DESC:FIELD OF INVENTION
The present invention relates to therapeutic proteins, particularly fusion protein formulations, and process of preparing the therapeutic protein formulations. In particular, the invention relates to process of making lyophilized fusion protein formulations.
BACKGROUND OF THE INVENTION
Over the past two decades, recombinant DNA technology has led to the commercialization of many proteins, particularly antibody therapeutics and fusion protein molecules.
Fusion proteins, in particular, Fc fusion protein molecules (in which Fc portion of human immunoglobulin (Ig) is conjugated to a particular portion of a receptor) are gaining significance, since their wide usage
in treatment of various oncological and immunological disorders. Etanercept (TNFR-IgFc), Aflibercept (VEGFR-IgFc) and Abatacept (CTLA4-IgFc) are among those Fc fusion proteins approved by Food and Drug Administration (FDA) to treat various disorders. The effectiveness of fusion protein molecule is majorly dependent on the stability, route of administration and their dosage forms and concentrations. This in turn, necessitates these protein molecules to be formulated appropriately to retain stability and activity.
Fc fusion proteins are known for their complexity and typically unstable in solution and sensitive to pH, temperature and oxidation and hence can undergo a variety of covalent and non-covalent reactions, modifications or degradations in solution. Further the fusion protein solutions, intended for long term storage or robust handling conditions may need to be lyophilized. “Lyophilization” or “freeze-drying” is a process whereby the substance to be lyophilized, is first frozen and then the solvent removed by primary and secondary drying, this technique is frequently employed as a formulation technique. Similar to selecting protein solvents and their components, excipients for lyophilization need to be carefully chosen so that the protein of interest is unaffected by the freezing process, and its stability not compromised during storage and subsequent use. Pikal, M., Biopharm. 3(9)26-30(1990) and Arkawa et, al., Pharm. Res. 8(3):285-291 (1991).
Thus, an optimal choice of buffer, excipients and technique of lyophilization can significantly enhance the stability, shelf life and cut storage and shipping costs, especially for therapeutic proteins, which otherwise demand cumbersome and expensive handling conditions.
CTLA4-Ig fusion protein molecules include Abatacept and Belatacept which are approved in liquid as well as lyophilized form. Lyophilized formulations of the fusion protein molecules are preferable as compared to liquid formulations due to less chances of degradation when the protein is stored in powder (lyo) form as compared to liquid formulation.
US8476239 patent discloses lyophilization process of CTLA4-Ig fusion protein formulations (column 28-30 of example 3 and 4 the specification. However, the lyophilization process disclosed in the Table 12 of US’ 239 takes at least 121 hours which is almost equivalent to five days. Hence, there is a need for shorter lyophilization cycle which saves time and it is important during manufacturing when the product needs to be produced at commercial scale continuously.
Hence, the objective of the present invention to develop an optimized shorter lyophilization cycle for CTLA4-Ig fusion protein which does not impact the quality attributes of lyophilized cake obtained from the said process.
SUMMARY OF THE INVENTION
The present invention discloses a method for obtaining a stable, freeze dried CTLA4-Ig fusion protein formulation comprising addition of at least 50 mg/ml of maltose during diafiltration and subjecting the said formulation to lyophilization process to obtain a dry, amorphous cake/powder. The lyophilized fusion protein formulation (or cake or powder) obtained by the inventive method comprises less than 1% of aggregate content and less than 1 % moisture content.
The invention additionally discloses a lyophilization process comprising the steps of freezing, annealing, refreezing, primary drying in stepwise approach, secondary drying and stoppering and wherein, the entire lyophilization process is accomplished in less than 80 hours, specifically in less than 72 hours.
The invention discloses shorter lyophilization cycle for CTLA4-Ig fusion protein molecules which does not impact the quality attributes of the product and the shorter lyophilization cycle obtained by introduction of annealing step and by optimizing primary drying step.
The disclosed freeze dried CTLA4-Ig fusion protein formulations of the invention are stable and contains less than 1 % of the fusion protein in aggregate form when stored at 30 ? for one month and at 25 ? for 6 months and less than 0.5 % when stored at 2-8 ?. The aggregate content of less than 0.5 % is maintained at least for 12 months when stored at 2-8 ?.
Further, the reconstitution time of the formulations obtained from the disclosed lyophilization process is less than 10 minutes and specifically, less than 5 minutes.
