DESC:RELATED APPLICATIONS
This application is related to Indian provisional application IN202421019444 filed 16th Mar. 2024 and is incorporated herein in its entirety.
FIELD OF THE INVENTION
The present invention relates to a stable liquid formulation of anti-PD-1 antibody for intravenous administration.
BACKGROUND OF THE INVENTION
Pembrolizumab is a programmed death receptor-1 (PD-1) blocking antibody. It is a humanized monoclonal IgG4 kappa antibody with an approximate molecular weight of 149 kDa. Pembrolizumab is produced in recombinant Chinese hamster ovary (CHO) cells.
Cancer immunotherapy has emerged as a ground-breaking approach in the treatment of various malignancies, offering new avenues for patients with advanced or metastatic cancers. Among the most promising agents in this field are immune checkpoint inhibitors, which target key regulatory pathways that govern the immune response against cancer cells. Pembrolizumab, a humanized monoclonal antibody that targets programmed cell death protein 1 (PD-1), has shown remarkable efficacy and durability in the treatment of multiple cancer types, including melanoma, non-small cell lung cancer, and others.
While pembrolizumab has demonstrated significant clinical benefits, challenges remain in optimizing its formulation to enhance its therapeutic efficacy, stability, and patient compliance. The formulation of pembrolizumab is crucial for ensuring its appropriate pharmacokinetic profile, tissue distribution, and immunomodulatory effects.
WO2012135408 discloses stable liquid/lyophilized pharmaceutical formulation comprising anti-PD-1 antibody, histidine buffer, sucrose and polysorbate 80.
WO2021123202 discloses stable liquid pharmaceutical formulation of Pembrolizumab comprising anti-PD-1 antibody, buffering agent, stabilizer and surfactant. More particularly, it discloses stable liquid pharmaceutical formulation comprising a mixture of an anti- PD-1 antibody, citrate or histidine buffer, trehalose or sugar alcohol and polysorbate 20 or polysorbate 80.
WO2020097141 discloses stable anti-PD1 formulation comprising 5mg/ml to 250mg/ml anti-PD1 antibody, 5mM to 20mM buffer, 1.5% to 8% w/v stabilizer, surfactant and 1mM to 30mM anti-oxidant.
The present invention provides a liquid pharmaceutical formulation for Pembrolizumab to develop improved formulation for wide range of patient population around the world.
OBJECTS OF THE INVENTION
The main object of the present invention is to provide a stable liquid pharmaceutical formulation of anti-PD1 antibody comprising: buffering agent, stabilizer/ bulking agent and non-ionic surfactant.
Another object of the present invention is to provide a stable liquid pharmaceutical formulation of Pembrolizumab comprising: a) anti-PD1 antibody; b) L-histidine/ histidine; c) trehalose dihydrate; d) mannitol; e) polysorbate 20, polysorbate 80 or poloxamer 188; and f) pH adjusting agent.
Another object of the present invention is to provide a stable liquid pharmaceutical formulation of Pembrolizumab comprising: a) 25 mg/mL anti-PD1 antibody; b) 5-50 mM L-histidine/ histidine as buffering agent; c) 60-120 mM trehalose dihydrate and 60-120 mM mannitol as stabilizer/ bulking agent; d) 0.1-1% polysorbate 20, polysorbate 80 or poloxamer 188 as non-ionic surfactant; and e) HCl or glacial acetic acid as pH adjusting agent.
Another object of the present invention is to provide a stable liquid pharmaceutical formulation of Pembrolizumab comprising: a) 25 mg/mL anti-PD1 antibody; b) 1-50 mg/mL L-histidine/ histidine; c) 5-150 mg/mL trehalose dihydrate and 5-150 mg/mL mannitol; d) 0.01-4 mg/mL polysorbate 20, polysorbate 80 or poloxamer 188; and at pH 5 to 6.
Another object of the present invention is to provide a stable liquid pharmaceutical formulation of Pembrolizumab comprising: a) 25 mg/mL anti-PD1 antibody; b) 10-20 mM L-histidine/histidine; c) 95 mM trehalose dihydrate, 96mM mannitol; d) 0.02% polysorbate 20, polysorbate 80 or poloxamer 188; and at pH 5.2 to 5.9.
Another object of the present invention is to provide a stable liquid pharmaceutical formulation of Pembrolizumab comprising: a) 25 mg/mL anti-PD-1 antibody; b) 1-5 mg/mL L-histidine/histidine; c) 36 mg/mL trehalose dihydrate, 17.5 mg/mL mannitol; d) 0.2 mg/mL polysorbate 20, polysorbate 80 or poloxamer 188; and at pH 5.2 to 5.9.
SUMMARY OF THE INVENTION
The main aspect of the present invention is to provide a stable liquid pharmaceutical formulation of anti-PD1 antibody comprising: buffering agent, stabilizer/ bulking agent and non-ionic surfactant.
Another aspect of the present invention is to provide a stable liquid pharmaceutical formulation of Pembrolizumab comprising: a) anti-PD1 antibody; b) L-histidine/ histidine; c) trehalose dihydrate, d) mannitol; e) polysorbate 20, polysorbate 80 or poloxamer 188; and f) pH adjusting agent.
Another aspect of the present invention is to provide a stable liquid pharmaceutical formulation of Pembrolizumab comprising: a) 25 mg/mL anti-PD1 antibody; b) 5-50 mM L-histidine/ histidine as buffering agent; c) 60-120 mM trehalose dihydrate and 60-120 mM mannitol as stabilizer/ bulking agent; d) 0.1-1% polysorbate 20, polysorbate 80 or poloxamer 188 as non-ionic surfactant; and e) HCl or glacial acetic acid as pH adjusting agent.
Another aspect of the present invention is to provide a stable liquid pharmaceutical formulation of Pembrolizumab comprising: a) 25 mg/mL anti-PD1 antibody; b) 1-50 mg/mL L-histidine/ histidine; c) 5-150 mg/mL trehalose dihydrate and 5-150 mg/mL mannitol; d) 0.01-4 mg/mL polysorbate 20, polysorbate 80 or poloxamer 188; and at pH 5 to 6.
Another aspect of the present invention is to provide a stable liquid pharmaceutical formulation of Pembrolizumab comprising: a) 25 mg/mL anti-PD1 antibody; b) 10-20 mM L-histidine/histidine; c) 95 mM trehalose dihydrate, 96mM mannitol; d) 0.02% polysorbate 20, polysorbate 80 or poloxamer 188; and at pH 5.2 to 5.9.
Another aspect of the present invention is to provide a stable liquid pharmaceutical formulation of Pembrolizumab comprising: a) 25 mg/mL anti-PD-1 antibody; b) 1-5 mg/mL L-histidine/histidine; c) 36 mg/mL trehalose dihydrate, 17.5 mg/mL mannitol; d) 0.2 mg/mL polysorbate 20, polysorbate 80 or poloxamer 188; and at pH 5.2 to 5.9.
BRIEF DESCRIPTION OF DRAWING
In order that disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figure with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure wherein:
Figure 1: pH trend analysis of Generic Formulation, AF 4, AF 5, AF 6 and AF 7 at 5°C
Figure 2: Protein concentration trend analysis of Generic Formulation, AF 4, AF 5, AF 6 and AF 7 at 5°C
Figure 3: Percentage HMW (SEC) analysis of Generic Formulation, AF 4, AF 5, AF 6 and AF 7 at 5°C
Figure 4: Percentage Purity (SEC) analysis of Generic Formulation, AF 4, AF 5, AF 6 and AF 7 at 5°C
Figure 5: Percentage Total pre-peak (HIC) analysis of Generic Formulation, AF 4, AF 5, AF 6 and AF 7 at 5°C
Figure 6: Percentage Total Purity (HIC) analysis of Generic Formulation, AF 4, AF 5, AF 6 and AF 7 at 5°C
Figure 7: Percentage Total probable oxidation (HIC) analysis of Generic Formulation, AF 4, AF 5, AF 6 and AF 7 at 5°C
Figure 8: Percentage Acidic (CEX HPLC) analysis of Generic Formulation, AF 4, AF 5, AF 6 and AF 7 at 5°C
Figure 9: Percentage Purity (CEX HPLC) analysis of Generic Formulation, AF 4, AF 5, AF 6 and AF 7 at 5°C
Figure 10: Percentage Basic (CEX HPLC) analysis of Generic Formulation, AF 4, AF 5, AF 6 and AF 7 at 5°C
Figure 11: pH trend analysis of Generic Formulation, AF 4, AF 5, AF 6, AF 7 and RMP at 25°C
Figure 12: Protein concentration trend analysis of Generic Formulation, AF 4, AF 5, AF 6, AF 7 and RMP at 25°C
Figure 13: Percentage HMW (SEC) analysis of Generic Formulation, AF 4, AF 5, AF 6, AF 7 and RMP at 25°C
