DESC:RELATED APPLICATIONS
This application is related to Indian Provisional Application IN202321055925 filed 21st Aug, 2023 and is incorporated herein in its entirely.
FIELD OF THE INVENTION
The present invention relates to stable liquid citrate free pharmaceutical formulation of an anti-a4ß7 antibody.
BACKGROUND OF THE INVENTION
Recombinant DNA techniques can be used to manufacture a range of proteins for medicinal applications thanks to advancements in biotechnology. The formulation of such proteins presents unique challenges because they are bigger and more complex than conventional organic and inorganic pharmaceuticals (i.e., they have many functional groups in addition to intricate three-dimensional structures). A formulation must protect the various functional groups of a protein from degradation while maintaining the structural integrity of at least a core sequence of the protein's amino acids for the protein to remain biologically active. Monoclonal and polyclonal antibodies, in particular, may be relatively unstable proteins.
A protein's stability may be affected by a variety of factors. In fact, the formation of such systems is made more difficult by the possibility of heterogeneous antibody structures even in the case of purified antibodies. Additionally, the excipients used in antibody formulations are designed to reduce any possible immunological reaction. Preservation of the conformational integrity is even more crucial in the case of antibodies. Protein degradation can occur through physical instability (i.e., modifications to the protein's higher order structure) or chemical instability (i.e., any process that modifies the protein through bond creation or cleavage resulting in a new chemical entity). De-amidation, isomerization, hydrolysis, oxidation, fragmentation, glycan beta elimination, and disulfide exchange are a few examples of how chemical instability manifests itself.
Recombinant DNA technology has facilitated the commercialization of numerous proteins during the past 20 years, most notably antibody therapies. These therapeutic antibodies' stability, method of administration, dose forms, and concentrations all have a significant role in how well they work. This calls for therapeutic antibodies to be correctly formulated in order to maintain their stability and activity. There may be distinct criteria for each dose type and mode of administration because of this. Liquid (aqueous) formulations are typically less stable than solid dose forms like lyophilized powders. However, compared to a liquid formulation, reconstitution of the lyophilized formulation involves high production costs, substantial vial overfilling, and careful handling. Although liquid formulations are advantageous in these situations and are typically preferred for injectable protein therapeutics (in terms of convenience for the end user and ease of preparation for the manufacturer), this form may not always be practical due to proteins' susceptibility to denaturation, aggregation, and oxidation under stresses such as temperature, pH changes, agitation, etc.
A therapeutic protein or antibody may lose its biological action as a result of any one of several stressors. High concentration liquid formulations in particular are prone to aggregation and/or deterioration. Nevertheless, when the frequency of administration and injection volume are decreased, high concentration formulations may be preferable for intravenous or subcutaneous routes of administration. On the other hand, a low concentration formulation may be needed for a specific treatment regimen and dose, and intravenous administration is preferred for more consistent delivery and full absorption of the therapeutic medication.
Because of this, creating a formulation that is stable at either high or low doses of the therapeutic protein or antibody and supports a variety of administration methods (intravenous or subcutaneous) presents a substantial developmental challenge. In addition, each protein or antibody has its own unique properties and degradation profile, which complicates the formulation process and may necessitate the development of a stable formulation.
A formulation with a higher concentration of protein and/or stabilizers may make the formulation more viscous, which may increase the injection time and discomfort at the injection site, as well as complicate the drug processing process. Therefore, an improved formulation must be developed, which has a minimum number and/or concentration of the excipients but stabilizes the active molecule at a broad range of concentrations.
Subcutaneous administration is used for a variety of drugs due to its high bioavailability and fast onset of effect. However, pain at the site of injection may reduce patient compliance. In addition to the direct effect of the drug itself, there are several factors that can affect pain which includes osmolality and the kind of excipients employed, including buffers and preservatives.
Ideally, injectable products should be formulated as isotonic solutions (osmolality of about 300 mOsm/kg) and no more than 600 mOsm/kg have to be used in order to prevent pain. Hence, the formulation used for parenteral route must have osmolality closer to the physiological osmolality i.e. osmolality of about 300 mOsm/kg. Buffers are frequently added to parenteral formulations to optimize solubility and stability by adjusting the pH; however, their strength should be kept as low as possible to avoid pain upon injection.
Literatures suggests that the pH of the injection site might not drastically change upon injection unless the formulation contains strong ions as buffering agents. This is supported by a study where it was reported that the pain patients experienced upon injection was more serious for citrate than for histidine or saline. Since citrate is a strong ion, whereas histidine and saline are not, it is possible that the pH shift within the SC tissue upon the administration of the buffers is more significant with citrate than with saline and histidine, resulting in a more painful injection. The data available recommend the concentration of citrate buffer should be lower than 7.3 mM to avoid an increased sensation of pain.
WO2012151247 discloses stable liquid pharmaceutical formulation of Vedolizumab comprising Citrate or EDTA as chelating agent or antioxidant. It further discloses free amino acid which is selected from the group consisting of histidine, alanine, arginine, glycine, glutamic acid and combinations thereof. More particularly, it discloses stable liquid pharmaceutical formulation comprising a mixture of an anti-a4ß7 antibody, citrate, histidine, arginine, and polysorbate 80.
A formulation combination with increased concentration of protein and /or excipients may increase the viscosity and osmolality of the formulation, in turn increasing the injection time and pain at the site of injection and also pose difficulties during processing of the drug substance. Hence, it is necessary to develop an improved formulation, which contains minimal number and/or concentration of excipients, yet stabilizing the drug at a wide range of its concentration.
OBJECTS OF THE INVENTION
The main object of the present invention is to provide stable liquid pharmaceutical formulation comprising an anti-a4ß7 antibody, a buffer, viscosity reducer and surfactant, wherein formulation does not contain any anti-oxidant.
Another object of the present invention is to provide stable liquid pharmaceutical formulation comprising an anti-a4ß7 antibody, L-histidine as a buffer, L-arginine hydrochloride as a viscosity reducer and polysorbate 80 as a surfactant, wherein formulation does not contain any anti-oxidant.