The method of obtaining the lyophilized protein formulation disclosed in the present invention is suitable/compatible for obtaining freeze-dried therapeutic and placebo compositions.
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses a method for obtaining a stable, freeze-dried formulation of CTLA4-Ig fusion protein formulation.
In one embodiment, the invention discloses a method of obtaining a stable, freeze-dried CTLA4-Ig fusion protein formulation comprising;
expressing and purifying CTLA4-Ig fusion protein,
subjecting the purified protein to one or more ultrafiltration and/or diafiltration steps with a buffer comprising at least 50 mg/ml of maltose in the filtration steps,
followed by freeze-drying the protein by a lyophilization process.
In another embodiment, the invention discloses a method of obtaining a stable, freeze-dried CTLA4-Ig fusion protein formulation comprising;
expressing and purifying CTLA4-Ig fusion protein,
subjecting the purified protein to one or more ultrafiltration and/or diafiltration steps with a buffer comprising at least 50 mg/ml of maltose in the filtration steps,
followed by freeze-drying the protein by a lyophilization process,
wherein the freeze-dried protein composition obtained by the method, contains less than 1% of aggregate content (pre and post the lyophilization process), and the freeze-dried powder/cake contains less than 1 % moisture content.
In the above mentioned embodiment, the lyophilization process comprises steps of;
(i) freezing the fusion protein formulation at a temperature, ranging fromabout-45° C to about -50° C, to transform the liquid formulation into a solid state,
(ii) annealing the frozen formulation obtained from step i) at a temperature, ranging from about -22° C to about -25° C,
(iii) refreezing the formulation obtained from step ii) at a temperature, ranging from about -45° C to about -50° C,
(iv) primary drying of the formulation obtained from step iii), at at least two different temperature, ranging from about -25° C to about 0° C and,
(v) secondary drying of the formulation obtained from step iv), at a temperature ranging from about 0° C to about 25° C.
In the above mentioned embodiment, the freezing rate maintained during step i), ii) and iii) is about 0.2-0.4 ?/minute, preferably 0.3 ?/minute.
In the above mentioned embodiments, the primary drying step is performed at a pressure range from 50-100 µ bar .and the secondary drying step is performed at a pressure range from 700-800 µ bar.
In any of the above mentioned embodiments, the CTLA4-Ig fusion protein in formulation is stable and contains less than 1 % of the aggregate content when stored at 30 ? for one month and at 25 ? for six months and less than 0.5 % when stored at 2-8 ?. The aggregate content of less than 0.5 % is maintained at least for 8 months when stored at 2-8 ?.
In another embodiment, the invention discloses a method of obtaining a stable, freeze-dried CTLA4-Ig fusion protein formulation comprising;
expressing and purifying CTLA4-Ig fusion protein,
subjecting the purified protein to one or more ultrafiltration and/or diafiltration steps with a buffer comprising at least 50 mg/ml of maltose in the filtration steps,
followed by freeze-drying the protein by a lyophilization process,
wherein the freeze-dried protein composition obtained by the method, is stable and contains less than 1% of the aggregate content when stored at 30? for one month and at 25 ? for six months and aggregate content of the fusion protein is less than 0.5% when stored at 2-8 ? for 8 months.
In the above mentioned embodiments, the CTLA4-Ig fusion protein formulation comprising maltose in one or more of the Ultrafiltration (UF) and/or diafiltration (DF) steps, comprises less than 1% of aggregate content, when analysed before and after the lyophilisation process. In other words, the aggregate content of the CTLA4-Ig fusion protein is less than 1%, pre and post lyophilization rocess, when maltose is included in-process filtration steps.
In the above mentioned embodiments, the CTLA4-Ig fusion protein formulation comprising maltose, comprises less than 1% of moisture content after lyophilisation.
In any of the above mentioned embodiment, the CTLA4-Ig fusion protein formulation comprising maltose, which is added during one or more ultrafiltration and/or diafiltration steps withstands freeze-thaw induced stress better, with less than 50 % of aggregate and moisture content, when compared to the CTLA4-Ig fusion protein formulation with maltose added at the time of lyophilisation.
In any of the above mentioned embodiments, the stable freeze-dried CTLA4-Ig fusion protein is reconstituted in less than 10 minutes, preferably in less than 5 minutes, more preferably in less than 3 minutes.
