DESC:RELATED APPLICATIONS
This application is related to Indian Provisional Application IN202421005075 filed on 25th Jan. 2024 and is incorporated herein in its entirety.
FIELD OF THE INVENTION
The present invention relates to a stable liquid high concentration formulation of anti-HER2 antibody and hyaluronidase enzyme for subcutaneous administration.
BACKGROUND OF THE INVENTION
Some breast cancer cells make too many copies of (overexpress) a particular gene known as HER2. The HER2 gene makes a protein known as a HER2 receptor. HER2 receptors are like ears, or antennae, on the surface of all cells. These HER2 receptors receive signals that stimulate the cell to grow and multiply. But breast cancer cells with too many HER2 receptors can pick up too many growth signals and start growing and multiplying too much and too fast. Breast cancer cells that overexpress the HER2 gene are said to be HER2-positive.
Pertuzumab and Trastuzumab are recombinant humanized immunoglobulin (Ig)G1? monoclonal antibodies (MAbs), which target the human epidermal growth factor receptor 2 (HER2, c-erbB2), a transmembrane glycoprotein with intrinsic tyrosine kinase activity. The MAbs bind to distinct HER2 epitopes, subdomains II and IV respectively, without competing, and they have complementary mechanisms for disrupting the HER2 signaling. Both active substances are approved and marketed by Genentech as individual products for IV injection under the names Perjeta (Pertuzumab) and Herceptin (Trastuzumab). Trastuzumab is additionally approved in a SC formulation in combination with hyaluronidase (Herceptin SC line extension). Pertuzumab IV and Trastuzumab IV/SC in combination with chemotherapy is already used clinically in free combination in the “Perjeta treatment regimen” for HER2-posivtive breast cancer. The proposed indication for the fixed-dose combination is therefore in line with currently approved therapeutic indications for patients with HER2-positive early or Metastatic Breast Cancer (EBC/MBC).
The approved Phesgo™ provides a subcutaneous route of administration for Pertuzumab and Trastuzumab over 5–8 minutes, every 3 weeks, thereby significantly shortening the amount of time required for patients to receive these HER2-targeted therapies, and could potentially be administered in a patients' home by a qualified health care provider.
The advantage of combine fixed doses of Trastuzumab and Pertuzumab, plus hyaluronidase, in one vial, can be injected subcutaneously. The loading dose takes about 8 minutes to inject and maintenance dose takes about 5 minutes, so the time to receive each dose is much shorter than getting an intravenous infusion of the drug.
The preparation of high concentration protein cocktail 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 proteins or antibodies 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.
The preparation of highly-concentrated antibody cocktail formulation 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 proteins. During development of a highly concentrated antibody cocktail 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 Pertuzumab and Trastuzumab plus hyaluronidase in accordance with the present invention is increased by the fact that three 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.
WO2011012637 discloses a stable liquid pharmaceutical formulation of Phesgo comprising HER2 antibody, buffering agent, stabilizer, surfactant and hyaluronidase enzyme. More specifically it discloses a formulation comprising Pertuzumab and Trastuzumab, Histidine, Trehalose, Sucrose, Methionine, Polysorbate 80 and hyaluronidase enzyme.
There is a desire to provide such highly concentrated, stable pharmaceutical formulations of Pertuzumab, Trastuzumab and hyaluronidase subcutaneous injection for wide range of patient population around the world. Hence, the present invention provides a novel formulation, which contains a minimal number and/or concentration of excipients.
OBJECTS OF THE INVENTION
The main object of the present invention is to provide a stable high concentration liquid pharmaceutical formulation of anti-HER2 antibody comprising buffer, chelating agent, sugar/stabilizer, surfactant, viscosity reducer or enzyme.
Another object of the present invention is to provide a stable high concentration liquid pharmaceutical formulation of Phesgo comprising: a) histidine/histidine HCl; b) DTPA or EDTA; c) trehalose, mannitol or sucrose; d) polysorbate 20; e) arginine HCl; and f) hyaluronidase.
Another object of the present invention is to provide a stable high concentration liquid pharmaceutical formulation of Phesgo comprising: a) 10-50 mM histidine/histidine HCl as buffer; b) 0-20 mM DTPA or EDTA as chelating agent; c) 10-500 mM trehalose, mannitol or sucrose as sugar/stabilizer; d) 0-2% polysorbate 20 as surfactant; e) 20-60 mM arginine HCl as viscosity reducer; and f) 1000-4000 units/ml hyaluronidase enzyme.
Another object of the present invention is to provide a stable high concentration liquid pharmaceutical formulation of Phesgo comprising: a) 10-50 mM histidine/histidine HCl as buffer; b) 0-10 mg/ml DTPA or EDTA as chelating agent; c) 10-150 mg/ml trehalose, mannitol or sucrose as sugar/stabilizer; d) 0-5 mg/ml polysorbate 20 as surfactant; e) 1-20 mg/ml arginine HCl as viscosity reducer; and f) 1000-4000 units/ml hyaluronidase enzyme.
Another object of the present invention is to provide a stable high concentration liquid pharmaceutical formulation of Phesgo comprising DTPA and/or EDTA as chelating agent.
Another object of the present invention is to provide a surfactant free stable high concentration liquid pharmaceutical formulation of Phesgo.
SUMMARY OF THE INVENTION
The main aspect of the present invention is to provide a stable high concentration liquid pharmaceutical formulation of anti-HER2 antibody comprising buffer, chelating agent, sugar/stabilizer, surfactant, viscosity reducer or enzyme.
Another aspect of the present invention is to provide a stable high concentration liquid pharmaceutical formulation of Phesgo comprising: a) histidine/histidine HCl; b) DTPA or EDTA; c) trehalose, mannitol or sucrose; d) polysorbate 20; e) arginine HCl; and f) hyaluronidase.