Figure 14: Percentage Purity (SEC) analysis of Generic Formulation, AF 4, AF 5, AF 6, AF 7 and RMP at 25°C
Figure 15: Percentage total pre-peaks (HIC) analysis of Generic Formulation, AF 4, AF 5, AF 6, AF 7 and RMP at 25°C
Figure 16: Percentage total purity (HIC) analysis of Generic Formulation, AF 4, AF 5, AF 6, AF 7 and RMP at 25°C
Figure 17: Percentage total probable oxidation (HIC) analysis of Generic Formulation, AF 4, AF 5, AF 6, AF 7 and RMP at 25°C
Figure 18: Percentage total acidic (CEX) analysis of Generic Formulation, AF 4, AF 5, AF 6, AF 7 and RMP at 25° C
Figure 19: Percentage Purity (CEX HPLC) analysis of Generic Formulation, AF 4, AF 5, AF 6, AF 7 and RMP at 25°C
Figure 20: Percentage basic (CEX HPLC) analysis of Generic Formulation, AF 4, AF 5, AF 6, AF 7 and RMP at 25°C
Figure 21: Trend analysis of pH of Batch 1, Batch 2, Batch 3 and RMP at 40°C
Figure 22: Trend analysis of Protein concentration of Batch 1, Batch 2, Batch 3 and RMP at 40°C
Figure 23: Percentage HMW (SE HPLC) analysis of Batch 1, Batch 2, Batch 3 and RMP at 40°C
Figure 24: Percentage % Purity (SE HPLC) analysis of Batch 1, Batch 2, Batch 3 and RMP at 40°C
Figure 25: Percentage Acidic variants (CEX HPLC) analysis of Batch 1, Batch 2, Batch 3 and RMP at 40°C
Figure 26: Percentage Purity (CEX HPLC) analysis of Batch 1, Batch 2, Batch 3 and RMP at 40°C
Figure 27: Percentage basic variants (CEX HPLC) analysis of Batch 1, Batch 2, Batch 3 and RMP at 40°C
Figure 28: Percentage total pre-peaks (HIC HPLC) analysis of Batch 1, Batch 2, Batch 3 and RMP at 40°C
Figure 29: Percentage purity (HIC HPLC) analysis of Batch 1, Batch 2, Batch 3 and RMP at 40°C
Figure 30: Percentage LMW (CE SDS- NR) analysis of Batch 1, Batch 2, Batch 3 and RMP at 40°C
Figure 31: Percentage Purity (CE SDS- NR) analysis of Batch 1, Batch 2, Batch 3 and RMP at 40°C
Figure 32: Trend analysis of pH of Batch 1, Batch 2 and Batch 3 at 5°C
Figure 33: Trend analysis of Protein concentration of Batch 1, Batch 2 and Batch 3 at 5°C
Figure 34: Percentage HMW (SE HPLC) analysis of Batch 1, Batch 2 and Batch 3 at 5°C
Figure 35: Percentage Purity (SE HPLC) analysis of Batch 1, Batch 2 and Batch 3 at 5°C
Figure 36: Percentage Acidic variants (CEX HPLC) analysis of Batch 1, Batch 2 and Batch 3 at 5°C
Figure 37: Percentage Purity (CEX HPLC) analysis of Batch 1, Batch 2 and Batch 3 at 5°C
Figure 38: Percentage basic variants (CEX HPLC) analysis of Batch 1, Batch 2 and Batch 3 at 5°C
Figure 39: Percentage Pre-peak (HIC HPLC) analysis of Batch 1, Batch 2 and Batch 3 at 5°C
Figure 40: Percentage Purity (HIC HPLC) analysis of Batch 1, Batch 2 and Batch 3 at 5°C
Figure 41: Percentage LMW (CE SDS - NR) analysis of Batch 1, Batch 2 and Batch 3 at 5°C
Figure 42: Percentage Purity (HIC HPLC) analysis of Batch 1, Batch 2 and Batch 3 at 5°C
Figure 43: Trend analysis of pH of Batch 1, Batch 2, Batch 3 and RMP at 25°C
Figure 44: Trend analysis of Protein concentration of Batch 1, Batch 2, Batch 3 and RMP at 25°C
Figure 45: Percentage HMW (SE HPLC) analysis of Batch 1, Batch 2, Batch 3 and RMP at 25°C
Figure 46: Percentage Purity (SE HPLC) analysis of Batch 1, Batch 2, Batch 3 and RMP at 25°C
Figure 47: Percentage Acidic variants (CEX HPLC) analysis of Batch 1, Batch 2, Batch 3 and RMP at 25°C
Figure 48: Percentage Purity (CEX HPLC) analysis of Batch 1, Batch 2, Batch 3 and RMP at 25°C
Figure 49: Percentage Basic variants (CEX HPLC) analysis of Batch 1, Batch 2, Batch 3 and RMP at 25°C
Figure 50: Percentage total pre-peaks (HIC HPLC) analysis of Batch 1, Batch 2, Batch 3 and RMP at 25°C
Figure 51: Percentage purity (HIC HPLC) analysis of Batch 1, Batch 2, Batch 3 and RMP at 25°C
Figure 52: Percentage LMW (CE SDS-NR) analysis of Batch 1, Batch 2, Batch 3 and RMP at 25°C
Figure 53: Percentage Purity (CE SDS-NR) analysis of Batch 1, Batch 2, Batch 3 and RMP at 25°C
DETAILED DESCRIPTION OF THE INVENTION
DEFINITION
The following definitions are provided to facilitate understanding of certain terms used throughout the specification.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of particular embodiments, preferred embodiments of compositions, methods and materials are described herein. For the purposes of the present disclosure, the following terms are defined below.
The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.
The words "comprise", "comprises", and "comprising" are to be interpreted inclusively rather than exclusively. The words "consist", "consisting", and its variants, are to be interpreted exclusively, rather than inclusively. While various embodiments in the specification are presented using “comprising” language, under other circumstances, a related embodiment is also intended to be interpreted and described using “consisting of’ or “consisting essentially of language.
The term "about" as used in the present patent specification is meant to specify that the specific value provided may vary to a certain extent, such as e.g. means that variations in the range of ± 10 %, preferably ± 5 %, most preferably ± 2 % are included in the given value.
The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile.
A "stable" formulation is one in which the antibody therein substantially retains its physical stability and/or chemical stability and/or its biological activity upon storage. In one aspect, the formulation substantially retains its physical and chemical stability, as well as its biological activity upon storage. The storage period is generally selected based on the intended shelf-life of the formulation.
A "sterile" formulation is aseptic or free from all living microorganisms and their spores.
The term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), nanobodies, modified antibodies, subunits of antibodies, antibody derivatives, artificial antibodies, combinations of antibodies with proteins and antibody fragments sufficiently long to display the desired biological activity, the monoclonal antibodies as used herein may be human antibodies. As used herein, an antibody, or antigen-binding fragment thereof, that has "binding specificity for the PD-1" binds to PD-1. Pembrolizumab is an example of an antibody that has binding specificity for the PD-1 target.
In the present invention antibody is selected from Pembrolizumab, Ranibizumab, Denosumab, Vedolizumab, Aflibercept, Daratumumab, Trastuzumab, Secukinumab, Pertuzumab, Nivolumab, Golimumab, Dupilumab, Etanercept, Atezolizumab, Risankizumab, Bevacizumab, or Rituximab. More preferably, antibody selected is Pembrolizumab.
As used herein the term "buffering agent providing a pH of 5.5 ± 2.0" refers to an agent which provides that the solution comprising it resists changes in pH by the action of its acid/base conjugate components. The buffer used in the formulations in accordance with the present invention has a pH in the range from about 5.0 to about 7.0, or from about 5.0 to about 6.5, or from 15 about 5.3 to about 5.8. A pH of about 5.5 to 5.7 has to be found to be most suitable. Examples of buffering agents that will control the pH in this range include acetate, succinate, gluconate, histidine, citrate, glycine and other organic acid buffers. The most suitable buffer in accordance with the present invention is a histidine buffer, such as e.g. L-histidine/ histidine.
A "histidine buffer" is a buffer comprising histidine ions. Examples of histidine buffers include histidine chloride, histidine acetate, histidine phosphate, histidine sulphate solutions. The histidine buffer or histidine-HCl buffer has a pH between about pH 5 to 6, about pH 5.2 to 5.8, or about pH 5.5 to 5.7.
