DESC:RELATED APPLICATIONS
This application is related to Indian Provisional Application IN202221016571 filed 24th March, 2022 and is incorporated herein in its entirety.
FIELD OF THE INVENTION
The present invention relates to stable high concentration liquid formulation of Anti-HER2 antibody (e.g. Trastuzumab) for subcutaneous injection.
BACKGROUND OF THE INVENTION
The pharmaceutical use of antibodies has increased over the past years. In many instances such antibodies are injected via the intravenous (IV) route. Unfortunately the amount of antibody that can be injected via the intravenous route is limited by the physico-chemical properties of the antibody, in particularly by its solubility and stability in a suitable liquid formulation and by the volume of the infusion fluid. Alternative administration pathways are subcutaneous or intramuscular injection. In order to increase the volume, and thereby the therapeutic dose, which can be safely and comfortably administered subcutaneously. It has been proposed to use glycosaminoglycanase enzyme(s) in order to increase the interstitial space into which the antibody formulation can be injected [WO2006/091871]. In addition to excipients, hyaluronidase enzyme as a permeation enhancer was added to formulation to facilitate delivery of higher volume of subcutaneous injection when co-administered with active drug.
While the antibody formulations have been found suitable for intravenous administration there is a desire to provide highly concentrated, stable pharmaceutical formulations of therapeutically active antibodies for subcutaneous injection. The advantage of subcutaneous injections is that it allows the medical practitioner to perform it in a rather short intervention with the patient. Moreover the patient can be trained to perform the subcutaneous injection by himself. Such self-administration is particularly useful during maintenance dosing because no hospital care is needed (reduced medical resource utilization). Usually injections via the subcutaneous route are limited to approximately 2 ml. For patients requiring multiple doses, several unit dose formulations can be injected at multiple sites of the body surface.
The injection of parenteral drugs into the hypodermis is generally limited to volumes of less than 2 mL due to the viscoelastic resistance to hydraulic conductance in the subcutaneous (SC) tissue, due to the generated backpressure upon injection [Aukland K. and Reed R., "Interstitial-Lymphatic Mechanisms in the control of Extracellular Fluid Volume", Physiology Reviews", 1993; 73:1-78], as well as due to the perceptions of pain. On account of limited injection volume, monoclonal antibody formulations often need to be developed at very high concentrations of 100-200 mg/mL or even higher in some case. The preparation of high concentration protein formulations is rather challenging and there is a need to adapt each formulation to the particular proteins used because each protein has a different aggregation behavior. Aggregates are suspected to cause immunogenicity of therapeutic proteins in at least some of the cases. Immunogenic reaction against protein or antibody aggregates may lead to neutralizing antibodies which may render the therapeutic protein or antibody ineffective. It appears that the immunogenicity of protein aggregates is most problematic in connection with subcutaneous injections, whereby repeated administration increases the risk of an immune response.
While antibodies have a very similar overall structure, such antibodies differ in the amino acid composition (in particular in the CDR regions responsible for the binding to the antigen) and the glycosylation pattern. Moreover there may additionally be post-translational modifications such as charge and glycosylation variants. In the particular case of anti-HER2 antibodies such post-translational modifications have been described e.g. for the humanized monoclonal antibody humMAb4D5-8 (= Trastuzumab). Particular purification methods for the removal of e.g. acidic variants have been developed and compositions comprising a reduced amount of acidic variants (predominantly deamidated variants wherein one or more asparagine residue(s) of the original polypeptide have been converted to aspartate, i.e. the neutral amide side chain has been converted to a residue with an overall acidic character) have first been provided by Basey, CD and Blank, G.S. in WO99/57134. Stable lyophilized antibody formulations comprising a lyoprotectant, a buffer and a surfactant have been described by Andya et al. (WO 97/04801 and US Patent Nos. 6,267,958, 6,685,940, 6,821,151 and 7,060,268).
WO 2006/044908 provides antibody formulations, including monoclonal antibodies formulated in histidine-acetate buffer, pH 5.5 to 6.5, preferably 5.8 to 6.2.
The preparation of highly-concentrated antibody formulations is challenging because of a potential increase in viscosity at higher protein concentration and a potential increase in protein aggregation, a phenomenon that is per se concentration-dependent. High viscosities negatively impact the process ability (e.g. pumping and filtration steps) of the antibody formulations and the administration (e.g. the syringe ability). By the addition of excipients high viscosities could be decreased in some cases. Control and analysis of protein aggregation is an increasing challenge. Aggregation is potentially encountered during various steps of the manufacturing process, which include fermentation, purification, formulation and during storage. Different factors, such as temperature, protein concentration, agitation stress, freezing and thawing, solvent and surfactant effects, and chemical modifications, might influence the aggregation behavior of a therapeutic protein. During development of a highly concentrated antibody formulation the aggregation tendency of the protein has to be monitored and controlled by the addition of various excipients and surfactants. The challenge to prepare suitable highly concentrated, stable pharmaceutical formulation Trastuzumab in accordance with the present invention is increased by the fact that two different proteins have to be formulated in one liquid formulation in such a way that the formulation remains stable over several weeks and the pharmaceutically active ingredients remain active during proper storage.
HERCEPTIN HYLECTA™ is the only available Trastuzumab antibody product for subcutaneous injection in the market. HERCEPTIN HYLECTA™ (Trastuzumab; Hyaluronidase-oysk) is a combination of Trastuzumab and hyaluronidase which is a sterile, preservative-free, colorless to yellowish, clear to opalescent solution supplied in single-dose vials for subcutaneous administration. It is supplied as 600 mg Trastuzumab and 10,000 units hyaluronidase per 5 mL in single dose vials. Each mL of solution contains Trastuzumab (120 mg), hyaluronidase (2,000 units), L-histidine (0.39 mg), L-histidine hydrochloride monohydrate (3.67 mg), L-methionine (1.49 mg), polysorbate 20 (0.4 mg), a, a trehalose dihydrate (79.45 mg), and Water for Injection.
Therefore there is a desire to provide such highly concentrated, stable pharmaceutical formulations of Trastuzumab subcutaneous injection for wide range of patient population around the world.