Another object of the present invention is to provide stable liquid pharmaceutical formulation comprising 150 mg/ml to 180 mg/ml anti-a4ß7 antibody, 0mM to 20mM L – histidine, 100mM to 170mM L-arginine hydrochloride and 0 to 0.1% w/v polysorbate 80 at pH 5.8 to 6.8, wherein formulation does not contain any anti-oxidant.
Another object of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml anti-a4ß7 antibody, 10mM L-histidine, 150mM L-arginine hydrochloride and 0.01% w/v polysorbate 80 at pH of 6.5, wherein formulation does not contain any anti-oxidant.
Another object of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8mg/ml Vedolizumab, 10mM L-histidine, 150mM L-arginine hydrochloride and 0.01% w/v polysorbate 80 at pH 6.5, wherein formulation does not contain any anti-oxidant.
Another object of the present invention is to provide stable liquid pharmaceutical formulation comprising 150 mg/ml to 180 mg/ml anti-a4ß7 antibody, 1mg/ml to 5mg/ml L-histidine, 20mg/ml to 50mg/ml L-arginine hydrochloride and 0mg/ml to 2mg/ml polysorbate 80 at pH 5.8 to 6.8, wherein formulation does not contain any anti-oxidant.
Another object of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml anti-a4ß7 antibody, 1mg/ml to 3mg/ml L-histidine, 28mg/ml to 32mg/ml L-arginine hydrochloride and 0.01mg/ml to 0.5mg/ml polysorbate 80 at pH of 6.5, wherein formulation does not contain any anti-oxidant.
Another object of the present invention is to provide stable liquid pharmaceutical formulation comprising an anti-a4ß7 antibody, a buffer, viscosity reducer and surfactant, wherein formulation is free of citrate.
Another object of the present invention is to provide stable liquid pharmaceutical formulation comprising 150 mg/ml to 180 mg/ml anti-a4ß7 antibody, 0mM to 20mM L – histidine, 100mM to 170mM L-arginine hydrochloride and 0 to 0.1% w/v polysorbate 80 at pH 5.8 to 6.8, wherein formulation is free of citrate.
Another object of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml anti-a4ß7 antibody, 10mM L-histidine, 150mM L-arginine hydrochloride and 0.01% w/v polysorbate 80 at pH of 6.5, wherein formulation is free of citrate.
Another object of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml Vedolizumab, 10mM L-histidine, 150mM L-arginine hydrochloride and 0.01% w/v polysorbate 80 at pH of 6.5, wherein formulation is free of citrate.
Another object of the present invention is to provide stable liquid pharmaceutical formulation comprising 150 mg/ml to 180 mg/ml anti-a4ß7 antibody, 1mg/ml to 5mg/ml L-histidine, 20mg/ml to 50mg/ml L-arginine hydrochloride and 0mg/ml to 2mg/ml polysorbate 80 at pH 5.8 to 6.8, wherein formulation is free of citrate.
Another object of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml anti-a4ß7 antibody, 1mg/ml to 3mg/ml L-histidine, 28mg/ml to 32mg/ml L-arginine hydrochloride and 0.01mg/ml to 0.5mg/ml polysorbate 80 at pH of 6.5, wherein formulation is free of citrate.
Another object of the present invention is to provide stable liquid pharmaceutical formulation comprising 150 mg/ml to 180 mg/ml anti-a4ß7 antibody, 0mM to 20mM L – histidine, 100mM to 170mM L-arginine hydrochloride, 80mM to 120mM Hydroxypropyl-beta-cyclodextrin (HPßCD) and 0 to 0.1% w/v polysorbate 80 at pH 5.8 to 6.8, wherein formulation is free of citrate.
Another object of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8mg/ml Vedolizumab, 10mM L-histidine, 150mM L-arginine hydrochloride, 100mM Hydroxypropyl-beta-cyclodextrin (HPßCD) and 0.01% w/v polysorbate 80 at pH 6.5, wherein formulation is free of citrate.
Another object of the present invention is to provide stable liquid pharmaceutical formulation comprising 150 mg/ml to 180 mg/ml anti-a4ß7 antibody, 0mM to 20mM L – histidine, 100mM to 170mM L-arginine hydrochloride, 0.1mM to 5mM Pentetic acid (DTPA) and 0 to 0.1% w/v polysorbate 80 at pH 5.8 to 6.8, wherein formulation is free of citrate.
Another object of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8mg/ml Vedolizumab, 10mM L-histidine, 150mM L-arginine hydrochloride, 1mM Pentetic acid (DTPA) and 0.01% w/v polysorbate 80 at pH 6.3, wherein formulation free of citrate.
Another object of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml anti-a4ß7 antibody, 1mg/ml to 3mg/ml L-histidine, 28mg/ml to 32mg/ml L-arginine hydrochloride, 150mg/ml to 155mg/ml Hydroxypropyl-beta-cyclodextrin (HPßCD) and 0.01mg/ml to 0.5mg/ml polysorbate 80 at pH of 6.5, wherein formulation is free of citrate.
Another object of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml anti-a4ß7 antibody, 1mg/ml to 3mg/ml L-histidine, 28mg/ml to 32mg/ml L-arginine hydrochloride, 0.1mg/ml to 1mg/ml Pentetic acid (DTPA) and 0.01mg/ml to 0.5mg/ml polysorbate 80 at pH of 6.3, wherein formulation is free of citrate.
Another object of the present invention is to provide a stable liquid formulation of an anti-a4ß7 antibody which is free of citrate and the osmolality of which is close to the iso-osmotic pressure of 300 mOsm/kg or more than 400mOsm/kg.
SUMMARY OF THE INVENTION
The main aspect of the present invention is to provide stable liquid pharmaceutical formulation comprising an anti-a4ß7 antibody, a buffer, viscosity reducer and surfactant, wherein formulation does not contain any anti-oxidant.
Another aspect of the present invention is to provide stable liquid pharmaceutical formulation comprising an anti-a4ß7 antibody, L-histidine as a buffer, L-arginine hydrochloride as a viscosity reducer and polysorbate 80 as a surfactant, wherein formulation does not contain any anti-oxidant.
Another aspect of the present invention is to provide stable liquid pharmaceutical formulation comprising 150 mg/ml to 180 mg/ml anti-a4ß7 antibody, 0mM to 20mM L – histidine, 100mM to 170mM L-arginine hydrochloride and 0 to 0.1% w/v polysorbate 80 at pH 5.8 to 6.8, wherein formulation does not contain any anti-oxidant.
Another aspect of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml anti-a4ß7 antibody, 10mM L-histidine, 150mM L-arginine hydrochloride and 0.01% w/v polysorbate 80 at pH of 6.5, wherein formulation does not contain any anti-oxidant.
Another aspect of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8mg/ml Vedolizumab, 10mM L-histidine, 150mM L-arginine hydrochloride and 0.01% w/v polysorbate 80 at pH 6.5, wherein formulation does not contain any anti-oxidant.
Another aspect of the present invention is to provide stable liquid pharmaceutical formulation comprising 150 mg/ml to 180 mg/ml anti-a4ß7 antibody, 1mg/ml to 5mg/ml L-histidine, 20mg/ml to 50mg/ml L-arginine hydrochloride and 0mg/ml to 2mg/ml polysorbate 80 at pH 5.8 to 6.8, wherein formulation does not contain any anti-oxidant.
Another aspect of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml anti-a4ß7 antibody, 1mg/ml to 3mg/ml L-histidine, 28mg/ml to 32mg/ml L-arginine hydrochloride and 0.01mg/ml to 0.5mg/ml polysorbate 80 at pH of 6.5, wherein formulation does not contain any anti-oxidant.
Another aspect of the present invention is to provide stable liquid pharmaceutical formulation comprising an anti-a4ß7 antibody, a buffer, viscosity reducer and surfactant, wherein formulation is free of citrate.
Another aspect of the present invention is to provide stable liquid pharmaceutical formulation comprising 150 mg/ml to 180 mg/ml anti-a4ß7 antibody, 0mM to 20mM L – histidine, 100mM to 170mM L-arginine hydrochloride and 0 to 0.1% w/v polysorbate 80 at pH 5.8 to 6.8, wherein formulation is free of citrate.
Another aspect of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml anti-a4ß7 antibody, 10mM L-histidine, 150mM L-arginine hydrochloride and 0.01% w/v polysorbate 80 at pH of 6.5, wherein formulation is free of citrate.