Another aspect of the present invention is to provide a stable high concentration liquid pharmaceutical formulation of Phesgo comprising: a) 10-50 mM histidine/histidine HCl as buffer; b) 0-20 mM DTPA or EDTA as chelating agent; c) 10-500 mM trehalose, mannitol or sucrose as sugar/stabilizer; d) 0-2% polysorbate 20 as surfactant; e) 20-60 mM arginine HCl as viscosity reducer; and f) 1000-4000 units/ml hyaluronidase enzyme.
Another aspect of the present invention is to provide a stable high concentration liquid pharmaceutical formulation of Phesgo comprising: a) 10-50 mM histidine/histidine HCl as buffer; b) 0-10 mg/ml DTPA or EDTA as chelating agent; c) 10-150 mg/ml trehalose, mannitol or sucrose as sugar/stabilizer; d) 0-5 mg/ml polysorbate 20 as surfactant; e) 1-20 mg/ml arginine HCl as viscosity reducer; and f) 1000-4000 units/ml hyaluronidase enzyme.
Another aspect of the present invention is to provide a stable high concentration liquid pharmaceutical formulation of Phesgo comprising DTPA and/or EDTA as chelating agent.
Another aspect of the present invention is to provide a surfactant free stable high concentration liquid pharmaceutical formulation of Phesgo.
BRIEF DESCRIPTION OF DRAWING
In order that disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figure with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure wherein:
Figure 1: pH data of Batch 1 to 3 at 25°C ± 2°C
Figure 2: pH data of Batch 3 to 4 at 25°C ± 2°C
Figure 3: Relative Potency of Transt. Batch 1 to 3 at 25°C ± 2°C
Figure 4: Relative Potency of Pert. Batch 1 to 3 at 25°C ± 2°C
Figure 5: Relative Potency of Trast. of Batch 4 to 6 at 25°C ± 2°C
Figure 6: Relative Potency of Pert. of Batch 4 to 6 at 25°C ± 2°C
Figure 7: Enzyme activity of Batch 1 to 3 at 25°C ± 2°C
Figure 8: Enzyme activity of Batch 4 to 6 at 25°C ± 2°C
Figure 9: % HMW of Batch 1 to 3 at 25°C ± 2°C
Figure 10: % HMW of Batch 4 to 6 at 25°C ± 2°C
Figure 11: % LMW of Batch 1 to 3 at 25°C ± 2°C
Figure 12: % LMW of Batch 4 to 6 at 25°C ± 2°C
Figure 13: % HMW of Batch 1 to 3 at 40°C ± 2°C
Figure 14: % HMW of Batch 4 to 6 at 40°C ± 2°C
Figure 15: % LMW of Batch 1 to 3 at 40°C ± 2°C
Figure 16: % LMW of Batch 4 to 6 at 40°C ± 2°C
DETAILED DESCRIPTION OF THE INVENTION
DEFINITION

The following definitions are provided to facilitate understanding of certain terms used throughout the specification.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of particular embodiments, preferred embodiments of compositions, methods and materials are described herein. For the purposes of the present disclosure, the following terms are defined below.
The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.
The words "comprise", "comprises", and "comprising" are to be interpreted inclusively rather than exclusively. The words "consist", "consisting", and its variants, are to be interpreted exclusively, rather than inclusively. While various embodiments in the specification are presented using “comprising” language, under other circumstances, a related embodiment is also intended to be interpreted and described using “consisting of’ or “consisting essentially of language.
The term "about" as used in the present patent specification is meant to specify that the specific value provided may vary to a certain extent, such as e.g. means that variations in the range of ± 10 %, preferably ± 5 %, most preferably ± 2 % are included in the given value.
The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile.
A "stable" formulation is one in which the antibody therein substantially retains its physical stability and/or chemical stability and/or its biological activity upon storage. In one aspect, the formulation substantially retains its physical and chemical stability, as well as its biological activity upon storage. The storage period is generally selected based on the intended shelf-life of the formulation.
A "sterile" formulation is aseptic or free from all living microorganisms and their spores.
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.
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; 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.
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.
A “Chelating agent” herein refers to an agents which reduce the rate of oxidation of proteins by scavenging the free metal ions before they oxidise the proteins. Examples of chelating agent herein includes EDTA, DTPA, penicillamine, trientine and dimercaprol.
An ‘anti-HER2 antibody’ herein refers to Pertuzumab, Trastuzumab, or combination of Pertuzumab and Trastuzumab.
The main embodiment of the present invention is to provide a stable high concentration liquid pharmaceutical formulation of anti-HER2 antibody comprising buffer, chelating agent, sugar/stabilizer, surfactant, viscosity reducer or enzyme.
Another embodiment of the present invention is to provide a stable high concentration liquid pharmaceutical formulation of Phesgo comprising: a) histidine/histidine HCl; b) DTPA or EDTA; c) trehalose, mannitol or sucrose; d) polysorbate 20; e) arginine HCl; and f) hyaluronidase.
Another embodiment of the present invention is to provide a stable high concentration liquid pharmaceutical formulation of Phesgo comprising: a) 10-50 mM histidine/histidine HCl as buffer; b) 0-20 mM DTPA or EDTA as chelating agent; c) 10-500 mM trehalose, mannitol or sucrose as sugar/stabilizer; d) 0-2% polysorbate 20 as surfactant; e) 20-60 mM arginine HCl as viscosity reducer; and f) 1000-4000 units/ml hyaluronidase enzyme.