A “Stabilizer/ Bulking agent” herein refers to an agent which helps to maintain protein stability during freeze-drying and storage by forming a protective glassy state and also helps increase the volume and mass of the lyophilized cake, improving its mechanical properties and reconstitution characteristics. For example, Sucrose, Trehalose, Mannitol, Sorbitol, Polyethylene glycol (PEG) and Hydroxypropyl methylcellulose (HPMC). Trehalose dihydrate and Mannitol respectively have been found to be particularly suitable in the formulations described herein.
A "surfactant" herein refers to an agent that lowers surface tension of a liquid. The surfactant can be a non-ionic surfactant. In one aspect, examples of surfactants herein include polysorbate (polyoxyethylene sorbitan monolaurate, for example, polysorbate 20 and polysorbate 80); poloxamer (e.g. poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, 8 linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol.
The main embodiment of the present invention is to provide a stable liquid pharmaceutical formulation of anti-PD1 antibody comprising: buffering agent, stabilizer/ bulking agent and non-ionic surfactant.
Another embodiment of the present invention is to provide a stable liquid pharmaceutical formulation of Pembrolizumab comprising: a) anti-PD1 antibody; b) L-histidine/ histidine; c) trehalose dihydrate, d) mannitol; e) polysorbate 20, polysorbate 80 or poloxamer 188; and f) pH adjusting agent.
Another embodiment of the present invention is to provide a stable liquid pharmaceutical formulation of Pembrolizumab comprising: a) 25 mg/mL anti-PD1 antibody; b) 5-50 mM L-histidine/ histidine as buffering agent; c) 60-120 mM trehalose dihydrate and 60-120 mM mannitol as stabilizer/ bulking agent; d) 0.1-1% polysorbate 20, polysorbate 80 or poloxamer 188 as non-ionic surfactant; and e) HCl or glacial acetic acid as pH adjusting agent.
Another embodiment of the present invention is to provide a stable liquid pharmaceutical formulation of Pembrolizumab comprising: a) 25 mg/mL anti-PD1 antibody; b) 1-50 mg/mL L-histidine/ histidine; c) 5-150 mg/mL trehalose dihydrate and 5-150 mg/mL mannitol; d) 0.01-4 mg/mL polysorbate 20, polysorbate 80 or poloxamer 188; and at pH 5 to 6.
Another embodiment of the present invention is to provide a stable liquid pharmaceutical formulation of Pembrolizumab comprising: a) 25 mg/mL anti-PD1 antibody; b) 10-20 mM L-histidine/histidine; c) 95 mM trehalose dihydrate, 96mM mannitol; d) 0.02% polysorbate 20, polysorbate 80 or poloxamer 188; and at pH 5.2 to 5.9.
Another embodiment of the present invention is to provide a stable liquid pharmaceutical formulation of Pembrolizumab comprising: a) 25 mg/mL anti-PD-1 antibody; b) 1-5 mg/mL L-histidine/histidine; c) 36 mg/mL trehalose dihydrate, 17.5 mg/mL mannitol; d) 0.2 mg/mL polysorbate 20, polysorbate 80 or poloxamer 188; and at pH 5.2 to 5.9.
In another embodiment of the present invention, concentration of L-histidine/ histidine is of about 0-200 mM, 10-190 mM, 20-180 mM, 30-170 mM, 40-160 mM, 50-150 mM, 60-140 mM, 70-130 mM, 80-120 mM, or 90-110 mM. More preferably the concentration of L-histidine/ histidine is about 5-50 mM.
In another embodiment of the present invention, concentration of L-histidine/ histidine is of about 0-10mg/mL, 0.2-9.8mg/mL, 0.4-9.6mg/mL, 0.6-9.4mg/mL, 0.8-9.2mg/mL, 1-9mg/mL, 1.2-8.8mg/mL, 1.4-8.6mg/mL, 1.6-8.4mg/mL, 1.8-8.2mg/mL, or 2.0-8mg/mL. More preferably the concentration of L-histidine/ histidine is about 1-5 mg/mL.
In another embodiment of the present invention, concentration of trehalose dihydrate and mannitol is of about 0-600 mM, 10-590 mM, 20-580 mM, 30-570 mM, 40-560 mM, 50-550 mM, 60-540 mM, 70-530 mM, 80-520 mM, 90-510 mM, 100-500 mM, 110-490 mM, 120-480 mM, 130-470 mM, 140-460 mM, 150-450 mM, 160-440 mM, 170-430 mM or 200-300 mM. More preferably the concentration of Trehalose dihydrate and mannitol is about 60-200 mM.
In another embodiment of the present invention, concentration of trehalose dihydrate and mannitol is of about 0-300 mg/ml, 10-290 mg/ml, 20-280 mg/ml, 30-270 mg/ml, 40-260 mg/ml, 50-250 mg/ml, 60-240 mg/ml, 70-230 mg/ml, 80-220 mg/ml, 90-210 mg/ml, 100-200 mg/ml, 110-190 mg/ml, 120-180 mg/ml,130-170 mg/ml, or 140-160 mg/ml. More preferably the concentration of Trehalose dihydrate and mannitol is about 5-150 mg/ml.
In another embodiment of the present invention, concentration of polysorbate 20, polysorbate 80 or poloxamer 188 is of about 0-20%, 1-19%, 2-18%, 3-17%, 4-16%, 5-15%, 6-14%, 7-13%, 8-12%, 9-11%, or 10-11%. More preferably the concentration of polysorbate 20, polysorbate, or poloxamer 188 is about 0.1-5%.
In another embodiment of the present invention, concentration of polysorbate 20, polysorbate 80 or Poloxamer 188 is of about 0-10 mg/ml, 1-9 mg/ml, 2-8 mg/ml, 3-7 mg/ml, 4-6 mg/ml, or 5-6 mg/ml. More preferably the concentration of polysorbate 20, polysorbate 80 or Poloxamer 188 is about 0.1-8 mg/ml.
In another embodiment of the present invention, pH of the liquid pharmaceutical formulation is maintained using acetic acid or hydrochloric acid.
In another embodiment of the present invention, the liquid pharmaceutical formulation of Pembrolizumab is free of sucrose.
The detailed description provided is exemplary in nature and is not intended to limit the application or uses of the invention. The following examples further illustrate the invention, but are not meant to limit its scope. Various changes and modifications may be made by those skilled in the art based on the description of the invention, and such changes and modifications are also encompassed within the scope of the present invention.
EXAMPLES
Experimental results related to the development of novel formulation for Pembrolizumab Drug Product (DP).
Different buffers were screened and resulting formulation trials were incepted on stress stability for faster screening to check impact on product quality. Details of executed trials of different buffers are mentioned in Table 1 and Table 6.
EXAMPLE: 1 SCREENING OF FORMULATION EXCIEPIENTS – TRIAL 1
Batch No. Ingredient AF 1 AF 2 AF 3 AF 4
Active Protein Pembrolizumab 25 mg/mL
Buffering Agent Histidine 10 mM (1.55 mg/mL)
Stabilizer/ Bulking agent Sorbitol 236 mM
(43 mg/mL) 236 mM
(43 mg/mL) -- --
Trehalose dihydrate - - 95 mM
(36 mg/mL) 95 mM
(36 mg/mL)
Mannitol - - - 96 mM
(17.5 mg/mL)
Non-ionic Surfactant Polysorbate 80 0.2 mg/mL - - -
Polysorbate 20 - 0.2 mg/mL 0.2 mg/mL 0.2 mg/mL
pH pH adjusted to 5.50 by 1N HCl pH adjusted to 5.50 by 1N HCl pH adjusted to 5.50 by 1% glacial acetic acid pH adjusted to 5.50 by 1% glacial acetic acid
Table 1: Composition of alternate formulations (AF) for Pembrolizumab DP (Trial 1)
The Pembrolizumab drug product was formulated in above mentioned different buffers and kept on stress stability (40 °C ± 2 °C) for 28 days.
Along with this, we also generated all data for Keytruda® (Innovator’s product) wherein DS and DP are as such of Keytruda®. Herein termed as “RMP” in below examples.
The samples were evaluated for analytical techniques as table 2.
Analytical Test Testing Time Point (Days)
0 D 7 D 14 D 28 D
Physical appearance X X X X
pH X X X X
Osmolality X X X X
Protein concentration X X X X
SE-HPLC X X X X
CEX-HPLC X X X X
HIC-HPLC X X X X
X: Testing time point
Table 2: Stability study plan for Stress condition (40 °C ± 2 °C) – Trial 1
A) Physical appearance
All the samples were found to be clear and colorless upto 28 days at stress condition (40 °C ± 2 °C).
B) pH, Osmolality, Protein concentration, SE-HPLC, CEX-HPLC, HIC-HPLC
The data generated from the above set of experiments (Trial 1) are presented in table 3 & 4.
Tests Time Points RMP
(W027953) AF 1 AF 2 AF 3 AF 4
pH Initial 5.52 5.60 5.60 5.60 5.60
7D 5.43 5.48 5.46 5.40 5.42
14D 5.43 5.49 5.50 5.43 5.43
28D 5.44 5.48 5.47 5.42 5.50