Another aspect of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml Vedolizumab, 10mM L-histidine, 150mM L-arginine hydrochloride and 0.01% w/v polysorbate 80 at pH of 6.5, wherein formulation is free of citrate.
Another aspect of the present invention is to provide stable liquid pharmaceutical formulation comprising 150 mg/ml to 180 mg/ml anti-a4ß7 antibody, 1mg/ml to 5mg/ml L-histidine, 20mg/ml to 50mg/ml L-arginine hydrochloride and 0mg/ml to 2mg/ml polysorbate 80 at pH 5.8 to 6.8, wherein formulation is free of citrate.
Another aspect of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml anti-a4ß7 antibody, 1mg/ml to 3mg/ml L-histidine, 28mg/ml to 32mg/ml L-arginine hydrochloride and 0.01mg/ml to 0.5mg/ml polysorbate 80 at pH of 6.5, wherein formulation is free of citrate.
Another aspect of the present invention is to provide stable liquid pharmaceutical formulation comprising 150 mg/ml to 180 mg/ml anti-a4ß7 antibody, 0mM to 20mM L – histidine, 100mM to 170mM L-arginine hydrochloride, 80mM to 120mM Hydroxypropyl-beta-cyclodextrin (HPßCD) and 0 to 0.1% w/v polysorbate 80 at pH 5.8 to 6.8, wherein formulation is free of citrate.
Another aspect of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8mg/ml Vedolizumab, 10mM L-histidine, 150mM L-arginine hydrochloride, 100mM Hydroxypropyl-beta-cyclodextrin (HPßCD) and 0.01% w/v polysorbate 80 at pH 6.5, wherein formulation is free of citrate.
Another aspect of the present invention is to provide stable liquid pharmaceutical formulation comprising 150 mg/ml to 180 mg/ml anti-a4ß7 antibody, 0mM to 20mM L – histidine, 100mM to 170mM L-arginine hydrochloride, 0.1mM to 5mM Pentetic acid (DTPA) and 0 to 0.1% w/v polysorbate 80 at pH 5.8 to 6.8, wherein formulation is free of citrate.
Another aspect of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8mg/ml Vedolizumab, 10mM L-histidine, 150mM L-arginine hydrochloride, 1mM Pentetic acid (DTPA) and 0.01% w/v polysorbate 80 at pH 6.3, wherein formulation free of citrate.
Another aspect of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml anti-a4ß7 antibody, 1mg/ml to 3mg/ml L-histidine, 28mg/ml to 32mg/ml L-arginine hydrochloride, 150mg/ml to 155mg/ml Hydroxypropyl-beta-cyclodextrin (HPßCD) and 0.01mg/ml to 0.5mg/ml polysorbate 80 at pH of 6.5, wherein formulation is free of citrate.
Another aspect of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml anti-a4ß7 antibody, 1mg/ml to 3mg/ml L-histidine, 28mg/ml to 32mg/ml L-arginine hydrochloride, 0.1mg/ml to 1mg/ml Pentetic acid (DTPA) and 0.01mg/ml to 0.5mg/ml polysorbate 80 at pH of 6.3, wherein formulation is free of citrate.
Another aspect of the present invention is to provide a stable liquid formulation of an anti-a4ß7 antibody which is free of citrate and the osmolality of which is close to the iso-osmotic pressure of 300 mOsm/kg or more than 400mOsm/kg.
BRIEF DESCRIPTION OF DRAWINGS
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 RMP & Batch 1, 2 & 3 at 40 °C
Figure 2: % HMW (SEC) trend analysis of RMP & Batch 1, 2 & 3 at 40 °C
Figure 3: % Purity (SEC) trend analysis of RMP & Batch 1, 2 & 3 at 40 °C
Figure 4: % LMW (SEC) trend analysis of RMP & Batch 1, 2 & 3 at 40 °C
Figure 5: % Acidic (CEX) trend analysis of RMP & Batch 1, 2 & 3 at 40 °C
Figure 6: % Purity (CEX) trend analysis of RMP & Batch 1, 2 & 3 at 40 °C
Figure 7: % Basic (CEX) trend analysis of RMP & Batch 1, 2 & 3 at 40 °C
Figure 8: % HMW (SEC) trend analysis of RMP & Batch 4, 5, & 6 at 40 °C
Figure 9: % Purity (SEC) trend analysis of RMP & Batch 4, 5, & 6 at 40 oC
Figure 10: % LMW (SEC) trend analysis of RMP & Batch 4, 5, & 6 at 40 oC
Figure 11: % HMW (SEC) trend analysis of RMP & Batch 7, 8, & 9 at 40 oC
Figure 12: % Purity (SEC) trend analysis of RMP & Batch 7, 8, & 9 at 40 oC
Figure 13: % LMW (SEC) trend analysis of RMP & Batch 7, 8, & 9 at 40 oC
DETAILED DESCRIPTION OF THE INVENTION
The following is a detailed description of embodiments of the invention. The embodiments are in such details as to clearly communicate the invention. However, the amount of details offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents of embodiments, and alternative falling within the spirit and scope of the present 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 "pharmaceutical formulation" refers to a preparation that contains an anti- a4ß7 antibody in such form as to permit the biological activity of the antibody to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
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.
Various analytical methods are available for measuring the physical and chemical degradation of the antibody in the pharmaceutical formulations. An antibody "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. An antibody 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 antibody of interest 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.
As used herein, "buffering agent" refers to a buffer that resists changes in pH by the action of its acid-base conjugate components. The buffering agent may be present in a liquid or solid formulation of the invention. In some embodiments, the buffering agent of this invention adjusts the pH of the formulation to about 5.8 to 6.8. In one aspect, examples of buffering agents that alone or in combination, will control the pH in the 5.8 to 6.8 range include acetate, succinate, gluconate, histidine, citrate, phosphate, maleate, cacodylate, 2-[N- morpholinojethanesulfonic acid (MES), bis(2- hydroxyethyl)iminotris[hydroxymethyl]methane (Bis-Tris), N-[2-acetamido]-2- iminodiacetic acid (ADA), glycylglycine and other organic acid buffers. In another aspect, the buffering agent herein is histidine.
A "histidine buffer" is a buffer comprising histidine ions. Examples of histidine buffers include histidine chloride, histidine acetate, histidine phosphate, histidine sulfate solutions. The histidine buffer or histidine-HCl buffer has a between about pH 5.8 to 6.8.
A "surfactant" herein refers to an agent that lowers surface tension of a liquid. The surfactant can be a nonionic surfactant. In one aspect, examples of surfactants herein include polysorbate (polyoxyethylene sorbitan monolaurate, for example, polysorbate 20 and, polysorbate 80) and poloxamer 188.
An "antioxidant" herein refers to an agent that inhibits the oxidation of other molecules. Examples of antioxidants herein include L-methionine, citrate, lipoic acid, uric acid, glutathione, tocopherol, carotene, lycopene, cysteine, phosphonate compounds, e.g., etidronic acid, desferoxamine and malate.
A "Viscosity reducer" herein refers to an agent which reduce surface tension of viscous materials. Examples of viscosity reducer herein includes phospholipid, such as phosphoglyceride; arginine hydrochloride; vegetable oil; and polycyclic aromatic hydrocarbon.
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 a4ß7 complex" binds to a4ß7. Vedolizumab is an example of an antibody that has binding specificity for the a4ß7 complex.
In the present invention antibody is selected from Ranibizumab, Denosumab, Pembrolizumab, Vedolizumab, Aflibercept, Daratumumab, Trastuzumab, Secukinumab, Pertuzumab, Nivolumab, Golimumab, Dupilumab, Etanercept, Atezolizumab, Risankizumab, Bevacizumab, Semaglutide, Dulaglutide or Rituximab. More preferably, antibody selected is Vedolizumab.
The main embodiment of the present invention is to provide stable liquid pharmaceutical formulation comprising an anti-a4ß7 antibody, a buffer, viscosity reducer and surfactant, wherein formulation does not contain any anti-oxidant.