Another embodiment of the present invention is to provide a stable high concentration liquid pharmaceutical formulation of Phesgo comprising: a) 10-50 mM histidine/histidine HCl as buffer; b) 0-10 mg/ml DTPA or EDTA as chelating agent; c) 10-150 mg/ml Trehalose, mannitol or sucrose as sugar/stabilizer; d) 0-5 mg/ml polysorbate 20 as surfactant; e) 1-20 mg/ml arginine HCl as viscosity reducer; and f) 1000-4000 units/ml hyaluronidase enzyme.
Another embodiment of the present invention is to provide a stable high concentration liquid pharmaceutical formulation of Phesgo comprising DTPA and/or EDTA as chelating agent.
Another embodiment of the present invention is to provide a surfactant free stable high concentration liquid pharmaceutical formulation of Phesgo.
In another embodiment of the present invention, concentration of histidine or histidine HCl is of about 10-200 mM, 20-190 mM, 30-180 mM, 40-170 mM, 50-160 mM, 60-150 mM, 70-140 mM, 80-130 mM, 90-120 mM, or 100-110 mM. More preferably, the concentration of histidine or histidine HCl is about 10-50 mM.
In another embodiment of the present invention, concentration of DTPA or EDTA is of about 0-100 mM, 10-90 mM, 20-80 mM, 30-70 mM, 40-60 mM, or 50-60 mM. More preferably, the concentration of DTPA or EDTA is about 0-20 mM.
In another embodiment of the present invention, concentration of DTPA or EDTA is of about 0-50 mg/ml, 5-45 mg/ml, 10-40 mg/ml, 15-35 mg/ml, 20-30 mg/ml, or 25-30 mg/ml. More preferably, the concentration of DTPA or EDTA is about 0-10 mg/ml.
In another embodiment of the present invention, concentration of mannitol, trehalose, or sucrose is of about 0-600 mM, 10-590 mM, 20-580 mM, 30-570 mM, 40-560 mM, 50-550 mM, 60-540 mM, 70-530 mM, 80-520 mM, 90-510 mM, 100-500 mM, 110-490 mM, 120-480 mM, 130-470 mM, 140-460 mM, 150-450 mM, 160-440 mM, 170-430 mM or 200-300 mM. More preferably, the concentration of Trehalose is 45-200 mM, Mannitol is 100-230mM and Sucrose is 1- 30 mM.
In another embodiment of the present invention, concentration of mannitol, trehalose, or sucrose is of about 0-300 mg/ml, 10-290 mg/ml, 20-280 mg/ml, 30-270 mg/ml, 40-260 mg/ml, 50-250 mg/ml, 60-240 mg/ml, 70-230 mg/ml, 80-220 mg/ml, 90-210 mg/ml, 100-200 mg/ml, 110-190 mg/ml, 120-180 mg/ml,130-170 mg/ml, or 140-160 mg/ml. More preferably, the concentration of Trehalose is 10-100 mg/ml, Mannitol is 10-80 mg/ml and Sucrose is 1- 50 mg/ml.
In another embodiment of the present invention, concentration of polysorbate 20 is of about 0-20%, 1-19%, 2-18%, 3-17%, 4-16%, 5-15%, 6-14%, 7-13%, 8-12%, 9-11%, or 10-11%. More preferably, the concentration of polysorbate 20 is about 0-2%.
In another embodiment of the present invention, concentration of polysorbate 20 is of about 0-10 mg/ml, 1-9 mg/ml, 2-8 mg/ml, 3-7 mg/ml, 4-6 mg/ml, or 5-6 mg/ml. More preferably, the concentration of polysorbate 20 is about 0-5 mg/ml.
In another embodiment of the present invention, concentration of arginine HCl is of about 10-100 mM, 15-95 mM, 20-90 mM, 25-85 mM, 30-80 mM, 35-75 mM, 40-70 mM, 45-65 mM, 50-60 mM, or 55-65 mM. More preferably, the concentration of arginine HCl is about 20-60 mM.
In another embodiment of the present invention, concentration of arginine HCl is of about 1-50 mg/ml, 5-45 mg/ml, 10-40 mg/ml, 15-35 mg/ml, 20-30 mg/ml, or 25-35 mg/ml. More preferably, the concentration of arginine HCl is about 1-20 mg/ml.
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 BUFFER & STABILIZER:
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 final formulation buffer are mentioned in Table 1.
Sr. no mM mg/mL Units/mL
Histidine-Histidine HCL buffer Mannitol Arginine HCl Polysorbate 20 Trehalose DTPA Sucrose EDTA Hyaluronidase
1 20 42 5 1 - - - - -
2 20 - - - - 0.4 - - 2000
3 20 - - - 17.0235 0.4 - - 2000
4 20 - 5 - 17.0235 0.4 - - 2000
5 20 - - 1 17.0235 0.4 - - 2000
6 20 42 5 1 - 0.4 - - 2000
7 20 - - - 17.0235 - - 0.4 2000
8 20 - - - - 0.4 5.1714 - 2000
9 20 18.22 5 - 37.84 0.4 - - 2000
10 20 45.54 5 1 - 0.4 - - 2000
11 20 - 5 - 75.67 0.4 - - 2000
12 25 - - - 94.58 0.4 - - 2000
13 20 - 5 - 66.21 0.4 - - 2000
14 20 - 5 - 66.21 0.1 - - 2000
15 20 - 5 - 66.21 0.05 - - 2000
Table 1: Screening of Formulation Buffers and Other Excipients
STABILITY STUDY PLAN OF FORMULATED COMPOSITION:
The Pertuzumab and Trastuzumab plus hyaluronidase formulation drug product was formulated in above mentioned different buffers and kept on the stress stability (at 40 °C ± 2 °C), accelerated stability (at 25 °C ± 2 °C), real time stability (at 5 °C ± 3 °C) and evaluated for analytical techniques as mentioned in Table 2.