In any of the above mentioned embodiments, the freeze-dried CTLA4-Ig fusion protein molecule is biologically active.
In any of the above mentioned embodiments, the CTLA4-Ig fusion protein formulation comprises pharmaceutically acceptable excipients such as buffers and salt.
In any of the above embodiments, the formulation subjected for lyophilization comprises, CTLA4-Ig fusion protein, maltose, phosphate buffer, sodium chloride and polysorbate and pH of the formulation is 7.1-7.5.
In the above mentioned embodiment, the buffers include organic buffer and inorganic buffer.
In any of the above mentioned embodiments, the pH of the CTLA4-Ig fusion protein formulation is 6-8.
In an embodiment, the invention disclosed a method for lyophilization of a mixture containing 20 mg/ml CTLA4-Ig fusion protein, 40 mg/ml maltose, sodium chloride, phosphate buffer comprising
(i) freezing the fusion protein formulation at a temperature, ranging fromabout-45° C to about -50° C, to transform the liquid formulation into a solid state,
(ii) annealing the frozen formulation obtained from step i) at a temperature, ranging from about -22° C to about -25° C,
(iii) refreezing the formulation obtained from step ii) at a temperature, ranging from about -45° C to about -50° C,
(iv) primary drying of the formulation obtained from step iii), at at least two different temperature, ranging from about -25° C to about 0° C at a pressures range from 50-100 µbar and,
(v) secondary drying of the formulation obtained from step iv), at a temperature ranging from about 0° C to about 25° C at a pressure range from 500-700 µ bar.
wherein, the freezing rate maintained during step i), ii) and iii) is about 0.2-0.4 ?/minute, preferably 0.3 ?/minute.
In the above mentioned embodiment, the lyophilized CTLA4-Ig fusion protein obtained from the process is stable at 25 ? for six months and at 30 ? for one month and contain less than 1% aggregate content and less than 1% moisture content.
In the above mentioned embodiment, the lyophilization method disclosed in the invention does not impact oxidation and deamidation of the product.
The present invention is specifically advantageous in saving time, resource as lyophilisation cycle to obtain freeze-dried CTLA4-Ig fusion protein formulation accomplished in = 80 hours and preferably in 72 hours, more preferably in 70 hours. The lyophilization process as disclosed in the current invention does not impact oxidation and deamidation of the fusion protein on formulation.
DEFINITIONS
The term "fusion protein" means a protein formed by fusing (i.e., joining) all or part of two polypeptides, which are not the same. Typically, fusion proteins are made using recombinant DNA techniques, by end to end joining of polynucleotides encoding the two polypeptides.
The terms “CTLA4-Ig” or “CTLA4-Ig molecule” or “CTLA4Ig molecule” are used interchangeably, and refer to a protein molecule that comprises a polypeptide having a CTLA4 extracellular domain or a portion thereof, and an immunoglobulin constant region or a portion thereof. The extracellular domain and the immunoglobulin constant region can be wild-type, or mutant or modified, and mammalian, including human or mouse. The polypeptide can further comprise additional protein domains. A CTLA4-Ig molecule can also refer to multimer forms of the polypeptide, such as dimers, tetramers, and hexamers. A CTLA4-Ig molecule also is capable of binding to CD80 and/or CD86.
The term “cake” or “powder” are used synonymously herein, and refers to a dry pellet or powder that results when a liquid formulation has been lyophilized or freeze-dried. As used herein, “dry cake or powder” refers to a cake or powder that comprises about 1% or less residual moisture content. In some embodiments of the invention, the moisture content of the dry cake is about 0.1% to about 1%.
The term "stable" formulation refers to the formulation, wherein the fusion protein molecule therein retains its physical stability and/or chemical stability and/or biological activity, upon storage.
Ultrafiltration (UF) and diafiltration (DF) are commonly used steps in downstream processing for product concentration and buffer exchange. Ultrafiltration may be used to increase the concentration of macromolecules in a solution and diafiltration is generally used for buffer exchange. UF and DF steps mentioned herein can be either sequential or simultaneous. These filtration steps may be performed or operated in tangential flow filtration or cross flow filtration mode or normal (direct) flow filtration mode. Addition of maltose ‘at the time of lyophilization’ means addition performed after the UF / DF or Tangential flow filtration steps, but while subjecting to lyophilization process.