Another embodiment of the present invention is to provide stable liquid pharmaceutical formulation comprising an anti-a4ß7 antibody, L-histidine as a buffer, L-arginine hydrochloride as a viscosity reducer and polysorbate 80 as a surfactant, wherein formulation does not contain any anti-oxidant.
Another embodiment of the present invention is to provide stable liquid pharmaceutical formulation comprising 150 mg/ml to 180 mg/ml anti-a4ß7 antibody, 0mM to 20mM L – histidine, 100mM to 170mM L-arginine hydrochloride and 0 to 0.1% w/v polysorbate 80 at pH 5.8 to 6.8, wherein formulation does not contain any anti-oxidant.
Another embodiment of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml anti-a4ß7 antibody, 10mM L-histidine, 150mM L-arginine hydrochloride and 0.01% w/v polysorbate 80 at pH of 6.5, wherein formulation does not contain any anti-oxidant.
In another embodiment of the present invention formulation of anti- a4ß7 antibody wherein buffer is less than 20mM.
In another embodiment of the present invention formulation of anti- a4ß7 antibody wherein L-arginine hydrochloride is used as viscosity reducer or free amino acid.
Another embodiment of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8mg/ml Vedolizumab, 10mM L-histidine, 150mM L-arginine hydrochloride and 0.01% w/v polysorbate 80 at pH 6.5, wherein formulation does not contain any anti-oxidant.
Another embodiment of the present invention is to provide stable liquid pharmaceutical formulation comprising 150 mg/ml to 180 mg/ml anti-a4ß7 antibody, 1mg/ml to 5mg/ml L-histidine, 20mg/ml to 50mg/ml L-arginine hydrochloride and 0mg/ml to 2mg/ml polysorbate 80 at pH 5.8 to 6.8, wherein formulation does not contain any anti-oxidant.
Another embodiment of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml anti-a4ß7 antibody, 1mg/ml to 3mg/ml L-histidine, 28mg/ml to 32mg/ml L-arginine hydrochloride and 0.01mg/ml to 0.5mg/ml polysorbate 80 at pH of 6.5, wherein formulation does not contain any anti-oxidant.
Another embodiment of the present invention is to provide stable liquid pharmaceutical formulation comprising an anti-a4ß7 antibody, a buffer, viscosity reducer and surfactant, wherein formulation is free of citrate.
Another embodiment of the present invention is to provide stable liquid pharmaceutical formulation comprising 150 mg/ml to 180 mg/ml anti-a4ß7 antibody, 0mM to 20mM L – histidine, 100mM to 170mM L-arginine hydrochloride and 0 to 0.1% w/v polysorbate 80 at pH 5.8 to 6.8, wherein formulation is free of citrate.
Another embodiment of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml anti-a4ß7 antibody, 10mM L-histidine, 150mM L-arginine hydrochloride and 0.01% w/v polysorbate 80 at pH of 6.5, wherein formulation is free of citrate.
Another embodiment of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml Vedolizumab, 10mM L-histidine, 150mM L-arginine hydrochloride and 0.01% w/v polysorbate 80 at pH of 6.5, wherein formulation is free of citrate.
Another embodiment of the present invention is to provide stable liquid pharmaceutical formulation comprising 150 mg/ml to 180 mg/ml anti-a4ß7 antibody, 1mg/ml to 5mg/ml L-histidine, 20mg/ml to 50mg/ml L-arginine hydrochloride and 0mg/ml to 2mg/ml polysorbate 80 at pH 5.8 to 6.8, wherein formulation is free of citrate.
Another embodiment of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml anti-a4ß7 antibody, 1mg/ml to 3mg/ml L-histidine, 28mg/ml to 32mg/ml L-arginine hydrochloride and 0.01mg/ml to 0.5mg/ml polysorbate 80 at pH of 6.5, wherein formulation is free of citrate.
Another embodiment of the present invention is to provide stable liquid pharmaceutical formulation comprising 150 mg/ml to 180 mg/ml anti-a4ß7 antibody, 0mM to 20mM L – histidine, 100mM to 170mM L-arginine hydrochloride, 80mM to 120mM Hydroxypropyl-beta-cyclodextrin (HPßCD) and 0 to 0.1% w/v polysorbate 80 at pH 5.8 to 6.8, wherein formulation is free of citrate.
Another embodiment of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8mg/ml Vedolizumab, 10mM L-histidine, 150mM L-arginine hydrochloride, 100mM Hydroxypropyl-beta-cyclodextrin (HPßCD) and 0.01% w/v polysorbate 80 at pH 6.5, wherein formulation is free of citrate.
Another embodiment of the present invention is to provide stable liquid pharmaceutical formulation comprising 150 mg/ml to 180 mg/ml anti-a4ß7 antibody, 0mM to 20mM L – histidine, 100mM to 170mM L-arginine hydrochloride, 0.1mM to 5mM Pentetic acid (DTPA) and 0 to 0.1% w/v polysorbate 80 at pH 5.8 to 6.8, wherein formulation is free of citrate.
Another embodiment of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8mg/ml Vedolizumab, 10mM L-histidine, 150mM L-arginine hydrochloride, 1mM Pentetic acid (DTPA) and 0.01% w/v polysorbate 80 at pH 6.3, wherein formulation free of citrate.
Another embodiment of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml anti-a4ß7 antibody, 1mg/ml to 3mg/ml L-histidine, 28mg/ml to 32mg/ml L-arginine hydrochloride, 150mg/ml to 155mg/ml Hydroxypropyl-beta-cyclodextrin (HPßCD) and 0.01mg/ml to 0.5mg/ml polysorbate 80 at pH of 6.5, wherein formulation is free of citrate.
Another embodiment of the present invention is to provide stable liquid pharmaceutical formulation comprising 158.8 mg/ml anti-a4ß7 antibody, 1mg/ml to 3mg/ml L-histidine, 28mg/ml to 32mg/ml L-arginine hydrochloride, 0.1mg/ml to 1mg/ml Pentetic acid (DTPA) and 0.01mg/ml to 0.5mg/ml polysorbate 80 at pH of 6.3, wherein formulation is free of citrate.
In another embodiment of the present invention formulation of anti- a4ß7 antibody wherein anti-oxidant is citrate.
In another embodiment of the present invention formulation of anti-a4ß7 antibody can be administered by subcutaneous route or intravenous route.
In another embodiment of the present invention formulation of anti-a4ß7 antibody is a Vedolizumab.
Another embodiment of the present invention is to provide a stable liquid formulation of an anti-a4ß7 antibody which is free of citrate and the osmolality of which is close to the iso-osmotic pressure of 300 mOsm/kg or more than 400mOsm/kg.
In another embodiment of the present invention formulation of anti- a4ß7 antibody composition wherein: buffer can be histidine or acetate; viscosity reducer can be L-arginine hydrochloride, arginine, lysine or glycine; stabilizer can be HPßCD, DTPA or proline; salt can be NaCl; sugar can be sorbitol or sucrose; or surfactant can be polysorbate 80.
The embodiments of the present invention are further described using specific examples herein after. The examples are provided for better understanding of certain embodiments of the invention and not, in any manner, to limit the scope thereof. Possible modifications and equivalents apparent to those skilled in the art using the teachings of the present description and the general art in the field of the invention shall also from the part of this specification and are intended to be included within the scope of it.
EXAMPLES:
EXAMPLE 1: SCREENING OF FORMULATION COMPONENTS
In the present invention a formulation that utilized the minimum number of excipients was developed. Remarkably, a formulation containing minimum number of excipients with a notably low concentration of surfactant while still obtaining promising stability results. Also, the developed formulation does not contain citrates in its composition which is advantageous for subcutaneous application of the formulation.
Table 1: Composition of Vedolizumab drug product with minimum number of excipients
Ingredient Concentration (mg/mL)
Vedolizumab DS 158.8
L- Histidine 1.55
L- Arginine hydrochloride 31.6
Polysorbate 80 0.1
HCl/NaOH Q.S. to pH 6.5
Water for Injection Q.S. to 1mL