Analytical Test Time Point (Days) for DP
0 D 14 D 28 D
Osmolality X NA NA
pH X NA NA
Protein concentration X NA NA
CEX-HPLC X X X
SEC-HPLC X X X
Relative Potency X NA X
CE-SDS (NR) X X X
Enzyme assay X NA NA
X: Testing time point
NA: Not Applicable
Table 2: Stability Study Plan at 40 °C ± 2 °C
From the above study, formulation comprising Histidine as a buffer, DTPA, Trehalose, Arginine, and Mannitol as stabilizers, Polysorbate 20 and Hyaluronidase shown closer degradation when compared to RMP.
Three different consistency batches BATCH 1 (P78F112Dm), BATCH 2 (P78F116Dm), & BATCH 3 (P78F120Dm) comprising 1200mg of Pertuzumab + 600mg of Trastuzumab and BATCH 4 (P78F113Dm), BATCH 5 (P78F117Dm), & BATCH 6 (P78F121Dm) comprising 600mg of Pertuzumab + 600mg of Trastuzumab were prepared using formulation F-13 given in table 1 to assess stress stability. Further, for comparative purpose innovator’s approved formulation was used as RMP A (B4471B08) & RMP B (B4471B05) comprising 1200mg of Pertuzumab + 600mg of Trastuzumab and RMP C (B4519B07) & RMP D (B4515B05) comprising 600mg of Pertuzumab + 600mg of Trastuzumab.
EXAMPLE 2: ACCELARATED STABILITY STUDY AT 25°C ± 2°C
A) Physical appearance:
All samples were observed to be Clear to opalescent and Colorless to slightly brownish solution
compliance with RMP.
B) pH:
Batch pH
Time Point 0M 1M 2M 3M
RMP A 5.49 - - 5.48
RMP B 5.49 - - 5.46
BATCH 1 5.52 5.58 5.56 5.60
BATCH 2 5.52 5.60 5.57 5.58
BATCH 3 5.50 5.60 5.57 5.57
RMP C 5.50 - - 5.46
RMP D 5.49 - - 5.48
BATCH 4 5.52 5.61 5.56 5.58
BATCH 5 5.52 5.58 5.59 5.56
BATCH 6 5.52 5.60 5.57 5.55
Table 3: pH data at 25°C ± 2°C
Observation: Based on 3 months stability data the pH of all the 6 batches was comparable with the reference formulation (RMP) as depicted in Figure 1 & 2.
C) Protein Concentration:
Batch Protein Concentration (mg/mL)
Time Point 0M 1M 2M 3M
Pert. Trast. Pert. Trast. Pert. Trast. Pert. Trast.
RMP A 75.7 40.6 - - - - 81.3 40.5
RMP B 76.7 40.6 - - - - 80.5 40.1
BATCH 1 82.9 42 86.5 49.8 86.2 40.1 85.1 39.8
BATCH 2 78.5 40.9 84.2 39.9 83.0 39.6 82.6 39.7
BATCH 3 80.1 41.5 82.9 38.6 83.5 39.3 82.2 38.9
RMP C 63.3 60.1 - - - - 63.2 64.2
RMP D 63.1 60.2 - - - - 60.80 61.3
BATCH 4 64.2 62.9 58.9 63.6 59.6 64.0 59.5 63.5
BATCH 5 60.3 58.6 56.0 59.9 58.5 62.1 56.6 59.9
BATCH 6 61.0 62.1 57.9 63.5 58.0 63.1 60.7 65.8s
Table 4: Protein concentration at 25°C ± 2°C
Observation: Based on 3 months stability data the protein concentration of Pertuzumab and Trastuzumab of all the 6 batches was comparable with the reference formulation (RMP) as depicted in Figure 3, 4, 5, & 6.
D) Relative potency:
Batch Relative potency (%) – anti proliferation assay
Time Point 0M 1M 2M 3M
Pert. Trast. Pert. Trast. Pert. Trast. Pert. Trast.
RMP A 99 109 - - - - 96 120
RMP B 101 105 - - - - 88 116
BATCH 1 90 97 99 99 78 99 86 104
BATCH 2 94 98 94 95 88 102 82 108
BATCH 3 93 96 89 100 79 97 76 88
RMP C 97 106 - - - - 89 112
RMP D 108 102 - - - - 88 113
BATCH 4 94 100 91 102 84 100 82 100
BATCH 5 98 104 92 112 80 96 89 104
BATCH 6 102 102 93 107 84 106 84 113
Table 5: Relative potency at 25°C ± 2°C
E) Enzyme activity:
Batch Enzyme activity
Time Point 0M 1M 2M 3M
RMP A 2035 - - 2219
RMP B 2261 - - 2203
BATCH 1 1981 2219 2003 1992
BATCH 2 2298 2034 1942 2196
BATCH 3 2367 2036 1941 1942
RMP C 2334 - - 2458
RMP D 2104 - - 2431
BATCH 4 1920 2096 2188 2342
BATCH 5 1730 1180 2147 2224
BATCH 6 1898 2003 2097 2189
Table 6: Enzyme activity at 25°C ± 2°C
F) SE-HPLC
Batch Main peak (%) HMW (%)
Time Point 0M 1M 2M 3M 0M 1M 2M 3M
RMP A 99.54 - - 98.16 0.37 - - 0.48
RMP B 99.54 - - 92.24 0.37 - - 0.44
BATCH 1 99.79 99.63 99.56 98.48 0.16 0.19 0.25 0.28
BATCH 2 99.72 99.62 99.54 98.53 0.22 0.21 0.27 0.31
BATCH 3 99.77 99.67 99.6 98.5 0.17 0.16 0.21 0.24
RMP C 99.56 - - 98.15 0.35 - - 0.47
RMP D 99.52 - - 98.54 0.38 - - 0.5
BATCH 4 99.78 99.6 99.53 98.48 0.18 0.22 0.28 0.32
BATCH 5 99.7 99.62 99.54 99.43 0.23 0.22 0.27 0.32
BATCH 6 99.8 99.65 99.58 98.46 0.16 0.17 0.22 0.26
Table 7: SE-HPLC data at 25°C ± 2°C
Observation: Based on 3 months Stress stability data the SE- HPLC of all the 6 batches was comparable with the reference formulation (RMP) as depicted in Figure 9 & 10.