Stability studies provide evidence of the quality fusion protein under the influence of various environmental factors during the course of time. ICH’s “Q1A: Stability Testing of New Drug Substances and Products,” states that data from accelerated stability studies can be used to evaluate the effect of short-term excursions higher or lower than label storage conditions that may occur during the shipping of the antibodies.
Various analytical methods are available for measuring the physical and chemical degradation of the fusion protein in the pharmaceutical formulations. A fusion protein "retains its physical stability" in a pharmaceutical formulation if it shows substantially no signs of aggregation, precipitation and/or denaturation upon visual examination of color and/or clarity, or as measured by UV light scattering or by size exclusion chromatography. A fusion protein is said to “retain its chemical stability” in a pharmaceutical formulation when its shows no or minimal formation of product variants which may include variants as a result of chemical modification of fusion protein such as deamination, oxidation etc. Analytical methods such as ion exchange chromatography and hydrophobic ion chromatography may be used to investigate the chemical product variants.
The monomer, dimer and high molecular weight (HMW) species of CTLA4Ig molecule may be separated by size exclusion chromatography (SEC). SEC separates molecules based on the molecular size. Separation is achieved by the differential molecular exclusion or inclusion as the molecules migrate along the length of the column. Thus, resolution increases as a function of column length. In order to maintain the appropriate activity of a fusion protein, it is desirable to reduce the formation of aggregate or fragmentation (monomer/low molecular weight species) of products and hence control the dimer content to a target value. Dimer is major form present in fusion proteins and elutes as main peak in size exclusion chromatography. CTLA4Ig molecule samples may be separated using a 2695 Alliance HPLC (Waters, Milford, Mass.) equipped with TSK Gel® G3000SWXL (300 mm×7.8 mm) and TSK Gel® G3000SWXL (40 mm×6.0 mm) columns (Tosoh Bioscience, Montgomery, Pa.).
Pharmaceutically acceptable excipients refer to the additives or carriers, which may contribute to stability of the fusion protein in formulation. The excipients may encompass stabilizers and tonicity modifiers. Examples of stabilizers and tonicity modifiers include, but not limited to, sugars, salts, surfactants, and derivatives and combination thereof.
The term “reconstitution time” refers to the time that is required to rehydrate a lyophilized formulation (cake or powder) so that the resulting reconstituted liquid formulation is dissolved.
Sugar/s herein include sugars and sugar alcohols such as polyols. Sugars can be referred to monosaccharides, disaccharides, and polysaccharides. Examples of sugars include, but are not limited to, sucrose, maltose, trehalose, glucose, dextrose, raffinose and others. Examples of polyols include, but are not limited to, mannitol, sorbitol, and others.
Surfactant refers to pharmaceutically acceptable excipients used to protect the protein formulations against various stress conditions, like agitation, shearing, exposure to high temperature etc. The suitable surfactants include but are not limited to polyoxyethylensorbitan fatty acid esters such as Tween 20™ or Tween 80™, polyoxyethylene-polyoxypropylene copolymer (e.g. Poloxamer, Pluronic), sodium dodecyl sulphate (SDS) and the like or combination thereof.
Examples of salts include, but not limited to, sodium chloride, potassium chloride, magnesium chloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zinc chloride and/or sodium acetate.
Certain specific aspects and embodiments of the invention are more fully described by reference to the following examples. However, these examples should not be construed as limiting the scope of the invention in any manner.
EXAMPLES
CTLA4-Ig fusion protein molecule, abatacept, suitable for storage in the present pharmaceutical composition is produced by standard methods known in the art. For example, abatacept is prepared by recombinant expression of CTLA4 fused with CH2 and CH3 portion of human IgG in a mammalian host cell such as Chinese Hamster Ovary cells. Further, the expressed abatacept is harvested and the crude harvest is subjected to standard downstream process steps that include purification, filtration and optionally dilution or concentration steps. For example, the crude harvest of abatacept may be purified using standard chromatography techniques such as affinity chromatography, ion-exchange chromatography and combinations thereof. The purified abatacept solution can additionally be subjected to one or more filtration steps, and the solution obtained is subjected to further formulation studies.