OBJECTS OF THE INVENTION
The main object of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation of Trastuzumab comprising mixture of two or more stabilizers.
Another object of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation of Trastuzumab for subcutaneous injection comprising mannitol, methionine and arginine HCl as stabilizers.
Another object of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 50 to 350 mg/ml Trastuzumab;
b) About 1 to 100 mM of a buffering agent;
c) About 1 to 500 mM of a mixture of two or more stabilizers wherein, stabilizers are selected from group consisting of mannitol, methionine and arginine HCl;
d) About 0.1 to 0.5 % of a nonionic surfactant; and
e) An effective amount of at least one hyaluronidase enzyme.
Another object of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 50 to 350 mg/ml Trastuzumab;
b) About 1 to 100 mM of a buffering agent providing a pH of 5.5 ± 2.0;
c) About 1 to 500 mM of mannitol, methionine and arginine HCl as stabilizers;
d) About 0.1 to 0.5 % of polysorbate 20; and
e) An effective amount of at least one hyaluronidase enzyme.
Another object of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 50 to 350 mg/ml Trastuzumab;
b) About 1 to 100 mM of a histidine buffer comprising histidine and histidine HCl providing a pH of 5.5 ± 2.0;
c) About 1 to 500 mM of mannitol, methionine and arginine HCl as stabilizers;
d) About 0.1 to 0.5 % of polysorbate 20; and
e) 2000 U/ml hyaluronidase enzyme.
Another object of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 50 to 350 mg/ml Trastuzumab;
b) About 1 to 100 mM of a histidine buffer comprising histidine and histidine HCl providing a pH of 5.5 ± 2.0;
c) About 150 to 300 mM of mannitol; about 5 to 15 mM methionine; and about 1 to 50 mM arginine HCl as stabilizers;
d) 0.1 % of polysorbate 20; and
e) 2000 U/ml hyaluronidase enzyme.
Another object of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 50 to 350 mg/ml Trastuzumab;
b) About 1 to 100 mM of a histidine buffer comprising histidine and histidine HCl providing a pH of 5.5 ± 2.0;
c) About 27 to 55 mg/ml of mannitol; about 0.7 to 2.3 mg/ml methionine; and about 0.2 to 17 mg/ml arginine HCl as stabilizers;
d) 0.1 % of polysorbate 20; and
e) 2000 U/ml hyaluronidase enzyme.
Another object of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 50 to 350 mg/ml of Trastuzumab;
b) About 1 to 100 mM of histidine buffer comprising histidine and histidine HCl providing a pH of 5.5 ± 2.0;
c) About 2.7 to 5.5 % of mannitol; about 0.07 to 0.23 % of methionine; and about 0.02 to 1.7 % of arginine HCl as stabilizers;
d) 0.1 % of polysorbate 20; and
e) 2000 U/ml hyaluronidase enzyme.
Another object of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 120 mg/ml Trastuzumab;
b) About 20 mM of a histidine buffer comprising of histidine and histidine HCl providing a pH of 5.5 ± 2.0;
c) About 165 to 296 mM of mannitol; about 10 mM methionine; and about 9 to 38 mM arginine HCl as stabilizers;
d) 0.1 % of polysorbate 20; and
e) 2000 U/ml hyaluronidase enzyme.
Another object of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 120 mg/ml Trastuzumab;
b) About 20 mM of a histidine buffer comprising of histidine and histidine HCl providing a pH of 5.5 ± 2.0;
c) About 231 mM of mannitol; about 10 mM methionine; and about 24 mM arginine HCl as stabilizers;
d) 0.1 % of polysorbate 20; and
e) 2000 U/ml hyaluronidase enzyme.
SUMMARY OF THE INVENTION
The main aspect of the present invention is to provide a novel high concentrated, stable liquid pharmaceutical formulation of Trastuzumab comprising mixture of two or more stabilizers.
Another aspect of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation of Trastuzumab for subcutaneous injection comprising mannitol, methionine and arginine HCl as stabilizers.
Another aspect of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 50 to 350 mg/ml Trastuzumab;
b) About 1 to 100 mM of a buffering agent;
c) About 1 to 500 mM of a mixture of two or more stabilizers wherein, stabilizers are selected from group consisting of mannitol, methionine and arginine HCl;
d) About 0.1 to 0.5 % of a nonionic surfactant; and
e) An effective amount of at least one hyaluronidase enzyme.
Another aspect of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 50 to 350 mg/ml Trastuzumab;
b) About 1 to 100 mM of a buffering agent providing a pH of 5.5 ± 2.0;
c) About 1 to 500 mM of mannitol, methionine and arginine HCl as stabilizers;
d) About 0.1 to 0.5 % of polysorbate 20; and
e) An effective amount of at least one hyaluronidase enzyme.
Another aspect of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 50 to 350 mg/ml Trastuzumab;
b) About 1 to 100 mM of a histidine buffer comprising histidine and histidine HCl providing a pH of 5.5 ± 2.0;
c) About 1 to 500 mM of mannitol, methionine and arginine HCl as stabilizers;
d) About 0.1 to 0.5 % of polysorbate 20; and
e) 2000 U/ml hyaluronidase enzyme.
Another aspect of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 50 to 350 mg/ml Trastuzumab;
b) About 1 to 100 mM of a histidine buffer comprising histidine and histidine HCl providing a pH of 5.5 ± 2.0;
c) About 150 to 300 mM of mannitol; about 5 to 15 mM methionine; and about 1 to 50 mM arginine HCl as stabilizers;
d) 0.1 % of polysorbate 20; and
e) 2000 U/ml hyaluronidase enzyme.
Another aspect of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 50 to 350 mg/ml Trastuzumab;
b) About 1 to 100 mM of a histidine buffer comprising histidine and histidine HCl providing a pH of 5.5 ± 2.0;
c) About 27 to 55 mg/ml of mannitol; about 0.7 to 2.3 mg/ml methionine; and about 0.2 to 17 mg/ml arginine HCl as stabilizers;
d) 0.1 % of polysorbate 20; and
e) 2000 U/ml hyaluronidase enzyme.
Another aspect of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 50 to 350 mg/ml of Trastuzumab;
b) About 1 to 100 mM of histidine buffer comprising histidine and histidine HCl providing a pH of 5.5 ± 2.0;
c) About 2.7 to 5.5 % of mannitol; about 0.07 to 0.23 % of methionine; and about 0.02 to 1.7 % of arginine HCl as stabilizers;
d) 0.1 % of polysorbate 20; and
e) 2000 U/ml hyaluronidase enzyme.
Another aspect of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 120 mg/ml Trastuzumab;
b) About 20 mM of a histidine buffer comprising of histidine and histidine HCl providing a pH of 5.5 ± 2.0;
c) About 165 to 296 mM of mannitol; about 10 mM methionine; and about 9 to 38 mM arginine HCl as stabilizers;
d) 0.1 % of polysorbate 20; and
e) 2000 U/ml hyaluronidase enzyme.
Another aspect of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 120 mg/ml Trastuzumab;
b) About 20 mM of a histidine buffer comprising of histidine and histidine HCl providing a pH of 5.5 ± 2.0;
c) About 231 mM of mannitol; about 10 mM methionine; and about 24 mM arginine HCl as stabilizers;
d) 0.1 % of polysorbate 20; and
e) 2000 U/ml hyaluronidase enzyme.
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: Comparative % HMW profile of the present invention and Innovator Formulation Buffer composition
Figure - 2: Comparative % HMW profile of the present invention (20 mM Histidine Buffer with Sorbitol 35 mg/mL and Mannitol 42 mg/mL), Innovator Buffer composition and RMP
Figure - 3: Comparative % HMW profile of the present invention (20 mM Histidine Buffer with Mannitol 42 mg/mL), Innovator Buffer composition and RMP
Figure - 4: Comparative CEX % peak 4 profile of the present invention (Histidine, Acetate and Succinate Buffer) and Innovator Buffer composition
Figure - 5: Comparative %HMW profile of confirmatory trial and Innovator Buffer composition
Figure - 6: Comparison of CEX % Peak 4 profile for confirmatory trial and Innovator Buffer composition
Figure - 7: SEC Results of Optimized Formulation with RMP
Figure - 8: CEX Results of Optimized Formulation with RMP
Figure – 9: Trend of Real time (RT) stability data of % HMW by SE-HPLC
Figure – 10: Trend of Real time (RT) stability data of % Purity by SE-HPLC
Figure – 11: Trend of Real time (RT) stability data of % LMW by CE-SDS
Figure – 12: Trend of Real time (RT) stability data of % Purity by CE-SDS
Figure – 13: Trend of Real time (RT) stability data of % Acidic by CEX-HPLC
Figure – 14: Trend of Real time (RT) stability data of % Peak 4 by CEX-HPLC
Figure – 15: Trend of Real time (RT) stability data of % Purity by CEX-HPLC
Figure – 16: Trend of Accelerated time (AT) stability data of % HMW by SE-HPLC
Figure – 17: Trend of Accelerated time (AT) stability data of % Purity by SE-HPLC
Figure – 18: Trend of Accelerated time (AT) stability data of % LMW by CE-SDS
Figure – 19: Trend of Accelerated time (AT) stability data of % purity by CE-SDS
Figure – 20: Trend of Accelerated time (AT) stability data of % Acidic peak by CEX-HPLC
Figure – 21: Trend of Accelerated time (AT) stability data of % peak 4 by CEX-HPLC
Figure – 22: Trend of Accelerated time (AT) stability data of % purity by CEX-HPLC
Figure – 23: % HMW by SEC (ST Study)
Figure – 24: % Acidic by CEX-HPLC (ST Stability)
Figure – 25: % Peak 4 by CEX-HPLC (ST Stability)
Figure – 26: % Purity by CEX-HPLC (ST Stability)
Figure – 27: % LMW by CE-SDS (ST Stability)
Figure – 28: % Purity by CE-SDS (ST Stability)
Figure – 29: % LMW by SE-HPLC (ST Stability)
Figure – 30: % Purity by SE-HPLC (ST Stability)