Osmolality (mOsmol/kg of water) Initial 248 254 255 141 241
7D 250 252 255 142 242
14D 251 255 257 142 245
28D 251 257 257 142 244

Protein concentration (mg/mL) Initial 26.44 24.61 25.46 24.18 25.55
7D 25.35 26.14 24.72 25.19 25.29
14D 26.13 26.14 26.24 25.36 26.62
28D 26.20 26.30 25.89 25.81 25.70
Table 3: Results of pH, Osmolality and Protein concentration of Trial 1 batches
Test Parameter Time point RMP (W027953) AF 1 AF 2 AF 3 AF 4
SE-HPLC % HMW Initial 0.275 0.07 0.07 0.07 0.06
7D 0.35 0.23 0.24 0.22 0.17
14D 0.43 0.39 0.32 0.35 0.26
28D 0.65 0.65 0.54 0.69 0.47

% Purity Initial 99.69 99.93 99.93 99.93 99.94
7D 99.59 99.72 99.71 99.72 99.77
14D 99.51 99.53 99.61 99.58 99.67
28D 99.26 99.21 99.33 99.15 99.41

CEX-HPLC % Acidic Variants Initial 15.62 13.3 13.27 13.38 13.45
7D 21.02 14.24 13.84 13.68 13.82
14D 23.98 23.46 22.48 24.54 23.41
28D 30.22 32.32 31.43 32.72 30.53

% Basic Variants Initial 21.92 7.06 6.93 6.89 6.87
7D 22.9 10.14 10.26 9.84 10.6
14D 22.54 10.53 10.81 10.73 10.64
28D 22.02 11.04 11.33 10.94 11.55

% Principal Peak Initial 62.46 79.65 79.8 79.73 79.68
7D 56.07 75.63 75.91 76.47 75.57
14D 53.48 65.99 66.73 64.72 65.95
28D 47.74 56.62 57.24 56.33 58.66

HIC-HPLC % Total Prepeaks Initial 11.55 9.28 9.74 9.75 10.09
7D 11.62 13.74 12.57 12.48 16.13
14D 12.04 14.27 16.64 16.09 14.99
28D 12.33 18.05 19.43 22.76 19.22

% Total Purity Initial 76.77 84.40 84.30 84.37 83.98
7D 76.14 79.51 80.68 81.96 82.58
14D 76.38 78.98 76.73 78.67 79.81
28D 75.73 76.11 74.50 72.40 75.77
Table 4: Results of Liquid Chromatographic tests of Trial 1 batches
Observation: Based on the study data presented in Table 3 and Table 4, the alternate formulation 4 (AF4) showed closer degradation pattern with RMP.
Category Excipients Alternate Formulation 4
Buffering agent L-histidine 10 mM
Stabilizer/ Bulking agent Trehalose dihydrate 95 mM
Mannitol 96 mM
Non-ionic Surfactant Polysorbate 20 0.02 %
Vehicle Water for injection q.s.
pH 5.50
Table 5: Lead formulation from Trial 1
EXAMPLE: 2 SCREENING OF FORMULATION EXCIEPIENTS – TRIAL 2
Taking into consideration the data from trial 1 set of experiments, few more combinations were designed taking the lead formulation - alternate formulation 4 as a base. Details of formulation composition are as table 6.
Batch No. Ingredient AF 4 (from trial 1 set) AF 5 AF 6 AF 7
Active Protein Pembrolizumab 25 mg/Ml
Buffering Agent Histidine 10 mM (1.55 mg/mL) 20 mM (3.10 mg/mL)
Stabilizer/ Bulking agent Trehalose dihydrate 95 mM
(36 mg/mL) 95 mM
(36 mg/mL) 95 mM
(36 mg/mL) 95 mM
(36 mg/mL)
Mannitol 96 mM
(17.5 mg/mL) 96 mM
(17.5 mg/mL) 96 mM
(17.5 mg/mL) 96 mM
(17.5 mg/mL)
Surfactant Polysorbate 80 - 0.2 mg/mL - 0.2 mg/mL
Polysorbate 20 0.2 mg/mL - - -
Poloxamer 188 - - 0.2 mg/mL -
pH pH adjusted to 5.50 by 1% glacial acetic acid pH adjusted to 5.50 by 1% glacial acetic acid pH adjusted to 5.50 by 1% glacial acetic acid pH adjusted to 5.70 by 1% glacial acetic acid
Table 6: Composition of Alternate formulations for Pembrolizumab drug product
The Pembrolizumab drug product was formulated in above mentioned different buffers and kept on stress stability (40 °C ± 2 °C) for 28 days.
Along with this, we also formulated our own In-house developed Pembrolizumab in generic buffer (Innovator’s approved formulation excipients) was also kept under similar conditions i.e. DS used here is In-house developed Pembrolizumab whereas DP is prepared as per Innovator’s approved formulation. Herein termed as “Pembrolizumab in generic formulation” in below examples.
Along with this, we also generated all data for Keytruda® (Innovator’s product) wherein DS and DP are as such of Keytruda®. Herein termed as “RMP” in below examples.