EXAMPLE-1 FREEZE-DRIED CTLA4-Ig FUSION PROTEIN FORMULATIONS
8-15 mg/ml concentration of abatacept fusion protein in acetate buffer obtained from chromatographic step was subjected for ultrafiltration to concentrate up to 20 mg/ml. Post which, the samples were subjected for diafiltration wherein the diafiltration medium contained 20 mM phosphate buffer (formulation buffer) with excipients such as at least 50 mg/ml maltose, 40 mM NaCl, and in another separate experiment, the diafiltration medium without sugar in the phosphate buffer was experimented. Post diafiltration, the samples were subjected for second ultrafiltration to concentrate up to 60 mg/ml to 80 mg/ml. The drug substance of abatacept comprising phosphate buffer, salt with and without maltose were subjected for lyophilisation process. Prior to lyophilisation process, all drug substances were prepared as formulated drug substance by adjusting the concentration of maltose to 80 mg/ml. Details of the final drug substance are given in Table 1. Post which, all samples were filed in vials and subjected for lyophilisation process. Details of the lyophilisation process are given in Table 2 and primary drying is effectively achieved by increasing the temperature from -19 ? to -10 ? (Ramp time: 1 hour and holding time: 20 hours) before increasing to 0 ?. Post lyophilisation, all the samples were checked for various quality attributes such as pH, appearance of cake, high molecular weight (HMW) content and moisture content. HMW content was measured using size exclusion chromatography and moisture content was measured using Fourier Transform Near infrared spectroscopy. Result of the quality attributes of freeze dried formulations are given in Table 3. Freeze-dried formulation of Aba-3 sample vial kept for stability at 30? for 1 month, at 25 ? for six months and at 2-8 ? 12 months and checked for HMW content. Results of the study are given in Table 4.
Table 1: Details of various abatacept formulations prior to lyophilization
Sample ID Composition of Drug substance post TFF Compensation buffer composition Formulated Drug substance composition (prior to lyophilization)
Aba-1 Abatacept (58 mg/ml), 20 mM phosphate buffer, 40 mM NaCl 260 mg/ml maltose stock Abatacept (40 mg/ml), maltose 80 mg/ml, 20 mM phosphate buffer, 40 mM NaCl, pH 7.0-8.0
Aba-2 Abatacept (58 mg/ml), 20 mM phosphate buffer, 40 mM NaCl 300 mg/ml maltose stock Abatacept (40 mg/ml), maltose 80 mg/ml, 20 mM phosphate buffer, 40 mM NaCl, pH 7.0-8.0
Aba-3 Abatacept (58 mg/ml), 50 mg/ml maltose, 20 mM phosphate buffer, 40 mM NaCl 160 mg/ml maltose stock Abatacept (40 mg/ml), maltose 80 mg/ml, 20 mM phosphate buffer, 40 mM NaCl, pH 7.0-8.0
Aba-4 Abatacept (58 mg/ml), 80 mg/ml maltose, 20 mM phosphate buffer, 40 mM NaCl NA Abatacept (40 mg/ml), maltose 80 mg/ml, 20 mM phosphate buffer, 40 mM NaCl, pH 7.0-8.0

Table 2: Lyophilization cycle for abatacept lyophilized product
Process parameter In-Process control
Loading temperature 5±3 ?
Freezing (ramp) 5 ? to -45 ? for 2.5 hours
Freezing (hold) -45? for 4 to 8 hours
Annealing -22 ? for 3 to 6 hours
Refreezing (ramp) -45? for 1 hour
Refreezing (hold) - 45? for 6 hours
Primary drying (Ramp) -19 ? for 2 hours at 100±20 µbar pressure
Primary drying (hold) -19 ? for 25 hours at 100±20 µbar pressure
Primary drying (Ramp) 0 ? for 1 hour at 100±20 µbar pressure
Primary drying (hold) 0 ? for 2 hours at 100±20 µbar pressure
Secondary drying (Ramp) 25 ? for 2.5 hours at 100±20 µbar pressure
Secondary drying (hold) 25 ? for 10 hours at 100±20 µbar pressure
Secondary drying 25 ? for 0.5 hour at 750±50 µbar pressure
Stoppering 25 ? at 750±20 µbar pressure

Table 3: Quality attributes of various freeze dried formulations prepared as per example 1
Sample ID Cake Appearance pH Reconstitution time Visual inspection HMW content Moisture content
Pre-lyo Post lyo
Aba-1 White, Crystalline 7.4 3 min. 10 sec Clear, colourless, free of visible particles 1.3 1.3 2.6 %
Aba-2 White, Crystalline 7.3 3 min. 55 sec Clear, colourless, free of visible particles 1.7 1.5 2.4 %
Aba-3 White, Intact 7.5 6 min Clear, colourless, free of visible particles 0.5 0.6 0.5%
Aba-4 White, intact, fragmented cake 7.5 2 min. 30 sec Clear, colourless, free of visible particles 0.8 0.7 1%

Table 4: HMW content of Aba-3 freeze dried CTLA4-Ig fusion protein formulation when stored at various temperature
Sample ID Temperature SEC Data
HMW Dimer
Aba-3 30 ? for 1 months 0.9 98.9
25 ? for 6 months 0.9 98.9
2-8 ? for 2 months 0.4 99.4
2-8 ? for 12 months 0.47 99.53

Aba-3 sample before and after lyophilization was subjected for LC-MS technique to evaluate the impact of lyophilization cycle on oxidation and deamidation of abatacept in formulation. It was observed that, there was no change in the oxidation/deamidation post lyophilization.