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 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 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 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 "sterile" formulation is aseptic or free from all living microorganisms and their spores.
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 techniques for measuring protein stability are available in the prior art.
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 about 5.3 to about 5.8. A pH of about 5.5 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/HCl.
A "histidine buffer" is a buffer comprising the amino acid histidine. Examples of histidine buffers include histidine chloride, histidine acetate, histidine phosphate, histidine sulfate. The histidine buffer identified in the examples as being most suitable is a histidine chloride buffer. Such histidine chloride buffer is prepared by titrating L-histidine (free base, solid) with diluted hydrochloric acid. In particular the histidine buffer or histidine chloride buffer is at pH of 5.5 ± 0.6, more particularly at a pH from about 5.3 to about 5.8, and most particularly has a pH of 5.5.
Herein, a "surfactant" refers to a surface-active agent, e.g. a nonionic surfactant. Examples of surfactants herein include polysorbate (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-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and the MONAQU AT™ series (Mona Industries, Inc., Paterson, New Jersey); polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68 etc); etc. Polysorbate 20 (PS20) and Polysorbate 80 (PS80), respectively have been found to be particularly suitable in the formulations described herein.
The main embodiment of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation of Trastuzumab comprising mixture of two or more stabilizers.
Another embodiment of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation of a Trastuzumab for subcutaneous injection, which is ready for use.
Another embodiment of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation of Trastuzumab for subcutaneous injection comprising mannitol, methionine and arginine HCl as stabilizers.
Another embodiment of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 50 to 350 mg/ml Trastuzumab;
b) About 1 to 100 mM of a buffering agent;
c) About 1 to 500 mM of a mixture of two or more stabilizers wherein, stabilizers are selected from group consisting of mannitol, methionine and arginine HCl;
d) About 0.1 to 0.5 % of a nonionic surfactant; and
e) An effective amount of at least one hyaluronidase enzyme.
Another embodiment of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 50 to 350 mg/ml Trastuzumab;
b) About 1 to 100 mM of a buffering agent providing a pH of 5.5 ± 2.0;
c) About 1 to 500 mM of mannitol, methionine and arginine HCl as stabilizers;
d) About 0.1 to 0.5 % of polysorbate 20; and
e) An effective amount of at least one hyaluronidase enzyme.
Another embodiment of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 50 to 350 mg/ml Trastuzumab;
b) About 1 to 100 mM of a histidine buffer comprising histidine and histidine HCl providing a pH of 5.5 ± 2.0;
c) About 1 to 500 mM of mannitol, methionine and arginine HCl as stabilizers;
d) About 0.1 to 0.5 % of polysorbate 20; and
e) 2000 U/ml hyaluronidase enzyme.
Another embodiment of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 50 to 350 mg/ml Trastuzumab;
b) About 1 to 100 mM of a histidine buffer comprising histidine and histidine HCl providing a pH of 5.5 ± 2.0;
c) About 150 to 300 mM of mannitol; about 5 to 15 mM methionine; and about 1 to 50 mM arginine HCl as stabilizers;
d) 0.1 % of polysorbate 20; and
e) 2000 U/ml hyaluronidase enzyme.
Another embodiment of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 50 to 350 mg/ml Trastuzumab;
b) About 1 to 100 mM of a histidine buffer comprising histidine and histidine HCl providing a pH of 5.5 ± 2.0;
c) About 27 to 55 mg/ml of mannitol; about 0.7 to 2.3 mg/ml methionine; and about 0.2 to 17 mg/ml arginine HCl as stabilizers;
d) 0.1 % of polysorbate 20; and
e) 2000 U/ml hyaluronidase enzyme.
Another embodiment of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 50 to 350 mg/ml of Trastuzumab;
b) About 1 to 100 mM of histidine buffer comprising histidine and histidine HCl providing a pH of 5.5 ± 2.0;
c) About 2.7 to 5.5 % of mannitol; about 0.07 to 0.23 % of methionine; and about 0.02 to 1.7 % of arginine HCl as stabilizers;
d) 0.1 % of polysorbate 20; and
e) 2000 U/ml hyaluronidase enzyme.
Another embodiment of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 120 mg/ml Trastuzumab;
b) About 20 mM of a histidine buffer comprising of histidine and histidine HCl providing a pH of 5.5 ± 2.0;
c) About 165 to 296 mM of mannitol; about 10 mM methionine; and about 9 to 38 mM arginine HCl as stabilizers;
d) 0.1 % of polysorbate 20; and
e) 2000 U/ml hyaluronidase enzyme.
Another embodiment of the present invention is to provide a novel high concentrated, stable pharmaceutical formulation comprising:
a) About 120 mg/ml Trastuzumab;
b) About 20 mM of a histidine buffer comprising of histidine and histidine HCl providing a pH of 5.5 ± 2.0;
c) About 231 mM of mannitol; about 10 mM methionine; and about 24 mM arginine HCl as stabilizers;
d) 0.1 % of polysorbate 20; and
e) 2000 U/ml hyaluronidase enzyme.
In another embodiment the present invention also provides pharmaceutical compositions consisting of a highly concentrated, stable pharmaceutical formulation of Trastuzumab and a suitable amount of at least one hyaluronidase enzyme in the form of a kit comprising both injection components and suitable instructions for their subcutaneous injection.
In one embodiment Trastuzumab concentration is 100 to 150 mg/ml, e.g. 120 ± 18 mg/ml, about 110 mg/ml, about 120 mg/ml or about 130 mg/ml.
In another embodiment the concentration of the buffering agent providing a pH of 5.5 ± 2.0 is 1 to 50 mM, e.g. 10 to 30 mM or about 20 mM. The buffering agent can be a histidine buffer, e.g. L-histidine/HCl. In a particular embodiment the pH of the L-histidine/HCl buffer is about 5.5 or about 6.0.
The highly concentrated, stable pharmaceutical formulation of Trastuzumab of the present invention may be provided in liquid form or may be provided in lyophilized form.