The samples were evaluated for analytical techniques as below:
Analytical Test Testing Time Point (Days)
0 D 7 D 14 D 28 D
Physical appearance X X X X
pH X X X X
Protein concentration X X X X
SE-HPLC X X X X
CEX-HPLC (with CpB) X X X X
HIC-HPLC X X X X
CE SDS (NR) X X X X
X: Testing time point
Table 7: Stability study plan for Stress condition (40 °C ± 2 °C) – Trial 2
A) Physical appearance:
All the samples were found to be clear and colorless up to 28 days at stress condition (40 °C ± 2 °C).
B) pH, Osmolality, Protein concentration, SE-HPLC, CEX-HPLC, HIC-HPLC
The data generated from the above set of experiments (Trial 2) are presented in table 8 & 9.
Tests Time Points RMP (W027953) Pembrolizumab in generic formulation AF 4 AF 5 AF 6 AF 7
pH Initial 5.5 5.5 5.6 5.6 5.6 5.8
7D 5.5 5.5 5.6 5.7 5.6 5.8
14D 5.6 5.5 5.7 5.6 5.6 5.8
28D 5.5 5.5 5.6 5.6 5.6 5.8

Protein concent-ration (mg/mL) Initial 25.11 25.1 24.82 23.67 24.37 24.47
7D 25.69 25.1 25.73 24.57 24.66 25.38
14D 25.5 25.4 25.77 24.1 24.52 25.47
28D 25.82 25.9 25.46 24.39 24.6 25.96
Table 8: Results of pH and Protein concentration of Trial 2 batches
Test Parameter Time point RMP (W027953) Pembroli-zumab in generic formulation AF 4 AF 5 AF 6 AF 7
SE-HPLC % HMW Initial 0.33 0.06 0.05 0.06 0.06 0.05
7D 0.52 0.12 0.08 0.09 0.08 0.09
14D 0.67 0.19 0.11 0.12 0.11 0.12
28D 0.94 0.47 0.19 0.21 0.17 0.19

% Purity Initial 99.62 99.95 99.95 99.95 99.94 99.95
7D 99.39 99.83 99.82 99.84 99.86 99.85
14D 99.26 99.74 99.82 99.81 99.79 99.82
28D 99.00 99.41 99.71 99.69 99.73 99.73

CEX-HPLC (with CpB) % Acidic Variants Initial 14.11 8.63 7.16 7.07 7.17 6.75
7D 19.51 10.22 11.65 11.51 11.89 12.05
14D 22.91 17.03 15.97 16.11 16.15 15.26
28D 29.86 26.42 24.63 24.8 24.92 25.60

% Principal Peak Initial 57.26 79.49 65.03 64.82 64.95 65.65
7D 52.55 76.22 61.56 61.54 61.14 61.61
14D 49.84 69.24 58.19 57.68 58.04 59.60
28D 44.88 59.45 51.20 51.30 51.21 53.21

HIC-HPLC % Total Prepeaks Initial 11.68 14.33 14.18 15.19 14.03 14.94
7D 18.55 14.99 15.06 16.62 14.59 16.05
14D 17.60 19.27 16.09 17.51 15.34 17.32
28D 19.56 29.70 17.85 19.77 17.01 19.73

% Total Purity Initial 77.71 80.32 80.61 79.29 80.55 79.66
7D 71.48 79.51 79.62 77.91 80.28 78.46
14D 72.13 75.31 78.39 77.29 79.27 77.36
28D 69.67 65.73 76.76 74.93 77.77 75.13

CE SDS (NR) % LMW Initial 2.37 1.16 1.51 1.30 1.37 1.52
7D 3.10 2.65 1.90 1.95 2.12 2.06
14D 3.39 3.15 2.03 2.26 2.73 2.48
28D 4.06 4.00 3.05 3.09 3.09 3.34
Table 9: Results of Liquid Chromatographic and CE SDS (NR) tests of Trial 2 batches
Observation: Based on the data obtained and trend analysis, the qualitative, quantitative and physiochemical attributes of present invention in the 4 alternate formulations (AF4, AF5, AF6 and AF7, refer Table 8) were found to be better than the pembrolizumab with generic buffer composition at stress condition (40 °C ± 2 °C). Also, all four alternate formulations (AF4, AF5, AF6 and AF7, refer Table 8) have degradation pathway similar to RMP.
From the above study, formulation comprising histidine, trehalose dihydrate, mannitol, and polysorbate 20, polysorbate 80 or poloxamer 188 shown closer degradation to RMP.
EXAMPLE 3: Stability Study at Real time (5 °C ± 3 °C) and Accelerated (25 °C ± 2 °C) condition
All the batches in alternate formulation (AF) considered in trial 2 were charged at Real time and accelerated stability also. Details of formulation composition are mentioned here below:
Batch No. Ingredient AF 4
(from trial 1 set) AF 5 AF 6 AF 7
Active Protein Pembrolizumab 25 mg/mL
Buffering Agent Histidine 10 mM (1.55 mg/mL) 20 mM (3.10 mg/mL)
Stabilizer/ Bulking agent Trehalose dihydrate 95 mM
(36 mg/mL) 95 mM
(36 mg/mL) 95 mM
(36 mg/mL) 95 mM
(36 mg/mL)
Mannitol 96 mM
(17.5 mg/mL) 96 mM
(17.5 mg/mL) 96 mM
(17.5 mg/mL) 96 mM
(17.5 mg/mL)
Surfactant Polysorbate 80 - 0.2 mg/mL - 0.2 mg/mL
Polysorbate 20 0.2 mg/mL - - -
Poloxamer 188 - - 0.2 mg/mL -
pH pH adjusted to 5.50 by 1% glacial acetic acid pH adjusted to 5.50 by 1% glacial acetic acid pH adjusted to 5.50 by 1% glacial acetic acid pH adjusted to 5.70 by 1% glacial acetic acid
Table 10: Composition of Alternate formulations for Pembrolizumab drug product
The Pembrolizumab drug product was formulated in above mentioned different buffers and charged on Real time (5 °C ± 3 °C) and accelerated (25 °C ± 2 °C) stability condition.
Along with this, we also formulated our own In-house developed Pembrolizumab in generic buffer (Innovator’s approved formulation excipients) was also kept under similar RT and AT conditions i.e. DS used here is In-house developed Pembrolizumab whereas DP is prepared as per Innovator’s approved formulation. Herein termed as “Pembrolizumab in generic formulation” in below examples.
Along with this, we also kept one lot at AT for Keytruda® (Innovator’s product) wherein DS and DP are as such of Keytruda®. Herein termed as “RMP” in below examples.
The samples at defined time points i.e., 1M, 2M and 3M were evaluated for analytical techniques as mentioned in Table 11.
Analytical Test Testing Time Point (Months)
Initial
(0 D ay) 1M 2M 3M
Physical appearance X X X X
pH X X X X
Protein concentration X X X X
SE-HPLC X X X X
CEX-HPLC (without CpB) X X X X
HIC-HPLC X X X X
X: Testing time point
Table 11: Stability study plan for RT (5 °C ± 3 °C) and AT (25 °C ± 2 °C)
A) Physical appearance:
All the samples formulated in different formulations (AF4- AF7) along with Intas DP in generic buffer were found to be clear and colorless upto 3 months of RT (5 °C ± 3 °C) and AT condition (25 °C ± 2 °C).
B) pH, Protein concentration:
The data generated for the set of experiments mentioned in Table 1 are presented in Table 12.
Tests Time Points Pembrolizumab in generic formulation AF 4 AF 5 AF 6 AF 7
pH Initial 5.5 5.6 5.5 5.6 5.7
1M 5.6 5.6 5.6 5.6 5.8
2M 5.6 5.6 5.6 5.7 5.8
3M 5.6 5.6 5.6 5.7 5.8

