.5 31.25 15.625
Sample % Cytotoxicity
Reference Standard 70.65 61.65 35.00
Week 1 67.69 49.13 24.35
Week 3 63.95 38.62 14.72
Table 48: Percentage cytotoxicity of samples stored at 37ºC by CDC assay
Following storage of the product at 370C, 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 370C 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 (table 48), although the monomer content was >95% (98.18).

Example 7: Hold time study
Earlier, freezing 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 against the limits set for the assay which should be within 70-130% of the standard. Low molecular weight proteins (LMWP) and high molecular weight proteins (HMWP) were calculated.
The study conducted at 2 different freezing temperatures: -200C and -800C. Thawing temperature in both cases was room temperature (RT) 22±20C.

Example 7.1 Stability following storage at -200C and -800C
Frozen state stability involved testing the ability of the monoclonal antibody to remain frozen for long periods of time. In this study, the monoclonal antibody in bags was left frozen at -200C and -800C for a total period of 12 months, and stability 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 refrozen.

Time point pH Description
T0 6.60 Clear, no particulate matter
-20°C
2 weeks 6.65 Clear, no particulate matter
3 weeks 6.60 Clear, no particulate matter
1 Month 6.58 Clear, no particulate matter
4 Months 6.55 Clear, no particulate matter
6.5 Months 6.58 Clear, no particulate matter
9 Months 6.62 Clear, no particulate matter
-80°C
1 week 6.79 Clear, no particulate matter
2 weeks 6.55 Clear, no particulate matter
3 weeks 6.62 Clear, no particulate matter
1 Month 6.47 Clear, no particulate matter
4 Months 6.55 Clear, no particulate matter
6.5 Months 6.57 Clear, no particulate matter
9 Months 6.66 Clear, no particulate matter
Table 49: pH, UV 280 data of samples stored at -20ºC and -80°C

BVX20 Bulk Stability Study Frozen State T0 0.870 0.7-1.3 of standard Potency Ratio
-20°C T 1 month 0.996
-80°C T 1 month 0.831
-20°C T 4 months 0.864
-80°C T 4 months 0.842
-20°C T 6.5 months 0.962
-80°C T 6.5 months 1.038
-20°C T 9 months 0.957
-80°C T 9 months 1.024
Table 50: CDC Assay results of formulated bulk stored at -20ºC and -80°C
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 T0.
There was a marginal increase in HMWP% and LMWP% from T0 at 4 Months at -20°C. After 4 months, the HMWP and LMWP % remains relatively stable. The distribution of HMWP, Monomer and LMWP in the frozen state stability -80°C samples is comparable to the T0 values (figure 15).
The distribution of acidic variant, main peak and basic variant in the frozen state stability (9 months) samples is comparable to the T0 values (figure 16)
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 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 -20°C in the production facility, 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.

Example 7.2 Stability following repeated freezing and thawing
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 -200C or -800C, 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 (T0) 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.

Time point pH Description
T0 6.6 Clear, no particulate matter
-20°C
1Week: FT cycle 1 6.67 Clear, no particulate matter
2Weeks: FT cycle 2 6.66 Clear, no particulate matter
3Weeks: FT cycle 3 6.55 Clear, no particulate matter
RT for 24 Hrs after FT3 6.57 Clear, no particulate matter
RT for 48 Hrs after FT3 6.51 Clear, no particulate matter
RT for 72 Hrs after FT3, 6.53 Clear, no particulate matter
-80°C
1Week: FT cycle 1 6.61 Clear, no particulate matter
2Weeks: FT cycle 2 6.61 Clear, no particulate matter
3Weeks: FT cycle 3 6.55 Clear, no particulate matter
RT for 24 Hrs after FT3 6.58 Clear, no particulate matter
RT for 48 Hrs after FT3 6.55 Clear, no particulate matter
RT for 72 Hrs after FT3, 6.49 Clear, no particulate matter
Table 51: pH data of formulated bulk upon FT (-20ºC/ RT)

Test Description Sample Potency Ratio Limits
BVX20 Bulk Stability Study T0 0.870 0.7-1.3 of standard Potency Ratio
FT3 -80°C 0.853
FT3 -20°C 0.873
72 hrs at RT after FT3 (-80°C) 0.822
72 hrs at RT after FT3 (-20°C) 0.814
Table 52: CDC Assay results of formulated bulk upon FT (-80ºC/ RT)
There was no significant difference in the distribution of monomer, HMWP and LMWP in the repeated freeze thaw samples (3 freeze thaw cycles at -20°C and -80°C) compared to the T0 samples (figure 17). The samples incubated at room temperature for 72 hours after undergoing 3 cycles of freeze-thaw at -20°C and -80°C showed comparable distribution of HMWP, monomer and LMWP 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 -20°C and -80°C) compared to the T0 samples (figure 18). The samples incubated at RT for 72 hours after undergoing 3 cycles of freeze-thaw at -20°C and -80°C 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).

Example 9: Final Formulation
The formulation was manufactured of BVX20 mAb finished product involves the buffer exchange of the antibody into buffer containing15 mM histidine at pH 6.5. The solution 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 -20ºC/-80ºC. In this case, the bulk is thawed at RT (22 ± 2ºC) prior to filling. The formulated bulk is then sterile filtered through 0.22µ 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. The vials are labeled and stored at 2-8ºC.
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 -20°C in the production facility, 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. Therefore the bulk of BVX-20 continues to be stored at 2-8°C.