The stabilizer (used synonymously with the term "stabilizing agent" in the present patent description) is e.g. a carbohydrate or saccharide or a sugar admitted by the authorities as a suitable additive or excipient in pharmaceutical formulations, e.g. Mannitol, Sorbitol, Methionine and Arginine HCl. The concentration of the stabilizer mannitol is 150 to 300 mM, or about 165 to 296 mM. Preferably about 231 mM. The formulation may contain a another stabilizer, whereby this another stabilizer can be methionine, e.g. in a concentration of 5 to 25 mM or in a concentration of 5 to 15 mM (e.g. methionine in a concentration of about 5mM, about 10 mM or about 15 mM). Preferably about 10 mM. Methionine can also act as an anti-oxidant. The formulation may further contain another stabilizer, whereby another stabilizer can be Arginine HCl, e.g. in a concentration of 1 to 50 mM or in a concentration of 9-38 mM. Preferably about 24 mM.
Suitable examples of pharmaceutically acceptable surfactants include polyoxyethylen- sorbitan fatty acid esters (Tween), polyethylene -polypropylene glycols, polyoxyethylene- stearates, polyoxyethylene alkyl ethers, e.g. polyoxyethylene monolauryl ether, alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulphate (SDS). Most suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). The nonionic surfactant can be a polysorbate, e.g. polysorbate 20 or polysorbate 80. The concentration of the nonionic surfactant is 0.1 to 0.5 % (w/v) or more; particularly about 0.1 % (w/v).
The concentration of the hyaluronidase enzyme depends on the actual hyaluronidase enzyme used in the preparation of the formulation in accordance with the invention. An effective amount of the hyaluronidase enzyme can easily be determined by the person skilled in the art based on the disclosure further below. It should be provided in sufficient amount so that an increase in the dispersion and absorption of the co-administered Trastuzumab is possible. The minimal amount of the hyaluronidase enzyme is > 150 U/ml. More particularly the effective amount of the hyaluronidase enzyme is about 1'000 to 16'000 U/ml, whereby the said amount corresponds to about 0.01 mg to 0.16 mg protein based on an assumed specific activity of 100'000 U/mg. Alternatively the concentration of the hyaluronidase enzyme is about 1'500 to 12000 U/ml, or more particularly about 2'000 U/ml or about 12000 U/ml. The amounts specified herein before correspond to the amount of hyaluronidase enzyme initially added to the formulation. The hyaluronidase enzyme is present either as a combined final formulation or for use for co-administration, e.g. as a co-formulation as further outlined below. The important issue for the formulation in accordance with the present invention is that at the time it is ready for use and/or is injected it has the composition as set out in the appended claims.
It has been proposed to facilitate the subcutaneous injection of therapeutic proteins and antibodies by using small amounts of soluble hyaluronidase glycoproteins (sHASEGPs). It has been shown that the addition of such soluble hyaluronidase glycoproteins (either as a combined formulation or by co-administration) facilitates the administration of therapeutic drug into the hypodermis. By rapidly depolymerizing hyaluronan HA in the extracellular space sHASEGP reduces the viscosity of the interstitium, thereby increasing hydraulic conductance and allowing for larger volumes to be administered safely and comfortably into the subcutaneous tissue. The increased hydraulic conductance induced by sHASEGP through reduced interstitial viscosity allows for greater dispersion, potentially increasing the systemic bioavailability of SC administered therapeutic drug.
The highly concentrated, stable pharmaceutical formulations of the present invention comprising a soluble hyaluronidase glycoprotein are therefore particularly suited for subcutaneous injection. It is clearly understood by the person skilled in the art that such a formulation comprising an Trastuzumab and a soluble hyaluronidase glycoprotein can be provided for administration in form of one single combined formulation or alternatively in form of two separate formulations which can be mixed just prior to the subcutaneous injection. Alternatively the Trastuzumab and the soluble hyaluronidase glycoprotein can be administered as separate injections at different sites of the body, preferably at sites which are immediately adjacent to each other. It is also possible to inject the therapeutic agents present in the formulation in accordance with the present invention as consecutive injections, e.g. first the soluble hyaluronidase glycoprotein followed by the injection of the Trastuzumab. These injections can also be performed in the reversed order, viz. by first injecting the Trastuzumab followed by injecting the soluble hyaluronidase glycoprotein. In case Trastuzumab and the soluble hyaluronidase glycoprotein are administered as separate injections, one or both of the proteins have to be provided with the buffering agent, the stabilizer(s) and the nonionic surfactant in the concentrations as specified in the appended claims but excluding the hyaluronidase enzyme.
As noted above the soluble hyaluronidase glycoprotein may be considered to be a further excipient in the Trastuzumab formulation. The soluble hyaluronidase glycoprotein may be added to the Trastuzumab formulation at the time of manufacturing the Trastuzumab formulation or may be added shortly before the injection. Alternatively the soluble hyaluronidase glycoprotein may be provided as a separate injection. In the latter case the soluble hyaluronidase glycoprotein may be provided in a separate vial either in lyophilized form which must be reconstituted with suitable diluents before the subcutaneous injection takes place, or may be provided as a liquid formulation by the manufacturer. Trastuzumab formulation and the soluble hyaluronidase glycoprotein may be procured as separate entities or may also be provided as kits comprising both injection components and suitable instructions for their subcutaneous administration.
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: FORMULATION BUFFER & STABILIZER SCREENING:
Different formulation buffers were screened and final optimized formulation buffer along with other excipients formulation trials were kept on stress stability for faster screening to check formulation composition impact on product quality. Details of executed trials of with final formulation buffer are mentioned in Table 1.