Protein concentration (mg/mL) Initial 25.1 25.0 25.1 25.3 25.1
1M 25.3 25.3 25.2 25.1 25.2
2M 24.7 24.6 24.9 24.8 25.0
3M 24.9 24.6 24.7 24.6 24.8
Table 12: Results of pH and Protein concentration at RT condition (5 °C ± 3 °C)
Tests Time Points RMP (X024363) Pembrolizumab in generic formulation AF 4 AF 5 AF 6 AF 7
pH Initial 5.4 5.5 5.6 5.5 5.6 5.7
1M 5.5 5.6 5.6 5.6 5.7 5.8
2M 5.5 5.6 5.7 5.6 5.7 5.8
3M 5.5 5.7 5.6 5.6 5.7 5.8

Protein Concentration (mg/mL) Initial 26.1 25.1 25.0 25.1 25.3 25.1
1M 25.2 25.3 25.2 25.3 25.2 25.4
2M 25.3 25.1 24.6 24.7 24.8 24.9
3M 25.2 24.8 24.8 24.6 24.8 24.7
Table 13: Results of pH and Protein concentration at AT condition (25 °C ± 2 °C)
Observation:
• Based on 3 month of RT and AT data, the pH of all the batches (AF4 to AF6) were found to be comparable with the RMP at AT condition and comparable with generic buffer formulation at RT condition.
• Also, based on 3 month of RT and AT data, the protein concentration of all batches (AF4 to AF7) were found to be comparable with the RMP at AT condition and comparable with generic buffer formulation at RT condition.
• The trends of pH and protein concentration are shown in Figure 1, 2, 11, & 12.
C) SE-HPLC, CEX-HPLC, HIC-HPLC
The data generated for the set of experiments mentioned in Table 10 are presented in Table 14 and Table 15.
Test Parameter Time point Pembrolizumab in generic formulation AF 4 AF 5 AF 6 AF 7
SE-HPLC % HMW Initial 0.10 0.04 0.04 0.04 0.05
1M 0.12 0.06 0.06 0.05 0.07
2M 0.12 0.05 0.06 0.05 0.08
3M 0.13 0.06 0.06 0.06 0.05

% Purity Initial 99.91 99.96 99.96 99.96 99.95
1M 99.88 99.95 99.94 99.95 99.94
2M 99.81 99.88 99.87 99.89 99.92
3M 99.80 99.89 99.88 99.89 99.91

CEX-HPLC (without CpB treatment) % Acidic Variants Initial 7.89 7.84 7.83 7.82 7.78
1M 7.44 7.10 7.38 7.38 7.34
2M 7.06 7.04 7.04 7.06 7.11
3M 7.01 7.21 7.32 7.38 7.36

% Principal Peak Initial 74.41 74.26 74.31 74.09 74.34
1M 73.58 73.69 73.24 73.36 73.70
2M 70.71 71.01 70.99 70.96 70.99
3M 71.54 71.97 70.96 70.86 71.14

HIC-HPLC % Total Pre-peaks Initial 11.73 11.54 11.45 11.45 11.51
1M 11.71 11.30 11.06 11.80 11.34
2M 11.55 11.39 11.29 11.24 11.12
3M 11.62 11.61 11.55 11.32 11.54

% Total Purity Initial 82.89 83.10 83.09 83.11 83.11
1M 82.81 83.63 83.69 82.95 83.34
2M 82.91 83.38 83.50 82.99 83.70
3M 83.27 82.88 82.87 83.14 83.06

Total probable oxidation Initial 2.26 2.09 2.04 1.95 2.13
1M 2.23 1.99 1.85 1.97 2.04
2M 2.18 2.17 1.97 1.91 1.96
3M 2.17 2.00 1.97 1.72 2.07
Table 14: Results of different Liquid Chromatographic tests at RT condition (5 °C ± 3 °C)
Test Parameter Time point RMP (X024363) Pembrolizumab in generic formulation AF 4 AF 5 AF 6 AF 7
SE-HPLC % HMW Initial 0.50 0.10 0.04 0.04 0.04 0.05
1M 0.57 0.21 0.15 0.16 0.15 0.15
2M 0.60 0.23 0.16 0.18 0.18 0.18
3M 0.69 0.27 0.23 0.23 0.22 0.22

% Purity Initial 99.46 99.91 99.96 99.96 99.96 99.95
1M 99.37 99.67 99.76 99.75 99.76 99.78
2M 99.33 99.63 99.71 99.69 99.65 99.68
3M 99.25 99.12 99.13 99.09 99.11 99.65

CEX-HPLC (without CpB treatment) % Acidic Variants Initial 15.30 7.89 7.84 7.83 7.82 7.78
1M 16.43 8.04 8.04 8.05 8.10 8.41
2M 21.50 13.57 12.22 13.26 13.30 13.07
3M 22.25 14.69 14.57 14.47 14.72 15.06

% Principal Peak Initial 64.85 74.41 74.26 74.31 74.09 74.34
1M 63.55 72.93 72.90 73.12 73.12 73.06
2M 56.10 64.73 66.59 65.41 65.73 66.34
3M 56.09 64.16 65.19 64.56 64.48 64.86

HIC-HPLC % Total Pre-peaks Initial 11.01 11.73 11.54 11.45 11.45 11.51
1M 11.65 12.50 11.92 11.84 11.67 12.36
2M 11.28 12.20 12.51 12.56 12.04 12.84
3M 12.35 12.80 12.96 13.34 12.63 13.75

% Total Purity Initial 80.99 82.89 83.10 83.09 83.11 83.11
1M 80.84 82.18 83.08 83.14 83.29 82.38
2M 81.03 82.59 82.15 82.36 82.80 82.13
3M 79.97 82.10 81.95 81.61 81.93 80.95