Method of Preparation:
Vedolizumab formulation was prepared in composition given in Table 1 by dissolving the excipients in water for injection. The protein concentration was set to 108 mg/0.68 mL (PFS)/ 158.8 mg/ mL and the pH of the formulation is set to 6.5.
0.68 mL of drug product solution was filled in three different types of 1 mL Syringe barrel-USP type I and stopper them with different types of plunger stopper and store the filled PFS in between 2°C and 8°C. Formulation were charged for stability at 40°C for 28 days. During stability study following tests were done:
Table 2: purpose of these tests
Analytical Test Purpose of the test
Physical appearance To monitor Physical appearance
pH To monitor pH of formulation
Protein Concentration To monitor Protein Concentration
CEX-HPLC To monitor Charge related impurities
SEC-HPLC To monitor aggregates (H.M.W. impurities)

EXAMPLE 2: STRESS STABILITY STUDY AT 40°C
Stress stability (40 ºC) were performed using composition given in Table 1 and method of preparation described above.
A) Physical appearance:
All the samples were observed to be clear or opaque and Colourless to yellow solution.

B) pH:
Table 3: pH data of Stress Stability (40 ºC)
Buffers pH
Time Points 0 D 7 D 14 D 21 D 28 D
RMP 6.52 6.50 6.53 6.53 6.51
Batch 1 6.54 6.65 6.65 6.65 6.67
Batch 2 6.54 6.66 6.67 6.69 6.66
Batch 3 6.54 6.67 6.66 6.67 6.65