Table 1: Screening of Formulation Buffers and Other Excipients

EXAMPLE 2: SCREENING OF BUFFERING AGENT WITH OTHER EXCIPIENTS
As mentioned in the table 1, three buffering agents (Histidine, Acetate and Succinate) were tried along with polyol (Sorbitol, Mannitol), Anti-Oxidant (L-Methionine), Amino Acid (L-Arginine HCl), Surfactant (Polysorbate 20) and Hyaluronidase enzyme (2000 units per mL). Formulation batches were manufactured and kept on stability study to generate DP quality data. To compare the stability of various formulated batches, Trastuzumab drug substance was formulated in innovator formulation buffer composition and kept on stability study in parallel with other batches. The RMP quality data was also generated with these batches on stability to compare the degradation profile and rate of degradation. The details formulation composition of various novel formulation buffer and innovator buffer are mentioned in Table 2. Details of drug product made using novel formulation buffer composition were enlisted with respective batch number and innovator buffer composition with respective batch number and henceforth referred to as Generic DP. In the below trials, Methionine (1.49 mg/mL), Polysorbate 20 (1 mg/mL) and Hyaluronidase enzyme (2000 U/mL) were added and kept similar in all the trials.

Table 2: Formulation Buffer Composition
Sr. No. Batch Name Histidine buffer (mM) Acetate buffer (mM) Succinate buffer (mM) Sorbitol/Trehalose/Mannitol L-Arginine HCl Theo. Osmo.
1 P26.1F98D20 20 NA NA 35
Sorbitol 20 430
2 P26.1F99D20 20 NA NA 30
Sorbitol 20 403
3 P26.1F100D20 20 NA NA 25
Sorbitol 20 375
4 P26.1F101D20 20 NA NA 25
Sorbitol 25 423
5 P26.1F102D20 (Generic DP) 20 NA NA 79.45 Trehalose 0 258
6 P26.1F103D21 NA 18 NA 35
Sorbitol 20 409
7 P26.1F104D21 NA 18 NA 30
Sorbitol 20 382
8 P26.1F105D21 NA 18 NA 25
Sorbitol 20 355
9 P26.1F106D21 NA 18 NA 25
Sorbitol 25 402
10 P26.1F107D22 NA NA 10 35
Sorbitol 20 407
11 P26.1F108D22 NA NA 10 30
Sorbitol 20 379
12 P26.1F109D22 NA NA 10 25
Sorbitol 20 352
13 P26.1F110D22 NA NA 10 25
Sorbitol 25 399
14 P26.1F111D20 20 NA NA 42 Mannitol 5 326
15 P26.1F112D21 NA 18 NA 42 Mannitol 5 326
16 P26.1F113D22 NA NA 10 42 Mannitol 5 326

Above batches with respective formulation composition were manufactured. One batch was prepared using innovator buffer composition (Generic DP) in order to rule out the impact of Trastuzumab protein if any on DP quality. Post manufacturing of DP using stated buffer composition of above table, samples were kept on stability study. The stress stability study samples were assessed by incubating samples at 50 °C and testing the samples at 0, 3 and 7 days . Forward charging was done in stress testing and 0 days, 3 days and 7 days DP data was assessed against RMP data of same time point and compared. Results of the study is given in below Table 3.

Table 3: Comparative data of 20 mM Histidine Buffer
P26.1F98D20
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water) CE-SDS
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic Main Peak % % LMW
0D 0.24 99.68 0.08 15.97 9.30 56.63 5.52 12.58 5.54 522 97.90 2.00
3D 0.63 99.09 0.28 13.75 8.79 41.32 11.93 24.23 NA NA 96.70 3.00
7D 0.85 98.52 0.63 18.23 9.53 27.46 18.58 26.19 N`A NA 95.30 4.30
P26.1F99D20
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water) CE-SDS
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic Main Peak % % LMW
0 0.24 99.68 0.08 16.1 9.39 56.89 5.36 12.27 5.53 510 97.90 2.00
3D 0.63 99.09 0.28 13.66 8.62 41.11 12.04 24.57 NA NA 96.70 2.80
7D 0.87 98.47 0.66 18.49 9.62 27.29 18.27 26.31 NA NA 95.40 4.20
P26.1F100D20
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water) CE-SDS
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic Main Peak % % LMW
0D 0.24 99.68 0.08 16.19 9.3 57.34 5.32 11.85 5.55 458 98.10 1.80
3D 0.64 99.08 0.28 13.65 8.71 41.52 12.03 24.08 NA NA 96.70 2.90
7D 0.88 98.49 0.63 18.36 9.94 27.35 17.95 26.39 NA NA 95.50 4.20
P26.1F101D20
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water) CE-SDS
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic Main Peak % % LMW
0D 0.24 99.68 0.09 16.23 9.31 56.99 5.25 12.2 5.55 512 98.10 2.00
3D 0.65 99.05 0.29 13.88 8.79 40.79 12.03 24.5 NA NA 96.80 3.00
7D 0.88 98.46 0.66 18.8 9.56 27.59 18.55 25.49 NA NA 95.50 4.20
P26.1F102D20
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water) CE-SDS
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic Main Peak % % LMW
0D 0.25 99.67 0.08 16.18 9.46 56.83 5.27 12.27 5.51 367 97.90 2.00
3D 0.5 99.24 0.26 13.01 9.11 40.49 12.38 25.01 NA NA 96.80 2.90
7D 0.77 98.64 0.59 16.61 10.48 27.16 19.03 26.7 NA NA 95.60 4.00
B1099B15, RMP
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water) CE-SDS
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic Main Peak % % LMW
0D 0.19 99.76 0.6 9.7 7 65.7 9.0 8.6 NA NA 97.50 2.40
3D 0.35 99.35 0.31 9.78 8.16 44.16 15.76 22.13 NA NA 96.90 2.90
7D 0.53 98.91 0.56 13.14 9.88 28.71 20.67 27.6 NA NA 95.60 4.30
B1097B15, RMP
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water) CE-SDS
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic Main Peak % % LMW
0D 0.2 99.74 0.6 9.4 6.9 64.7 9.4 9.4 NA NA 97.70 2.30
3D 0.36 99.33 0.31 9.9 8.13 43.77 15.97 22.24 NA NA 96.90 3.0
7D 0.56 98.88 0.58 12.73 10.14 28.51 20.61 27.99 NA NA 95.5 4.5