% Total Probable oxidation Initial 4.64 2.26 2.09 2.04 1.95 2.13
1M 5.11 2.60 2.37 2.31 2.07 2.63
2M 5.00 2.55 2.60 2.74 2.19 2.94
3M 5.89 2.50 2.74 3.05 2.33 3.43
Table 15: Results of Liquid Chromatographic tests at AT condition (25 °C ± 2 °C)
Observation:
Based on the comparative data analysis, it was observed that the trend of increase in total pre-peaks and probable oxidized impurities for AF6 was lower compared to RMP and other alternate formulations at AT condition (25 °C ± 2 °C) (refer Figure 15 & 17).
Furthermore, as evident from overall data across RT and AT condition (refer Table 4 to Table 6) and trend analysis as depicted in Figure 1 to Figure 20, the qualitative, quantitative and physiochemical attributes of Alternate Formulation 6 (AF 6) were found to be superior than the Pembrolizumab with a generic buffer composition and other alternate formulations listed in Table 10 (AF4, AF 5 and AF 7) under both RT (5 °C ± 3 °C) and AT condition (25 °C ± 2 °C) conditions.
From the above study, formulation comprising histidine, trehalose dihydrate, mannitol, and poloxamer 188 (AF 6) exhibited degradation characteristics similar to RMP.
Category Excipients Alternate Formulation 6
Buffering agent L-histidine 10 mM
Stabilizer/ Bulking agent Trehalose dihydrate 95 mM
Mannitol 96 mM
Non-ionic Surfactant Poloxamer 188 0.02 %
Vehicle Water for injection q.s.
pH 5.50
Table 16: Final alternate formulation for Pembrolizumab
The pH of formulation needs to be adjusted to 5.50 ± 0.10 using 1% Glacial acetic acid
EXAMPLE 4: REAL TIME (5 °C ± 3 °C), ACCELARATED TIME (25° C) AND STRESS DATA (40 °C) OF BATCHES FORMULATED USING ALTERNATE FORMULATION 6
Three batches of Pembrolizumab drug product were formulated in the final Alternate Formulation 6 buffer (AF 6) and are referred as Batch 1, batch 2 and Batch 3, here after. These batches were charged on real time condition (5 °C ± 3 °C), accelerated condition (25 °C ± 2 °C) and stress stability (40 °C ± 2 °C) .The stability plan for the batches are mentioned in Table 17 and Table 18.
Along with these DP batches, one lot of RMP (Y000337) was also charged under AT (25 °C ± 2 °C) and ST (40 °C ± 2 °C). The samples were evaluated for analytical techniques at defined time points as mentioned in Table 17 and Table 18.
Along with this, we also generated all data for Keytruda® (Innovator’s product) under AT and ST, wherein DS and DP are as such of Keytruda®. Herein termed as “RMP” in below examples.
Method of Preparation:
• Pembrolizumab formulation was prepared in composition given in Table 16 by dissolving the excipients in water for injection.
• The protein concentration was set to 25 mg/mL and the pH of the formulation is set to 5.5 using glacial acetic acid.
• 4.3 mL of drug product solution was filled in 10 mL USP type I glass vial and stoppered with 20 mm flurotec coated chlorobutyl rubber stopper.
• Three such DP batches were formulated and then were charged for stability at real time (5 °C ± 3 °C), accelerated (25 °C ± 2 °C) and stress (40 °C ± 2 °C) condition.
• The stability data for the three DP batches are mentioned in subsequent section.
Analytical Test Testing Time Point (Months)
Initial
(0 D ay) 1 M 2 M 3 M
Physical appearance X X X X
pH X X X X
Protein concentration X X X X
SE-HPLC X X X X
CEX-HPLC (without CpB) X X X X
HIC-HPLC X X X X
CE SDS (NR) X X X X
X: Testing time point
Table 17: Stability study plan for three DP batches formulated in the final alternate formulation (AF6) at Real time condition (5 °C ± 3 °C) and AT condition (25 °C ± 3 °C)
Analytical Test Testing Time Point (Days)
Initial (0 Day) 7 D 14 D 28 D
Physical appearance X X X X
pH X X X X
Protein concentration X X X X
SE-HPLC X X X X
CEX-HPLC (without CpB) X X X X
HIC-HPLC X X X X
CE SDS (NR) X X X X
X: Testing time point
Table 18: Stability study plan for three DP batches formulated in the final alternate formulation (AF6) at Stress condition (40 °C ± 2 °C) - AF 6
1. Data at Stress condition (40 °C ± 2 °C)
A) Physical appearance:
The samples from all three DP batches were found to be clear and colorless upto 28 days at stress condition (40 °C ± 2 °C).
B) pH, Protein concentration
The data generated from the 3 Pembrolizumab batches at ST condition, formulated in Alternate Formulation 6, are presented in Table 19.
Tests Time Points RMP (Y000337) Batch 1 Batch 2 Batch 3
pH Initial 5.5 5.4 5.5 5.5
7D 5.5 5.5 5.5 5.5
14D 5.5 5.5 5.5 5.5
28D 5.5 5.5 5.5 5.5

Protein
concentration (mg/mL) Initial 24.7 24.7 24.1 25.3
7D 25.1 24.9 25.1 25.2
14D 25.1 25.1 25.0 25.2
28D 25.1 25.3 24.9 25.2
Table 19: Results of pH and protein concentration of AF 6 batches at ST condition (40 °C ± 2 °C)
Observation:
Based on 28 days ST data, the pH and protein concentration of all the 3 batches was found to be comparable with the reference formulation (RMP) as depicted in Figure 21 & Figure 22.
C) SE-HPLC, CEX-HPLC, HIC-HPLC and CE SDS (NR)
The data generated from the three Pembrolizumab batches at ST condition, formulated in Alternate Formulation 6, are presented in Table 20.
Test Parameter Time point RMP (Y000337) Batch 1 Batch 2 Batch 3
SE-HPLC % HMW Initial 0.21 0.03 0.03 0.04
7D 0.29 0.06 0.07 0.07
14D 0.35 0.09 0.11 0.10
28D 0.53 0.18 0.19 0.19

% Purity Initial 99.76 99.97 99.97 99.96
7D 99.66 99.89 99.89 99.90
14D 99.58 99.83 99.84 99.85
28D 98.38 99.70 99.74 99.74

CEX-HPLC (without CpB treatment) % Acidic Variants Initial 15.34 7.07 8.19 7.90
7D 18.63 8.19 9.27 8.53
14D 23.36 16.01 16.77 16.68
28D 30.59 25.28 25.29 25.41

% Principal Peak Initial 62.21 76.35 71.79 74.66
7D 59.50 73.16 68.95 72.95
14D 56.08 66.25 62.43 65.18
28D 49.50 58.11 55.17 57.49

% Basic Variants Initial 22.46 16.59 20.02 17.46
7D 21.87 18.64 21.78 18.52
14D 20.55 17.74 20.81 18.15
28D 19.92 16.61 19.54 17.10

HIC-HPLC % Total Pre-peaks Initial 11.27 9.67 14.19 11.17
7D 11.12 10.56 15.20 11.72
14D 11.00 11.63 15.82 12.77
28D 11.18 12.86 17.59 14.55

% Total Purity Initial 79.87 85.97 80.64 83.49
7D 80.51 84.83 79.67 82.67
14D 80.76 83.89 79.05 81.96
28D 80.46 82.75 77.37 80.07

CE SDS (NR) % LMW Initial 2.86 2.31 2.09 2.14
7D 2.85 2.12 2.29 2.75
14D 2.90 2.99 2.51 3.20
28D 3.71 2.83 3.27 3.76

% Total Purity Initial 97.15 97.70 97.91 97.85
7D 97.15 97.88 97.70 97.25
14D 97.10 97.02 97.50 96.80
28D 96.29 97.17 96.74 96.23
Table 20: Results of Liquid Chromatographic and CE SDS (NR) tests of AF 6 batches at ST condition (40 °C ± 2 °C)
Observation:
Based on 28 days ST data, the qualitative and quantitative attributes of the three Pembrolizumab DP batches formulated in Alternate Formulation buffer 6 are found to be comparable with the reference formulation (RMP) as depicted in Figure 23 & Figure 31.
2. Data at Real time condition (5 °C ± 3 °C)
A) Physical appearance:
All the samples from three DP batches were found to be clear and colorless upto 3 months at RT condition (5 °C ± 3 °C).
B) pH, Protein concentration
The data generated from the three Pembrolizumab batches, formulated in Alternate Formulation 6, are presented in Table 21 and Table 22.
Tests Time Points Batch 1 Batch 2 Batch 3
pH Initial 5.4 5.5 5.5
1 M 5.5 5.5 5.5
2 M 5.5 5.5 5.5
3 M 5.5 5.5 5.5

Protein concentration (mg/mL) Initial 24.7 24.1 25.3
1 M 25.2 24.9 25.2
2 M 25.1 25.0 25.1
3 M 25.1 25.1 25.2
Table 21: Results of pH and Protein concentration of AF 6 batches at RT condition (5 °C ± 3 °C)
Observation:
Based on 3 month of RT condition, the pH and protein concentration of all the 3 batches were found to be consistent as depicted in Figure 32 & Figure 33.
C) SE-HPLC, CEX-HPLC, HIC-HPLC and CE SDS (NR)
The data generated from the three Pembrolizumab batches at RT condition, formulated in Alternate Formulation 6, are presented in Table 13.
Test Parameter Time point Batch 1 Batch 2 Batch 3
SE-HPLC % HMW Initial 0.03 0.03 0.04
1 M 0.03 0.04 0.04
2 M 0.03 0.04 0.04
3 M 0.04 0.04 0.04

% Purity Initial 99.97 99.97 99.96
1 M 99.97 99.96 99.96
2 M 99.97 99.96 99.96
3 M 99.96 99.96 99.96

CEX-HPLC (without CpB) % Acidic Variants Initial 7.07 8.19 7.90
1 M 6.69 8.13 7.63
2 M 6.54 7.97 7.52
3 M 6.35 7.62 7.29

% Principal Peak Initial 76.35 71.79 74.66
1 M 75.23 70.23 74.04
2 M 73.28 68.04 71.52
3 M 71.79 67.68 71.23