G) CEX:
Batch Before Pert. Main peak (%) Pert. Main peak (%) Between Pert. & Trast. Main peak (%)
Time Point 0M 1M 2M 3M 0M 1M 2M 3M 0M 1M 2M 3M
RMP A 11.5 - - 16.16 45.24 - - 42.45 8.07 - - 9.84
RMP B 11.26 - - 16.23 45.02 - - 42.37 8.3 - - 9.79
BATCH 1 7.82 9.61 11.47 13.33 56.99 55.84 52.79 51.16 11.81 10.87 11.17 11.53
BATCH 2 9.09 11.41 12.71 14.09 53.50 51.98 50.74 49.68 11.1 10.26 10.61 10.99
BATCH 3 6.96 8.89 10.33 12.46 56.98 55.04 51.1 51.06 10.37 11.06 11.43 11.27
RMP C 8.78 - - 12.38 34.54 - - 33.03 9.07 - - 10.71
RMP D 7.99 - - 12.68 36.52 - - 33.08 8.37 - - 10.71
BATCH 4 7.0 8.18 8.89 10.28 42.29 41.87 40.63 40.11 15.66 13.47 13.45 13.92
BATCH 5 7.82 8.45 9.5 10.81 40.25 40.62 39.64 38.75 13.92 12.19 12.59 13.0
BATCH 6 6.0 5.48 8.93 10.19 43.45 46.23 40.88 40.06 13.12 10.94 12.52 13.0
Table 8 (a): CEX data at 25°C ± 2°C
Batch Trast. Main peak (%) After Trast. Main peak (%) Purity (%)
Time Point 0M 1M 2M 3M 0M 1M 2M 3M 0M 1M 2M 3M
RMP A 28.17 - - 18.13 7.02 - - 13.43 73.41 - - 60.58
RMP B 28.0 - - 18.10 7.42 - - 13.49 73.02 - - 60.47
BATCH 1 20.82 18.15 15.78 13.45 2.56 6.53 8.79 10.52 77.81 72.99 68.57 64.61
BATCH 2 22.65 19.65 16.89 14.43 3.68 6.70 9.03 10.81 76.2 71.63 67.63 64.11
BATCH 3 21.78 19.02 16.05 13.83 3.92 6.01 11.07 11.37 78.75 74.06 67.15 64.89
RMP C 37.16 - - 23.27 10.45 - - 20.32 71.7 - - 56.30
RMP D 38.49 - - 22.65 8.64 - - 20.89 75.01 - - 55.73
BATCH 4 30.09 26.52 22.97 19.74 4.94 9.93 14.05 15.96 72.38 68.39 63.6 59.85
BATCH 5 32.63 29.03 24.89 21.1 5.37 9.7 13.38 16.36 72.88 69.65 64.53 59.85
BATCH 6 30.64 29.14 23.3 19.89 6.79 8.21 14.38 16.85 74.09 75.37 64.18 59.95
Table 8 (b): CEX data at 25°C ± 2°C
Observation: Based on 3 months Stress stability data the CEX of all the 6 batches was comparable with the reference formulation (RMP).
H) CE-SDS:

Batch Main peak (%) LMW (%)
Time Point 0M 1M 2M 3M 0M 1M 2M 3M
RMP A 97.2 - - 96.2 2.4 - - 3.5
RMP B 97.1 - - 96.4 2.8 - - 3.3
BATCH 1 98.6 98.60 97.90 97.70 1.5 1.30 2.10 2.30
BATCH 2 98.5 98.50 97.90 97.50 1.5 1.40 1.90 2.40
BATCH 3 98.6 98.60 98.10 97.70 1.2 1.40 1.90 2.20
RMP C 97.10 - - 96.4 2.5 - - 3.4
RMP D 97.6 - - 96.5 2.4 - - 3.2
BATCH 4 98.6 98.50 98.00 97.60 1.6 1.50 1.90 2.40
BATCH 5 98.5 98.30 97.90 97.40 1.4 1.70 2.10 2.50
BATCH 6 98.7 98.40 98.10 97.80 1.1 1.40 1.80 2.10
Table 9: CE-SDS data at 25°C ± 2°C
Observation: Based on 3 months Stress stability data the CE-SDS of all the 6 batches was comparable with the reference formulation (RMP) as depicted in Figure 11 & 12.
EXAMPLE 3: SRTRESSED STABILITY STUDY AT 40°C ± 2°C
A) Physical appearance:
All samples were observed to be Clear to opalescent and Colorless to slightly brownish solution
compliance with RMP.
B) pH:
Batch pH
Time Point 0M 7D 14D 28D
RMP A 5.49 5.52 5.52 5.50
RMP B 5.49 5.52 5.52 5.53
BATCH 1 5.52 5.58 5.59 5.60
BATCH 2 5.52 5.58 5.60 5.60
BATCH 3 5.50 5.58 5.59 5.61
RMP C 5.50 5.51 5.51 5.51
RMP D 5.49 5.51 5.51 5.52
BATCH 4 5.52 5.58 5.60 5.60
BATCH 5 5.52 5.59 5.59 5.60
BATCH 6 5.52 5.58 5.59 5.59
Table 10: pH of all batches at 40°C ± 2°C
Observation: Based on 3 months stability data the pH of all the 6 batches was comparable with the reference formulation (RMP).