Table 4: Comparative data of 18 mM Acetate Buffer
P26.1F103D21
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water)
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic
0D 0.27 99.66 0.08 16.25 9.49 57.16 5.06 12.03 5.49 510
3D 0.8 98.91 0.29 15.05 8.89 40.03 12.6 23.43 NA NA
7D 1.23 98.08 0.69 20.39 10.38 26.23 17.82 25.19 NA NA
P26.1F104D21
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water)
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic
0 0.27 99.66 0.08 16.15 9.41 56.98 5.27 12.18 5.48 466
3D 0.79 98.92 0.29 14.93 9 40.26 12.42 23.4 NA NA
7D 1.2 98.14 0.67 20.16 10.46 26.48 18.31 24.61 NA NA
P26.1F105D21
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water)
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic
0D 0.26 99.66 0.08 16.48 9.26 56.98 5.25 12.03 5.48 425
3D 0.79 98.93 0.29 14.91 8.97 40.04 12.4 23.67 NA NA
7D 1.2 98.11 0.69 20.3 10.49 26.48 18.25 24.49 NA NA
P26.1F106D21
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water)
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic
0D 0.26 99.66 0.08 16.31 9.28 57.29 4.96 12.16 5.49 480
3D 0.79 98.91 0.3 14.85 8.94 40.09 12.33 23.78 NA NA
7D 1.17 98.13 0.71 20.3 10.35 26.15 18.13 25.06 NA NA

Table 5: Comparative data of 10 mM Succinate Buffer
P26.1F107D22
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water)
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic
0D 0.25 99.67 0.08 16.41 9.46 57.40 4.92 11.81 5.34 480
3D 0.84 98.82 0.34 13.36 8.94 39.07 11.77 26.86 NA NA
7D 1.14 98.05 0.81 16.89 10.3 25.30 17.81 29.7 NA NA
P26.1F108D22
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water)
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic
0 0.25 99.68 0.08 16.43 9.4 57.49 5.01 11.67 5.33 443
3D 0.85 98.82 0.34 13.52 8.96 39.4 11.75 26.37 NA NA
7D 1.17 98.01 0.83 17 10.42 25.63 16.96 29.98 NA NA
P26.1F109D22
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water)
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic
0D 0.26 99.66 0.08 16.35 9.64 57.36 4.91 11.74 5.35 408
3D 0.86 98.8 0.34 13.43 9.14 39.51 11.68 26.24 NA NA
7D 1.17 98.01 0.82 16.96 10.47 25.98 17.93 28.66 NA NA
P26.1F110D22
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water)
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic
0D 0.24 99.68 0.08 16.47 9.41 57.49 5 11.64 5.32 458
3D 0.9 98.73 0.37 13.4 8.89 39.48 11.62 26.61 NA NA
7D 1.17 97.95 0.88 16.85 10.39 26.2 17.82 28.74 NA NA

Table 6: Comparative data of Histidine, Acetate and Succinate buffer with Mannitol
P26.1F111D20
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water) CE-SDS
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic Main Peak % % LMW
0D 0.27 99.64 0.09 16.87 9.4 57.57 5.14 11.0 5.50 430 97.90 2.00
3D 0.51 99.19 0.29 13.62 8.81 40.84 12.39 24.33 NA NA 96.90 2.90
7D 0.8 98.6 0.59 17.23 10.42 27.4 18.8 26.17 NA NA 95.60 4.10
P26.1F112D21
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water) CE-SDS
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic Main Peak % % LMW
0 0.3 99.61 0.09 16.87 9.26 58.08 4.71 11.08 5.44 436 NA NA
3D 0.65 99.04 0.31 15.17 9.58 39.35 13.19 22.72 NA NA NA NA
7D 1.12 98.26 0.62 19.27 11.53 26.49 18.21 24.21 NA NA NA NA
P26.1F113D22
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water) CE-SDS
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic Main Peak % % LMW
0D 0.3 99.61 0.09 16.99 9.15 57.65 5.05 11.13 5.34 400 NA NA
3D 0.7 98.98 0.33 14.06 9.37 38.29 12.3 25.97 NA NA NA NA
7D 1.07 98.16 0.77 17.56 11.27 25.18 18.08 27.92 NA NA NA NA

From the above analytical data and Figure 1 to Figure 4, it was observed that the formulation composition of sorbitol and L-arginine in Histidine buffer shows lower degradation rate than the same composition when used with acetate and succinate buffer. Additionally, the formulation composition trials of histidine buffer with mannitol and L-arginine HCl is showing slightly less degradation (% HMW) in comparison to that of histidine buffer with trehalose. The rate of degradation of formulations of the present invention were found similar compared to the Trastuzumab DS when formulated with innovator formulation composition. For the trial sample containing mannitol and L-arginine HCl. the results showed only a 0.2% difference in high molecular weight (HMW) as compared to the reference material's (RMP) HMW value at day 7 at 50 °C, and the % increase in aggregation was found to be comparable to that of RMP. To visually demonstrate the results, initial batch data, incorporating histidine buffer, acetate buffer, and succinate buffer, were depicted in graphical format. Based on the trend analysis, it was determined that the composition containing histidine buffer, mannitol, and L-arginine HCl demonstrates improved colloidal stability of the molecule in comparison to the innovator formulation. In size exclusion chromatography (CEX) evaluation, Peak 4 was selected for analysis as it exhibits biological activity.
EXAMPLE 3: CONFIRMATORY TRIALS FOR RESULTS OBTAINED IN EXAMPLE 2
To confirm the obtained results in example 2, two additional batches were manufactured one with formulation of present invention and other with innovator formulation composition (Generic DP). After manufacturing, these batches were kept on stability study at 50 ?C and generated data for initial, 3 days and 7 days.
Table 7: Formulation Composition of Lead Buffer
Experiment No. Histidine (mM) Trehalose dihydrate Mannitol Arginine Met Polysorbate 20 Hyaluronidase (2000 IU / mL)
P26.1F114D23 20 mM - 42 5 1.49 1 2000
P26.1F115D23
(Generic DP) 20 mM 79.45 - - 1.49 0.4 2000