Observation: Based on 28 days stress stability data the pH of all the 3 batches was comparable with the reference formulation (RMP) as depicted in Figure 1.
C) SEC- HPLC
Table 4: SEC data of Stress Stability (40 ºC)
Buffers % HMW % Purity % LMW
Time Points 0D 7D 14D 21D 28D 0D 7D 14D 21D 28D 0D 7D 14D 21D 28D
RMP 0.80 0.90 1.00 1.40 1.60 99.00 98.80 98.50 98.00 97.70 0.10 0.30 0.40 0.60 0.80
Batch 1 0.31 0.73 1.02 1.26 1.39 99.66 99.08 98.65 98.27 98.01 0.03 0.19 0.33 0.47 0.61
Batch 2 0.31 0.71 0.93 1.14 1.30 99.66 99.11 98.75 98.40 98.10 0.03 0.18 0.32 0.47 0.60
Batch 3 0.31 0.71 0.95 1.16 1.37 99.66 99.10 98.73 98.38 98.01 0.03 0.19 0.31 0.46 0.63

Observation: Based on 28 days stress stability data the SEC- HPLC results of all the 3 batches were comparable with the reference formulation (RMP) as depicted in Figure 2, 3 & 4.
D) CEX- HPLC
Table 5: CEX data of Stress Stability (40 ºC)
Buffers % Acidic % Purity % Basic
Time Points 0D 7D 14D 21D 28D 0D 7D 14D 21D 28D 0D 7D 14D 21D 28D
RMP 24.30 29.30 37.18 43.71 47.99 71.60 62.30 58.59 51.71 48.36 4.20 8.30 4.23 4.58 3.64
Batch 1 17.08 22.80 28.98 34.25 38.73 78.68 68.96 63.79 58.94 54.97 4.24 8.23 7.23 6.80 6.30
Batch 2 17.08 23.04 28.69 34.40 38.49 78.68 68.98 64.12 59.00 55.27 4.24 7.98 7.19 6.60 6.23
Batch 3 17.08 23.50 29.28 34.44 39.19 78.68 68.70 63.64 58.94 54.50 4.24 7.80 7.08 6.61 6.29