C) Protein Concentration:
Batch Protein Concentration (mg/mL)
Time Point 0M 7D 14D 28D
Pert. Trast. Pert. Trast. Pert. Trast. Pert. Trast.
RMP A 72.70 40.60 79.60 39.20 80.0 39.80 82.10 41.30
RMP B 76.70 40.60 79.60 39.20 80.30 38.50 81.60 41.10
BATCH 1 82.90 42.0 85.20 39.4 82.50 38.4 84.3 40.0
BATCH 2 78.5 40.9 81.6 38.8 79.8 38.1 78.9 38.4
BATCH 3 80.1 41.5 81.7 38. 82.0 38.6 80.0 38.5
RMP C 60.1 63.3 60.2 58.2 59.4 57.7 61.9 60.5
RMP D 60.2 63.1 60.6 59.3 60.0 59.1 61.7 61.1
BATCH 4 62.9 64.2 63.2 57.5 61.7 57.6 60.3 56.8
BATCH 5 58.6 60.3 58.3 55.0 58.8 54.8 59.9 55.8
BATCH 6 62.1 61.0 62.8 57.4 32.0 29.6 62.4 57.8
Table 11: Protein concentration at 40°C ± 2°C

D) Relative potency:
Batch Relative potency (%) – anti proliferation assay
Time Point 0M 7D 14D 28D
Pert. Trast. Pert. Trast. Pert. Trast. Pert. Trast.
RMP A 99 109 - - 90 - 75 120
RMP B 101 105 - - 94 - 68 109
BATCH 1 90 97 - - 83 - 65 111
BATCH 2 94 98 - - 82 - 68 118
BATCH 3 93 96 - - 81 - 69 111
RMP C 97 106 - - 87 - 85 102
RMP D 108 102 - - 96 - 81 107
BATCH 4 94 100 - - 78 - 75 91
BATCH 5 98 104 - - 82 - 76 100
BATCH 6 102 102 - - 81 - 84 107
Table 12: Relative potency at 40°C ± 2°C
E) Enzyme activity:
Batch Enzyme activity
Time Point 0M 7D 14D 28D
RMP A 2035 Out of Std. range
RMP B 2261 Out of std. range
BATCH 1 1981 1549 1452 1270
BATCH 2 2298 1358 1428 1337
BATCH 3 2267 1596 1469 1306
RMP C 2334 Out of Std. range
RMP D 2104 Out of Std. range
BATCH 4 1920 1505 1513 1215
BATCH 5 1730 1418 1430 1276
BATCH 6 1898 1495 1599 1296
Table 13: Enzyme activity at 40°C ± 2°C
F) SE-HPLC
Batch Main peak (%) HMW (%)
Time Point 0M 7D 14D 28D 0M 7D 14D 28D
RMP A 99.54 99.40 97.43 96.19 0.37 0.42 0.53 0.67
RMP B 99.54 99.40 97.37 95.50 0.37 0.42 0.53 0.67
BATCH 1 99.79 99.59 97.77 96.71 0.16 0.23 0.28 0.41
BATCH 2 99.72 99.58 97.75 96.64 0.22 0.25 0.30 0.43
BATCH 3 99.77 99.64 97.78 96.68 0.17 0.18 0.26 0.38
RMP C 99.56 99.40 97.39 96.40 0.35 0.41 0.53 0.67
RMP D 99.52 99.33 97.35 96.29 0.38 0.47 0.57 0.70
BATCH 4 99.78 99.55 97.74 96.73 0.18 0.27 0.31 0.45
BATCH 5 99.70 99.59 97.81 96.72 0.23 0.25 0.30 0.38
BATCH 6 99.80 99.60 97.74 96.65 0.16 0.22 0.28 0.41
Table 14: SE-HPLC data at 40°C ± 2°C
Observation: Based on 3 months Stress stability data the SEC- HPLC of all the 6 batches was comparable with the reference formulation (RMP) as depicted in Figure 13 & 14.
G) CEX:
Batch Before Pert. Main peak (%) Pert. Main peak (%) Between Pert. & Trast. Main peak (%)
Time Point 0M 7D 14D 28D 0M 7D 14D 28D 0M 7D 14D 28D
RMP A 11.50 15.11 17.59 22.01 45.24 44.06 42.31 36.23 8.07 8.64 10.19 12.61
RMP B 11.26 14.98 17.59 22.02 45.02 44.10 42.37 36.00 8.30 8.61 10.21 12.71
BATCH 1 7.82 11.91 15.64 21.05 56.99 53.09 50.02 42.81 11.81 11.26 12.17 14.15
BATCH 2 9.09 12.92 16.35 21.67 53.50 51.26 48.12 40.95 11.10 10.53 12.03 14.14
BATCH 3 6.96 10.88 15.05 19.95 56.98 53.48 50.22 42.75 10.37 10.68 11.74 14.28
RMP C 8.78 10.15 12.54 17.79 34.54 35.42 33.87 28.24 9.07 9.40 11.25 14.39
RMP D 7.99 10.44 12.89 17.98 36.52 35.65 34.59 28.16 8.37 9.43 11.13 14.19
BATCH 4 7.00 9.18 12.04 16.17 42.29 41.63 39.74 33.97 15.66 13.23 14.59 16.69
BATCH 5 7.82 9.80 12.60 16.54 40.25 40.10 38.42 32.96 13.92 12.19 13.89 15.97
BATCH 6 6.00 10.36 11.68 15.94 43.45 40.02 40.64 33.95 13.12 12.90 13.62 16.26
Table 15 (a): CEX data at 40°C ± 2°C
Batch Trast. Main peak (%) After Trast. Main peak (%) Purity (%)
Time Point 0M 7D 14D 28D 0M 7D 14D 28D 0M 7D 14D 28D
RMP A 28.17 22.78 18.68 13.45 7.02 9.44 11.20 15.70 73.41 66.84 60.99 49.68
RMP B 28.00 22.86 18.65 13.40 7.42 9.46 11.17 15.85 73.02 66.96 61.02 49.40
BATCH 1 20.82 16.88 13.53 9.57 2.56 6.85 8.62 12.43 77.81 69.97 63.55 52.38
BATCH 2 22.62 18.26 14.45 9.93 3.68 6.97 9.05 13.30 76.12 69.55 62.57 50.88
BATCH 3 21.78 17.57 13.82 9.76 3.92 7.39 9.16 13.27 78.75 71.05 64.04 52.51
RMP C 37.16 30.90 24.69 16.54 10.45 14.12 17.65 23.03 71.70 66.32 58.56 44.78
RMP D 38.49 30.27 24.41 16.27 8.64 14.22 16.96 23.39 75.01 65.92 59.00 44.43
BATCH 4 30.09 25.51 20.22 13.77 4.94 10.47 13.42 19.38 72.38 67.14 59.96 47.74
BATCH 5 32.63 27.38 21.53 14.73 5.37 10.54 13.54 19.79 72.88 67.48 59.95 47.69
BATCH 6 30.64 25.06 20.47 13.98 6.79 11.63 13.57 19.88 74.09 65.08 61.11 47.93
Table 15 (b): CEX data at 40°C ± 2°C
Observation: Based on 3 months Stress stability data the CEX of all the 6 batches was comparable with the reference formulation (RMP).