Table 8: Analytical results of DP batches
P26.1F114D23
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water)
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic
0D 0.26 99.62 0.11 18.28 10.27 58.09 5.08 8.29 5.53 406
3D 0.59 99.03 0.38 13.56 9.72 40.27 11.59 24.86 NA NA
7D 0.88 98.5 0.61 15.5 10.45 26.75 17.46 29.82 NA NA
P26.1F115D23
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water)
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic
0D 0.26 99.61 0.13 18.24 10.21 58.32 4.97 8.25 5.4 365
3D 0.6 99.02 0.38 13.59 9.67 40.19 11.57 24.98 NA NA
7D 0.84 98.54 0.62 14.88 10.72 26.48 17.52 30.41 NA NA
Lead formulation composition containing mannitol and arginine exhibited similar results in DP quality tests as earlier data in confirmatory trial (Figure 5 & Figure 6). This lead composition was considered for further optimization of desired level of excipients through DoE trials.
EXAMPLE 4: DoE OPTIMIZATION TRIALS
In order to optimize further level of excipients, DoE trials were designed to get the desired level of excipients in order to reduce the % HMW in order to match the degradation profile with RMP. Total nine batches (as shown in Table 9) were manufactured with formulation of the present invention and one batch with innovator formulation composition (Generic DP). After manufacturing, these batches were kept on stability study at 50 °C and analytical data was generated for initial, 3 days and 7 days at 50 °C. The results of the same are presented in Table 10 to Table 13.
Table 9: DoE Formulation Batches
Experiment No. Histidine (mM) Mannitol Arginine HCl Methionine Poly. 20 Hyaluronidase (2000 IU / mL)
P26.1F129D24 20 30 2 1.49 1 2000
P26.1F130D24 20 30 5 1.49 1 2000
P26.1F131D24 20 30 8 1.49 1 2000
P26.1F132D24 20 42 2 1.49 1 2000
P26.1F133D24 20 42 5 1.49 1 2000
P26.1F134D24 20 42 8 1.49 1 2000
P26.1F135D24 20 54 2 1.49 1 2000
P26.1F136D24 20 54 5 1.49 1 2000
P26.1F137D24 20 54 8 1.49 1 2000
P26.1F138D24
Generic DP 20 79.45_Trehalose 0 1.49 0.4 2000
B3007B10- RMP NA

Table 10: Analytical results of DoE Batches containing Mannitol 30 mg/mL
P26.1F129D24
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water)
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic
0D 0.2 99.71 0.07 18 10.25 58.93 4.9 7.92 5.48 284
3D 0.62 99.07 0.31 14.14 9.99 40.33 11.85 23.69 NA NA
7D 1.13 98.27 0.6 17.4 11 26.7 17.2 27.8 NA NA
P26.1F130D24
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water)
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic
0 0.21 99.71 0.07 18.07 10.21 59.03 4.84 7.83 5.49 324
3D 0.64 99.05 0.31 14.2 9.92 40.76 11.86 23.25 NA NA
7D 1.17 98.23 0.61 17.5 10.9 26.8 17.3 27.7 NA NA
P26.1F131D24
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water)
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic
0D 0.21 99.71 0.08 18.05 10.22 58.9 4.86 7.94 5.51 349
3D 0.64 99.07 0.28 14.2 9.83 40.84 11.83 23.29 NA NA
7D 1.17 98.22 0.62 17.5 10.7 27.0 17.3 27.5 NA NA

Table 11: Analytical results of DoE Batches containing Mannitol 42 mg/mL
P26.1F132D24
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water) CE-SDS
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic Main Peak % % LMW
0D 0.21 99.71 0.08 18.02 10.28 58.88 4.9 7.94 5.52 375 98.4 1.5
3D 0.57 99.12 0.32 14.05 9.95 40.67 11.91 23.42 NA NA NA NA
7D 0.98 98.43 0.6 17.1 10.9 26.8 17.2 28.1 NA NA 95.6 4.0
P26.1F133D24
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water) CE-SDS
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic Main Peak % % LMW
0 0.2 99.72 0.07 18.01 10.23 59.02 4.87 7.88 5.53 398 NA NA
3D 0.58 99.1 0.32 14.08 9.9 40.75 11.84 23.41 NA NA NA NA
7D 1.01 98.39 0.61 17.3 10.8 26.9 17.3 27.7 NA NA NA NA
P26.1F134D24
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water) CE-SDS
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic Main Peak % % LMW
0D 0.21 99.71 0.07 18.04 10.25 59.05 4.88 7.78 5.53 432 NA NA
3D 0.61 99.08 0.32 14.11 9.76 40.66 11.87 23.59 NA NA NA NA
7D 1.04 98.35 0.62 17.5 10.7 26.9 17.4 27.7 NA NA NA NA

Table 12: Analytical results of DoE Batches containing Mannitol 54 mg/mL
P26.1F135D24
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water)
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic
0D 0.22 99.71 0.07 18.07 10.24 58.99 4.88 7.83 5.5 419
3D 0.58 99.11 0.31 14.07 10.02 40.4 11.88 23.63 NA NA
7D 1.02 98.38 0.59 17.5 11.1 26.4 17.0 28 NA NA
P26.1F136D24
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water)
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic
0D 0.21 99.71 0.08 18.08 10.18 59.01 4.87 7.88 5.51 451
3D 0.57 99.07 0.36 14.03 9.88 40.69 11.89 23.52 NA NA
7D 1.01 98.38 0.61 17.3 10.9 26.6 17.2 28.1 NA NA
P26.1F137D24
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water)
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic
0D 0.21 99.72 0.07 18.01 10.25 59.05 4.86 7.82 5.53 487
3D 0.61 99.07 0.31 14.12 9.86 40.63 11.87 23.17 NA NA
7D 1.07 98.31 0.63 17.5 10.8 26.7 17.2 27.9 NA NA

Table 13: Analytical results of Innovator Buffer and RMP
P26.1F138D24
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water) CE-SDS
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic Main Peak % % LMW
0D 0.21 99.71 0.07 18.04 10.22 59.05 4.83 7.86 5.48 378 98.4 1.6
3D 0.6 99.09 0.32 14.01 10.09 40.31 11.99 23.6 NA NA NA NA
7D 0.98 98.4 0.61 17.1 11 26.5 17.4 28 NA NA 96.2 3.4
B3007B10_RMP
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm /kg of water) CE-SDS
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic Main Peak % % LMW
0D 0.28 99.63 0.1 10.15 6.5 58.93 13.58 10.83 NA NA 97.7 2.1
3D 0.4 99.32 0.28 10.35 8.39 39.4 17.86 24 NA NA NA NA
7D 0.57 98.82 0.62 13.2 9.9 26.5 20.9 29.4 NA NA 95.9 3.8

From the above analytical data (Table 10, 11, 12 and 13), it was observed that the formulation composition of mannitol and L-arginine at various levels in Histidine buffer does not show any significant impact on drug product quality parameters. As such there is no critical impact of the variation in level of these excipients on % HMW when charged at 50 °C for 7 day.