Observation: Based on 28 days stress stability data the CEX- HPLC of all the 3 batches was comparable with the reference formulation (RMP) as depicted in Figure 5, 6 & 7.
EXAMPLE 3: STABILITY STUDY AT REAL TIME (2-8°C) AND ACCELERATED TIME (25°C)
Real time (2-8 ºC) and Accelerated time (25 ºC) study were performed using Composition given in Table 1 and Method of preparation described above.
A. Physical appearance:
All the samples were observed to be clear or opaque and colourless to yellow solution.
B. pH:
Table 6: pH data of Batch 1, 2, and 3 at RT (2-8 ºC)
Buffers pH
Time Points Initial 1M 3M 6M
Batch 1 6.54 6.63 6.62 6.66
Batch 2 6.54 6.63 6.63 6.64
Batch 3 6.54 6.63 6.62 6.66

Table 7: pH of Batch 1, 2, and 3 at AT (25 ºC)
Buffers pH
Time Points Initial 1MAT 3MAT 6MAT
Batch 1 6.54 6.65 6.66 6.65
Batch 2 6.54 6.65 6.65 6.64
Batch 3 6.54 6.65 6.66 6.65

C. SEC- HPLC
Table 8: SEC data of batch 1, 2, and 3 at RT (2-8 ºC)
Buffers % HMW % Purity % LMW
Time Points Initial 1M 3M 6M Initial 1M 3M 6M Initial 1M 3M 6M
Batch 1 0.31 0.36 0.54 0.76 99.66 99.59 99.32 99.08 0.03 0.04 0.14 0.17
Batch 2 0.31 0.35 0.51 0.76 99.66 99.60 99.37 99.07 0.03 0.04 0.12 0.17
Batch 3 0.31 0.35 0.53 0.78 99.66 99.61 99.35 99.05 0.03 0.05 0.13 0.17

Table 9: SEC data of batch 1, 2, and 3 at AT (25 ºC)
Buffers % HMW % Purity % LMW
Time Points Initial 1M 3M 6M Initial 1M 3M 6M Initial 1M 3M 6M
Batch 1 0.31 0.66 1.15 1.96 99.66 99.20 98.49 97.10 0.03 0.14 0.37 0.95
Batch 2 0.31 0.65 1.29 1.91 99.66 99.21 98.31 97.17 0.03 0.14 0.40 0.92
Batch 3 0.31 0.68 1.09 2.03 99.66 99.18 98.54 97.02 0.03 0.14 0.36 0.96

D. CEX- HPLC
Table 10: CEX data of 1, 2, and 3 at RT (2-8 ºC)
Buffers % Acidic % Purity % Basic
Time Points Initial 1M 3M 6M Initial 1M 3M 6M Initial 1M 3M 6M
Batch 1 17.08 18.28 18.87 19.37 78.68 75.69 75.50 75.56 4.24 6.04 5.63 5.07
Batch 2 17.08 18.07 18.88 19.34 78.68 75.94 75.46 75.66 4.24 6.00 5.66 4.99
Batch 3 17.08 18.18 18.88 19.46 78.68 75.58 75.38 75.63 4.24 6.24 5.75 4.91

Table 11: CEX data of 1, 2, and 3 at AT (25 ºC)
Buffers % Acidic % Purity % Basic
Time Points Initial 1M 3M 6M Initial 1M 3M 6M Initial 1M 3M 6M
Batch 1 17.08 21.59 28.52 39.70 78.68 72.48 66.65 56.55 4.24 5.93 4.83 3.76
Batch 2 17.08 21.53 28.90 39.50 78.68 72.45 66.31 56.79 4.24 6.03 4.79 3.71
Batch 3 17.08 21.61 28.66 39.82 78.68 72.05 66.59 56.44 4.24 6.34 4.76 3.75

EXAMPLE 4: FORMULATION COMPOSITION CONTAINING HPßCD
Trials were conducted using Hydroxypropyl beta-cyclodextrin and pentetic acid within the composition described in table 1 to further control size variants. This led to the development of two distinct formulations, each offering different controlled size variants for further evaluation.
Table 12: Composition of Vedolizumab drug product with Hydroxypropyl beta-cyclodextrin
Ingredient Concentration (mg/mL)
Vedolizumab DS 158.8
L- Histidine 1.55
L- Arginine hydrochloride 31.6
Polysorbate 80 0.1
Hydroxypropyl beta-cyclodextrin (HPßCD) 154.15
HCl/NaOH Q.S. to pH 6.5
Sterile water for Injection Q.S. to 1mL

Method of Preparation:
Vedolizumab formulation was prepared in composition given in table 12 by dissolving the excipients in water for injection. The protein concentration was set to 108 mg/0.68 mL (PFS)/ 158.8 mg/ mL and the pH of the formulation is set to 6.5.
0.68 mL of drug product solution were filled in 1 mL Syringe barrel-USP type I and stopper them with plunger stopper and store the filled PFS in between 2oC and 8oC. Formulation were charged for stability at 40 oC for 28 days. During stability SEC-HPLC test was done:
Table 13: SEC data of Stress Stability (40 ºC)
Buffers % HMW % Purity % LMW
Time Points 0D 7D 14D 21D 28D 0D 7D 14D 21D 28D 0D 7D 14D 21D 28D
RMP 0.80 0.90 1.00 1.40 1.60 99.00 98.80 98.50 98.00 97.70 0.10 0.30 0.40 0.60 0.80
Batch 4 0.42 0.69 0.83 0.95 1.00 99.49 98.89 98.55 98.21 98.06 0.09 0.42 0.62 0.84 0.94
Batch 5 0.53 0.75 0.93 1.13 1.25 99.39 98.94 98.52 98.21 97.88 0.08 0.31 0.55 0.66 0.87
Batch 6 0.39 0.75 0.87 1.02 1.10 99.56 99.05 98.82 98.54 98.32 0.05 0.20 0.32 0.44 0.58