,CLAIMS:CLAIMS
We Claim:
1) A method of obtaining a stable, freeze-dried CTLA4-Ig fusion protein formulation comprising;
expressing and purifying CTLA4-Ig fusion protein,
subjecting the purified protein to one or more ultrafiltration and/or diafiltration steps with a buffer comprising at least 50 mg/ml of maltose in the filtration steps,
followed by freeze-drying the protein by a lyophilization process.
2) The method as claimed in 1, freeze-dried CTLA4-Ig fusion protein formulation obtained by the method is stable at 25 ? for 6 months and at 30 ? for one months, contains less than 1% of aggregate content (pre and post the lyophilization process), and the freeze-dried powder/cake contains less than 1 % moisture content.
3) The method as claimed in 1, wherein the lyophilization process accomplishes in less than 72 hours and comprises steps of;
(i) freezing the fusion protein formulation at a temperature, ranging from about-45° C to about -50° C
(ii) annealing the frozen formulation obtained from step i) at a temperature, ranging from about -22° C to about -25° C,
(iii) refreezing the formulation obtained from step ii) at a temperature, ranging from about -45° C to about -50° C,
(iv) primary drying of the formulation obtained from step iii), at two different temperature, ranging from about -25° C to about 0° C and,
(v) secondary drying of the formulation obtained from step iv), at a temperature ranging from about 0° C to about 25° C
wherein, the freezing rate maintained during step i), ii) and iii) is about 0.2-0.4 ?/minute.
4) The lyophilization process as claimed in 2, wherein the primary drying step is performed at a pressure range from 50-100 µ bar and the secondary drying step is performed at a pressure range from 700-800 µ bar.
5) The method as claimed in 1, the stable freeze-dried CTLA4-Ig fusion protein is reconstituted in less than 10 minutes.
6) The method as claimed in 1, the pH of the CTLA4-Ig fusion protein formulation is 6-8.
7) A method for lyophilization of a mixture containing CTLA4-Ig fusion protein, maltose, sodium chloride, phosphate buffer comprising the steps of;
(i) freezing the fusion protein formulation at a temperature, ranging from about-45° C to about -50° C
(ii) annealing the frozen formulation obtained from step i) at a temperature, ranging from about -22° C to about -25° C,
(iii) refreezing the formulation obtained from step ii) at a temperature, ranging from about -45° C to about -50° C,
(iv) primary drying of the formulation obtained from step iii), at at least two different temperature, ranging from about -25° C to about 0° C, at a pressure ranging from 50-100 µbar and,
(v) secondary drying of the formulation obtained from step iv), at a temperature ranging from about 0° C to about 25° C, at a pressure ranging from 500-700 µ bar
wherein, the freezing rate maintained during step i), ii) and iii) is about 0.2-0.4 ?/minute.
8) The method as claimed in 7, the lyophilized CTLA4-Ig fusion protein obtained from the process is stable at 25 ? for six months and at 30 ? for one month, and contains less than 1% aggregate content and less than 1% moisture content.
9) The method as claimed in 1 or 7, the lyophilization method does not impact oxidation and deamidation of the product.
10) The method as claimed in 1 or 7, wherein the lyophilization cycle time is less than 72 hours.