H) CE-SDS:
Batch Main peak (%) LMW (%)
Time Point 0M 7D 14D 28D 0M 7D 14D 28D
RMP A 97.20 95.80 95.80 94.70 2.40 3.90 3.90 5.10
RMP B 97.10 95.80 95.80 95.10 2.80 3.90 3.90 4.50
BATCH 1 98.60 97.80 97.10 96.10 1.50 2.10 2.80 3.70
BATCH 2 98.50 97.80 97.30 96.30 1.50 2.20 2.70 3.50
BATCH 3 98.60 97.30 97.30 96.40 1.20 2.50 2.50 3.50
RMP C 97.10 96.00 96.00 95.10 2.50 3.80 3.80 4.60
RMP D 97.60 96.20 96.20 95.10 2.40 3.70 3.70 4.60
BATCH 4 98.50 97.70 97.10 96.40 1.60 2.30 2.80 3.40
BATCH 5 98.50 97.70 97.20 96.30 1.40 2.20 2.70 3.40
BATCH 6 98.70 97.50 97.50 96.40 1.10 2.30 2.30 3.30
Table 16: CE-SDS data at 40°C ± 2°C
Observation: Based on 3 months Stress stability data the CE-SDS of all the 6 batches was comparable with the reference formulation (RMP) as depicted in Figure 15 & 16.
EXAMPLE 4: REAL TIME STABILITY STUDY AT 5°C ± 3°C
A) Physical appearance:
All samples were observed to be Clear to opalescent and Colorless to slightly brownish solution compliance with RMP.
B) pH:
Batch pH
Time Point 0M 1M 2M 3M
BATCH 1 5.52 5.60 5.60 5.56
BATCH 2 5.52 5.60 5.55 5.56
BATCH 3 5.50 5.60 5.55 5.59
BATCH 4 5.52 5.59 5.59 5.55
BATCH 5 5.52 5.56 5.57 5.56
BATCH 6 5.52 5.56 5.57 5.56
Table 17: pH of all batches at 5°C ± 3°C
C) Protein Concentration:
Batch Protein Concentration (mg/mL)
Time Point 0M 1M 2M 3M
Pert. Trast. Pert. Trast. Pert. Trast. Pert. Trast.
BATCH 1 82.90 42.0 84.7 38.8 84.5 38.9 84.2 38.7
BATCH 2 78.5 40.9 83.40 39.3 83.0 39.3 81.0 38.3
BATCH 3 80.1 41.5 83.7 38.9 82.2 38.3 82.8 38.6
BATCH 4 64.2 62.9 58.2 62.9 59.4 64.0 58.0 62.4
BATCH 5 60.3 58.6 54.1 62.8 58.2 61.6 56.3 60.1
BATCH 6 61.0 62.1 57.9 63.6 58.4 63.8 57.3 62.6
Table 18: Protein concentration at 5°C ± 3°C
D) Relative potency:
Batch Relative potency (%) – anti proliferation assay
Time Point 0M 1M 2M 3M
Pert. Trast. Pert. Trast. Pert. Trast. Pert. Trast.