% Basic Variants Initial 16.59 20.02 17.46
1 M 18.07 21.67 18.33
2 M 20.08 23.98 20.97
3 M 21.87 24.71 21.48

HIC-HPLC % Total Pre-peaks Initial 9.67 14.19 11.17
1 M 9.89 14.39 11.19
2 M 10.15 14.53 11.37
3 M 10.23 14.52 11.07

% Total Purity Initial 85.97 80.64 83.49
1 M 85.57 80.56 83.72
2 M 84.88 79.99 82.94
3 M 84.83 79.93 83.12

CE SDS (NR) % LMW Initial 2.31 2.09 2.14
1 M 1.59 1.88 1.93
2 M 1.90 1.78 1.12
3 M 2.45 1.94 1.44

% Total Purity Initial 97.70 97.91 97.85
1 M 98.41 98.13 98.07
2 M 98.09 98.21 98.89
3 M 97.55 98.07 98.56
Table 22: Results of Liquid Chromatographic and CE SDS (NR) tests of AF 6 batches at RT condition
Observation:
Based on 3 month of RT condition, the qualitative and quantitative attributes of all the 3 batches were found to be comparable among each other over stability as depicted in Figure 34 to Figure 42.
3. Data at Accelerated condition (25 °C ± 2 °C)
A) Physical appearance:
All the samples from three DP batches were found to be clear and colorless upto 3 months at AT condition (25 °C ± 2 °C).
B) pH, Protein concentration
The data generated from the 3 Pembrolizumab batches, formulated in Alternate Formulation 6, are presented in Table 23.
Tests Time Points RMP (Y000337) Batch 1 Batch 2 Batch 3
pH Initial 5.5 5.4 5.5 5.5
1 M 5.5 5.5 5.5 5.5
2 M 5.5 5.5 5.5 5.5
3 M 5.5 5.5 5.5 5.5

Protein concentration (mg/mL) Initial 24.7 24.7 24.1 25.3
1 M 25.2 25.2 24.9 25.1
2 M 25.3 25.1 25.0 25.0
3 M 25.4 25.0 25.1 24.9
Table 23: Results of pH and Protein concentration of AF 6 batches at AT condition
Observation:
Based on 3 month of AT condition, the pH and protein concentration of all the 3 batches were found to be comparable with the reference formulation (RMP) as depicted in Figure 43 & Figure 44.
C) SE-HPLC, CEX-HPLC, HIC-HPLC and CE SDS (NR)
The data generated from the three Pembrolizumab batches at AT condition, formulated in Alternate Formulation 6, are presented in Table 24.
Test Parameter Time point RMP (Y000337) Batch 1 Batch 2 Batch 3
SE-HPLC % HMW Initial 0.21 0.03 0.03 0.04
1 M 0.24 0.06 0.06 0.05
2 M 0.26 0.08 0.08 0.07
3 M 0.29 0.09 0.08 0.09

% Purity Initial 99.76 99.97 99.97 99.96
1 M 99.72 99.91 99.92 99.92
2 M 99.70 99.89 99.89 99.90
3 M 99.64 99.85 99.87 99.87

CEX- HPLC (without CpB) % Acidic Variants Initial 15.34 7.07 8.19 7.90
1 M 16.18 7.35 8.70 8.33
2 M 19.42 11.67 12.36 12.13
3 M 21.40 13.62 14.45 14.19

% Principal Peak Initial 62.21 76.35 71.79 74.66
1 M 61.71 75.22 70.17 73.61
2 M 56.20 68.44 64.15 66.73
3 M 54.94 66.81 62.61 65.45

% Basic Variants Initial 22.46 16.59 20.02 17.46
1 M 22.09 17.42 21.13 18.08
2 M 24.37 19.88 23.49 21.15
3 M 23.65 19.59 22.94 20.33

HIC-HPLC % Total Pre-peaks Initial 11.27 9.67 14.19 11.17
1 M 11.11 10.60 14.93 11.50
2 M 10.88 11.00 15.34 12.38
3 M 11.56 11.51 16.17 12.07

% Total Purity Initial 79.87 85.97 80.64 83.49
1 M 80.49 85.01 79.58 83.33
2 M 79.81 84.28 79.05 82.06
3 M 79.07 83.33 77.97 82.18

CE SDS (NR) % LMW Initial 2.86 2.31 2.09 2.14
1 M 2.84 2.66 1.95 2.15
2 M 2.60 2.21 2.56 2.29
3 M 3.78 3.01 2.79 3.48

% Total Purity Initial 97.15 97.70 97.91 97.85
1 M 97.16 97.34 98.05 97.85
2 M 97.40 97.79 97.43 97.72
3 M 96.22 96.98 97.11 96.53
Table 24: Results of Liquid Chromatographic and CE SDS (NR) tests of AF 6 batches at AT condition
Observation:
Based on 3 month of AT condition, the trend of increase in impurities of all the three batches were found to be comparable with the reference formulation (RMP) at accelerated condition as depicted in Figure 45 to Figure 53.
Conclusion:
Based on the data obtained for three DP batches and trend analysis, the qualitative, quantitative and physiochemical attributes of present invention in the Alternate Formulation 6 was found to be comparable to reference standard at AT and ST condition. The batches exhibited similar degradation pathway as RMP. The data obtained at RT condition for the batches shows that the Alternate formulation developed for Pembrolizumab is stable over indented real time storage condition.
From the above different trials and stability studies, it has been confirmed that the Alternate Formulation 6 (AF 6) comprising histidine, trehalose dihydrate, mannitol, and poloxamer 188 (pH adjusted to 5.5 using Glacial acetic acid) is highly comparable with RMP. ,CLAIMS:We Claim,
1. A stable liquid pharmaceutical formulation of Pembrolizumab comprising: buffering agent, stabilizer/bulking agent and non-ionic surfactant; wherein formulation is free of sucrose.
2. The pharmaceutical formulation of claim 1, wherein the liquid pharmaceutical formulation of Pembrolizumab comprising: a) anti-PD1 antibody; b) L-histidine/ histidine; c) trehalose dihydrate; d) mannitol; e) polysorbate 20, polysorbate 80 or poloxamer 188; and f) pH adjusting agent.
3. A stable liquid pharmaceutical formulation of Pembrolizumab comprising: a) 25 mg/mL anti-PD1 antibody; b) 1-50 mg/mL L-histidine/histidine; c) 5-150 mg/mL trehalose dihydrate and 5-150 mg/mL mannitol; d) 0.01-4 mg/mL polysorbate 20, polysorbate 80 or poloxamer 188; and at pH 5 to 6.
4. The pharmaceutical formulation according to any of previous claims, wherein the formulation comprising: a) 25 mg/mL anti-PD1 antibody; b) 5-50 mM L-histidine/histidine as buffering agent; c) 60-120 mM trehalose dihydrate and 60-120 mM mannitol as stabilizer/ bulking agent; d) 0.1-1% polysorbate 20, polysorbate 80 or poloxamer 188 as non-ionic surfactant; and e) HCl or glacial acetic acid as pH adjusting agent.
5. A stable liquid pharmaceutical formulation of Pembrolizumab comprising: a) 25 mg/mL anti-PD-1 antibody; b) 1-5 mg/mL L-histidine/histidine; c) 36 mg/mL trehalose dihydrate, 17.5 mg/mL mannitol; d) 0.2 mg/mL polysorbate 20, polysorbate 80 or poloxamer 188; and at pH 5.2 to 5.9.
6. The pharmaceutical formulation according to claim 5, wherein the formulation comprising: a) 25 mg/mL anti-PD1 antibody; b) 10-20 mM L-histidine/histidine; c) 95 mM trehalose dihydrate, 96mM mannitol; d) 0.02% polysorbate 20, polysorbate 80 or poloxamer 188; and at pH 5.2 to 5.9.