Observation: Based on 28 days stress stability study at 40°C the SEC-HPLC results of all the 3 batches were comparable with the reference formulation (RMP) as depicted in figure 8, 9 & 10.
EXAMPLE 5: FORMULATION COMPOSITION CONTAINING PENTETIC ACID
Table 14: Composition of Vedolizumab drug product with Pentetic acid
Ingredient Concentration (mg/mL)
Vedolizumab DS 158.8
L- Histidine 1.55
L- Arginine hydrochloride 31.6
Polysorbate 80 0.1
Pentetic acid (DTPA) 0.39
HCl/NaOH Q.S. to pH 6.5
Sterile water for Injection Q.S. to 1mL

Vedolizumab formulation was prepared in composition given in table 14 by dissolving the excipients in water for injection. The protein concentration was set to 108 mg/0.68 mL (PFS)/ 158.8 mg/ mL and the pH of the formulation is set to 6.5.
0.68 mL of drug product solution were filled in 1 mL Syringe barrel-USP type I and stopper them with plunger stopper and store the filled PFS in between 2oC and 8oC. Formulation were charged for stability at 40 oC for 28 days. During stability SEC-HPLC test was done:
Table 15: SEC data of Stress Stability (40 ºC)
Buffers % HMW % Purity % LMW
Time Points 0D 7D 14D 21D 28D 0D 7D 14D 21D 28D 0D 7D 14D 21D 28D
RMP 0.80 0.90 1.00 1.40 1.60 99.00 98.80 98.50 98.00 97.70 0.10 0.30 0.40 0.60 0.80
Batch 7 0.42 0.69 0.88 1.01 1.06 99.50 98.03 98.69 98.39 98.28 0.09 0.28 0.43 0.60 0.67
Batch 8 0.52 0.75 0.90 1.02 1.11 99.40 98.03 98.78 98.51 97.27 0.08 0.21 0.33 0.48 0.61
Batch 9 0.40 0.75 0.88 1.00 1.10 99.56 99.10 98.87 98.65 98.45 0.05 0.16 0.25 0.35 0.45

Observation: Based on 28 days stress stability study at 40°C the SEC-HPLC results of all the 3 batches were comparable with the reference formulation (RMP) as depicted in figure 11, 12 & 13.
EXAMPLE 6: OSMOLALITY RESULTS FOR FORMULATIONS
Osmolality of the developed formulations were measured to evaluate the isotonicity of the composition. The results for the osmolality are tabulated in the Table 16.
Buffers Osmolality (mOsm/kg)
RMP 403
Batch-1 287
Batch-6 470
Batch-9 291
Table 16: Osmolality data of different buffer composition

Based on the data presented in above examples it is confirmed that the formulation of present invention is highly comparable with RMP.

,CLAIMS:We Claim,

1. A stable liquid pharmaceutical formulation comprising an anti-a4ß7 antibody, a buffer, viscosity reducer and surfactant, wherein formulation does not contain any anti-oxidant.
2. The stable liquid pharmaceutical formulation of claim 1, wherein formulation comprising an anti-a4ß7 antibody, L-histidine as a buffer, L-arginine hydrochloride as a viscosity reducer and polysorbate 80 as a surfactant, wherein formulation does not contain any anti-oxidant.
3. The stable liquid pharmaceutical formulation of claim 1, wherein buffer is L-Histidine, having concentration of 0mM to 20 mM, more preferably about 10mM.
4. The stable liquid pharmaceutical formulation of claim 1, wherein viscosity reducer is L-arginine hydrochloride, having concentration of 100mM to 170 mM, more preferably about 150mM.
5. The stable liquid pharmaceutical formulation of claim 1, wherein surfactant is Polysorbate 80, having concentration of 0 to 0.1% w/v, more preferably about 0.01% w/v.
6. A stable liquid pharmaceutical formulation comprising 158.8 mg/ml Vedolizumab, 10mM L-histidine, 150mM L-arginine hydrochloride and 0.01% w/v polysorbate 80 at pH of 6.5, wherein formulation is free of citrate.
7. A stable liquid pharmaceutical formulation comprising 158.8mg/ml Vedolizumab, 10mM L-histidine, 150mM L-arginine hydrochloride, 100mM Hydroxypropyl-beta-cyclodextrin (HPßCD) and 0.01% w/v polysorbate 80 at pH 6.5, wherein formulation is free of citrate.
8. A stable liquid pharmaceutical formulation comprising 158.8mg/ml Vedolizumab, 10mM L-histidine, 150mM L-arginine hydrochloride, 1mM Pentetic acid (DTPA) and 0.01% w/v polysorbate 80 at pH 6.3, wherein formulation is free of citrate.
9. The stable pharmaceutical formulation according any of the preceding claims wherein formulation comprising 1mg/ml to 3mg/ml L-histidine, 28mg/ml to 32mg/ml L-arginine hydrochloride and 0.01mg/ml to 0.5mg/ml polysorbate 80 at pH of 6.5, wherein formulation is free of citrate.