BATCH 1 90 97 89 96 92 99 93 103
BATCH 2 94 98 106 94 97 103 96 109
BATCH 3 93 96 102 93 92 105 90 96
BATCH 4 94 100 89 106 92 98 99 125
BATCH 5 98 104 117 110 93 115 103 104
BATCH 6 102 102 107 105 93 97 93 104
Table 19: Relative potency at 5°C ± 3°C
E) Enzyme activity:
Batch Enzyme activity
Time Point 0M 1M 2M 3M
BATCH 1 1981 2090 2129 2281
BATCH 2 2298 1916 1969 1709
BATCH 3 2367 1873 2023 1809
BATCH 4 1920 1926 1955 2213
BATCH 5 1730 1970 1919 2153
BATCH 6 1898 1954 1812 2262
Table 20: Enzyme activity at 5°C ± 3°C
F) SE-HPLC
Batch Main peak (%) HMW (%)
Time Point 0M 1M 2M 3M 0M 1M 2M 3M
BATCH 1 99.79 99.75 99.77 99.75 0.16 0.14 0.17 0.18
BATCH 2 99.72 99.74 99.76 99.74 0.22 0.16 0.18 0.20
BATCH 3 99.77 99.78 99.80 99.78 0.17 0.11 0.14 0.15
BATCH 4 99.78 99.73 99.75 99.73 0.18 0.16 0.19 0.20
BATCH 5 99.70 99.74 99.76 99.75 0.23 0.16 0.18 0.20
BATCH 6 99.80 99.70 99.78 99.78 0.16 0.13 0.15 0.16
Table 21: SE-HPLC data at 5°C ± 3°C
G) CEX:
Batch Before Pert. Main peak (%) Pert. Main peak (%) Between Pert. & Trast. Main peak (%)
Time Point 0M 1M 2M 3M 0M 1M 2M 3M 0M 1M 2M 3M
BATCH 1 7.82 8.90 8.81 8.55 56.99 55.88 56.00 56.30 11.81 11.62 11.42 11.35
BATCH 2 9.09 10.04 10.01 10.10 53.50 53.60 53.52 53.75 11.10 10.60 10.60 10.20
BATCH 3 6.96 8.02 8.01 7.88 56.98 55.84 55.67 56.14 10.37 10.42 10.53 10.39
BATCH 4 7.00 6.92 7.42 7.19 42.29 43.21 42.78 43.04 15.66 14.75 14.84 14.34
BATCH 5 7.82 8.17 8.08 7.94 40.25 41.15 41.02 41.29 13.92 13.09 13.23 12.92
BATCH 6 6.00 6.70 6.81 6.57 43.45 43.23 43.04 43.44 13.12 12.78 12.95 12.52
Table 22 (a): CEX data at 5°C ± 3°C
Batch Trast. Main peak (%) After Trast. Main peak (%) Purity (%)
Time Point 0M 1M 2M 3M 0M 1M 2M 3M 0M 1M 2M 3M
BATCH 1 20.82 20.05 20.45 20.29 2.56 3.56 3.32 3.52 77.81 75.93 76.45 76.59
BATCH 2 22.62 22.38 22.66 22.52 3.68 3.38 3.19 3.44 76.12 75.98 76.18 76.27
BATCH 3 21.78 21.37 21.37 21.34 3.92 4.34 4.41 4.23 78.75 77.21 77.04 77.48
BATCH 4 30.09 30.08 29.97 29.96 4.94 5.04 5.01 5.48 72.38 73.29 72.75 73.00
BATCH 5 32.63 32.84 32.90 32.96 5.37 4.76 4.78 4.87 72.88 73.99 73.92 74.25
BATCH 6 30.64 31.13 31.14 31.06 6.79 6.16 6.05 6.42 74.09 74.36 74.18 74.50
Table 22 (b): CEX data at 5°C ± 3°C
H) CE-SDS:
Batch Main peak (%) LMW (%)
Time Point 0M 1M 2M 3M 0M 1M 2M 3M
BATCH 1 98.60 98.90 98.40 98.50 1.50 1.20 1.50 1.50
BATCH 2 98.50 98.60 98.60 98.60 1.50 1.30 1.20 1.40
BATCH 3 98.60 98.90 98.60 98.60 1.20 1.20 1.30 1.40
BATCH 4 98.50 98.60 98.50 98.40 1.60 1.40 1.50 1.60
BATCH 5 98.50 98.60 98.60 98.60 1.40 1.40 1.20 1.50
BATCH 6 98.70 98.80 98.70 98.70 1.10 1.20 1.40 1.30
Table 23: CE-SDS data at 5°C ± 3°C
Based on the stability data provided above it shows that there is no change in terms of protein concentration /stability between formulation of present invention and RMP formulation. This means that formulation of the present invention is highly comparable with RMP formulation. ,CLAIMS:We Claim,
1. A stable high concentration liquid pharmaceutical formulation of Phesgo comprising buffer, chelating agent, sugar/stabilizer, surfactant, viscosity reducer or enzyme.
2. The stable high concentration liquid pharmaceutical formulation of claim 1, wherein the formulation comprising: a) histidine/histidine HCl; b) DTPA or EDTA; c) trehalose, mannitol or sucrose; d) polysorbate 20; e) arginine HCl; and f) hyaluronidase.
3. A stable high concentration liquid pharmaceutical formulation of Phesgo comprising: a) 10-50 mM histidine/histidine HCl as buffer; b) 0-10 mg/ml DTPA or EDTA as chelating agent; c) 10-150 mg/ml trehalose, mannitol or sucrose as sugar/stabilizer; d) 0-5 mg/ml polysorbate 20 as surfactant; e) 1-20 mg/ml arginine HCl as viscosity reducer; and f) 1000-4000 units/ml hyaluronidase enzyme.
4. The stable high concentration liquid pharmaceutical formulation of claim 3, wherein the formulation of Phesgo comprising: a) 20-25 mM histidine/histidine HCl; b) 0.1-1mM DTPA or 1.37mM EDTA; c) 45-200 mM trehalose, 100-230mM mannitol or 15mM sucrose; d) 0-0.1% polysorbate 20; e) 23.73mM arginine HCl; and f) 2000units/ml hyaluronidase enzyme.
5. The stable high concentration liquid pharmaceutical formulation of any of preceding claims, wherein formulation is free of surfactant.
6. A stable liquid high concentration pharmaceutical formulation of Phesgo comprising histidine/histidine HCl, arginine HCl, DTPA, Trehalose, and hyaluronidase enzyme.
7. The stable high concentration pharmaceutical formulation of claim 6, wherein the formulation comprising: a) 20mM histidine/histidine HCl; b) 0.1-1 mM DTPA; c) 175 mM trehalose; d) 23.73mM arginine HCl; and e) 2000 units/ml hyaluronidase enzyme.