EXAMPLE 5: CONFIRMATION OF FINAL FORMULATION
Two confirmatory trials were designed and charged on the stability to evaluate the DP quality. Two batches were manufactured with formulation of the present invention and one batch with innovator formulation composition. After manufacturing, these batches were kept on the stability study at 50 °C and generated data for initial, 3 days and 7 days (Table 14). The results of the same are presented in Table 15.
Table 14: Optimized Formulation Buffer Composition
Experiment No. Histidine (mM) Mannitol Arginine Met Polysorbate 20 Hyaluronidase (2000 IU / mL)
P26.1F139D27 20 mM 42 5 1.49 1 2000
P26.1F149D27 20 mM 42 5 1.49 1 2000
P26.1F143D27
Generic DP 20 mM 79.45 0 1.49 0.4 2000

Table 15: Analytical Results of Optimized Formulation Buffer Composition
P26.1F139D27
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm / kg of water) CE-SDS
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic Main Peak % % LMW
0D 0.17 99.73 0.1 18.1 10.08 59.76 4.48 7.59 5.59 418 NA NA
3D 0.48 99.21 0.31 16.13 9.61 41.32 12.91 20.02 NA NA NA NA
7D 0.63 98.85 0.51 20.91 10.71 27.25 18.65 22.48 NA NA NA NA
P26.1F149D27
Time Point Size related impurities by SEC Charge related impurities by CEX pH Osmolality (mOsm / kg of water) CE-SDS
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic Main Peak % % LMW
0D 0.26 99.67 0.7 18.06 10.08 59.84 4.49 7.59 5.63 416 97.1 2.9
3D 0.47 99.24 0.31 15.98 9.68 41.56 13.1 19.68 NA NA NA NA
7D 0.64 98.87 0.5 20.97 10.91 27.23 18.58 22.32 NA NA 94.2 5.8
P26.1F143D27
Time Point Size related impurities by SEC Charge related impurities by CEX pH
Osmolality (mOsm / kg of water) CE-SDS
%HMW % Purity % LMW %Acidic Peak3* Main Peak 3 Peak 4 % Basic Main Peak % % LMW
0D 0.18 99.72 0.11 18.04 10.03 59.9 4.47 7.57 5.52 367 97.2 2.8
3D 0.47 99.25 0.3 15.88 9.86 41.16 13.21 19.87 NA NA NA NA
7D 0.59 98.84 0.46 20.27 11.14 27.08 18.9 22.61 NA NA 94.4 5.6

From the above analytical data, it was observed that the formulation composition of mannitol and L-arginine in Histidine buffer able to control the % HMW in the formulation (Figure 7 and Figure 8). These results were confirmed about the earlier data generated with the same excipient composition level. These batches were additionally kept on the stability study at 40 ?C for 14 days and 28 days and evaluated for quality data. Results from this stressed study is given in below Table 16 and Table 17.

Table 16: SEC data for Stress stability samples

Table 17: CEX data for Stress stability samples

This final formulation demonstrates comparable stability to the reference product.

EXAMPLE 5: STABILITY STUDY OF FINAL FORMULATION
Further evaluation of stability was carried out by preparing three batches of final formulation (P26.1F156D31, P26.1F161D34, P26.1F162D35) and evaluated at different temperature conditions (RT, AT & ST) against RMP formulation batch (B2013B06). Table 18, Table 19 and Table 20 shows RT, AT and ST stability results respectively.

Table 18: RT Stability Data for Final formulation batches (P26.1F156D31, P26.1F161D34, and P26.1F162D35) and RMP (B2013B06).

Figure 9 to Figure 15 represents RT stability study results. As it can be seen by above table and figures, No significant changes were observers between final formulation batches and RMP.
Table 19: AT Stability Data for Final formulation batches (P26.1F156D31, P26.1F161D34, and P26.1F162D35) and RMP (B2013B06).

Figure 16 to Figure 22 represents AT stability study results. As it can be seen by above table and figures, No significant changes were observers between final formulation batches and RMP.
Table 20: ST Stability Data for Final formulation batches (P26.1F156D31, P26.1F161D34, and P26.1F162D35) and RMP (B2013B06)

? = Complies
Figure 23 to Figure 29 represents ST stability study results. As it can be seen by above table and figures, No significant changes were observers between final formulation batches and RMP. ,CLAIMS:We Claim,
1. A high concentrated, stable pharmaceutical formulation of Trastuzumab for subcutaneous injection comprising stabilizer or mixture of stabilizers thereof, selected from the group consisting of: mannitol, methionine and arginine HCl.
2. A high concentrated, stable pharmaceutical formulation comprising:
a) About 50 to 350 mg/ml Trastuzumab;
b) About 1 to 100 mM of a buffering agent;
c) About 1 to 500 mM of a mixture of two or more stabilizers wherein, stabilizers are selected from group consisting of mannitol, methionine and arginine HCl;
d) About 0.1 to 0.5 % of a nonionic surfactant; and
e) An effective amount of at least one hyaluronidase enzyme.
3. The high concentrated, stable pharmaceutical formulation of Trastuzumab according to any of the preceding claim, wherein the Trastuzumab concentration is 100 to 150 mg/ml, 120 ± 18 mg/ml, about 110 mg/ml, about 120 mg/ml, or about 130 mg/ml, respectively.

4. The high concentrated, stable pharmaceutical formulation of Trastuzumab any of the preceding claim, wherein said formulation further comprises 20 mM of histidine and histidine monohydrochloride buffer.

5. The high concentrated, stable pharmaceutical formulation according to claim 6, wherein the formulation comprises about 150 to 300 mM of mannitol; about 5 to 15 mM methionine; and about 1 to 50 mM arginine HCl as stabilizers.

6. The high concentrated, stable pharmaceutical formulation of Trastuzumab any of the preceding claim, wherein said formulation comprises 0.1% of polysorbate 20 as a non-ionic surfactant.

7. The high concentrated, stable pharmaceutical formulation of Trastuzumab any of the preceding claim, wherein said formulation comprises 2000 U/mL hyaluronidase enzyme.

8. A high concentrated, stable pharmaceutical formulation comprising:
a) About 50 to 350 mg/ml of Trastuzumab;
b) About 1 to 100 mM of histidine buffer comprising histidine and histidine HCl providing a pH of 5.5 ± 2.0;
c) About 2.7 to 5.5 % of mannitol; about 0.07 to 0.23 % of methionine; and about 0.02 to 1.7 % of arginine HCl as stabilizers;
d) 0.1 % of polysorbate 20; and
e) 2000 U/ml hyaluronidase enzyme.
9. A high concentrated, stable pharmaceutical formulation comprising:
a) About 120 mg/ml Trastuzumab;
b) About 20 mM of a histidine buffer comprising of histidine and histidine HCl providing a pH of 5.5 ± 2.0;
c) About 165 to 296 mM of mannitol; about 10 mM methionine; and about 9 to 38 mM arginine HCl as stabilizers;
d) 0.1 % of polysorbate 20; and
e) 2000 U/ml hyaluronidase enzyme.
10. The high concentration formulation according to any of the preceding claims, wherein said formulation comprises about 231 mM of Mannitol, 10 mM of methionine, and about 24 mM of arginine HCl as stabilizers.