A method for the production of recombinant fusion protein

Abstract

The present disclosure relates to a method for high yield expression of recombinant fusion proteins and a system thereof. The invention encompasses the development and utilization of a novel expression vector construct that significantly enhances the expression and yield of the protein of interest. The expression vector construct encodes nucleotide sequence fusion protein and other regulatory elements. Further, the expression vector construct provides high expression more than 2 gm / L with desired quality of fusion protein.

Description

[0001] A METHOD FOR THE PRODUCTION OF RECOMBINANT FUSION PROTEIN

[0002] FIELD OF INVENTION

[0003] The present invention relates to a method for high yield expression of recombinant fusion proteins and a system to express the fusion protein. This invention encompasses the development and utilization of a novel expression vector construct that significantly enhances the expression and yield of the protein of interest. The invention provides a novel upstream process for the production of a recombinant fusion protein.

[0004] BACKGROUND OF THE INVENTION

[0005] Lifestyle diseases and metabolic disorders have become a significant global health concern, affecting millions of people worldwide. These diseases, include conditions such as diabetes, obesity, cardiovascular diseases, and hypertension, are primarily caused by unhealthy lifestyle choices such as poor diet, lack of physical activity, and excessive stress. According to the World Health Organization (WHO), non-communicable diseases (NCDs) account for approximately 71% of all deaths globally, with cardiovascular diseases alone responsible for 17.9 million deaths each year. Diabetes, a major metabolic disorder, affects over 422 million people worldwide and is a leading cause of blindness, kidney failure, heart attacks, and lower limb amputations. The prevalence of obesity has nearly tripled since 1975, with over 1.9 billion adults classified as overweight, and of these, more than 650 million are obese. These statistics highlight the urgent need for effective prevention and management strategies to combat the growing burden of lifestyle and metabolic diseases on humankind.

[0006] The biopharmaceuticals market size stood at $389.3 billion in 2021, and it is expected to grow at a CAGR of 7.1% during 2021-2030, to reach $720.8 billion by 2030. This increased demand of biologicals is primarily due to increasing global prevalence of chronic diseases, such as diabetes, cancer, and cardiovascular diseases. Recombinant DNA technology is nowadays in routine practice to produce biologicals to achieve products of high purity, and well-defined safety and physicochemical characteristics in comparison to extraction of products from tissues or body fluids.

[0007] Moreover, the prevalence of metabolic disorders and lifestyle diseases such as diabetes, obesity, amongst others is rapidly growing, non-discriminatory disorders that affects people of all ages and ethnicities. Pharmacological treatment of such disorders including diabetes, obesity have changed dramatically over the past few years, with new classes of drugs now available. Many of the medications used in the treatment of these disorders can be used in combination with various other oral therapies to achieve better glycemic control. Some can be given once weekly, an option that provides an avenue to limit polypharmacy and noncompliance, which otherwise may not have been achieved with daily monotherapy.

[0008] Amongst metabolic disorders, such as diabetes type-2 diabetes, or non-insulin-dependent diabetes, or adult-onset diabetes, or maturity-onset diabetes is the most common type of diabetes, and patients commonly rely on both oral and injectable agents to control it. With respect to the injectable agents, Glucagon-like peptide-1 (GLP-1) agonists are growing in popularity. GLP-1 agonists, also known as “incretin mimetics,” increases insulin secretion, decreases glucagon secretion, slows gastric emptying, improve satiety, and may result in weight loss (Dulagluttde (Trulicity): The Third Once-Weekly GLP-1 Agonist. P T. 2016 Jun;41(6):357-60. PMID: 27313432; PMCID: PMC4894510).

[0009] In recent years, the field of expression vectors for the expression of desired proteins has seen significant advancements to cure such disorders / diseases. Researchers have been focusing on developing robust and efficient expression vector constructs that can achieve high expression levels and yields of proteins in mammalian cells. These vectors often incorporate hybrid promoters, signal peptides, and other vector elements to optimize protein expression. Additionally, the use of chemically defined and serum-free media (Bandaranayake and Almo 2014; Kim et al. 2012; Kuystermans and Al-Rubeai 2015a; Spearman and Butler 2015) has become more prevalent, allowing for better control over the cell culture environment and reducing the risk of contamination. However, despite these advancements, there are still several challenges and drawbacks. One major issue is the variability in protein expression levels, which can be influenced by factors such as vector design, host cell line, and culture conditions. Another challenge is the production of proteins with desired quality attributes, such as proper glycosylation patterns and minimal impurities. Furthermore, the scalability of the production process and the cost of manufacturing remain significant hurdles for the biopharmaceutical industry.

[0010] Therefore, providing a method and an appropriate expression vector construct or vector for gene / protein expression and quality is essential. There is a constant need to enhance and develop robust vectors that optimize protein expression in mammalian cells with effective method of its expression.

[0011] Accordingly, it is an object of the invention to provide a method for expressing the recombinant fusion protein having an expression vector construct that significantly enhances the expression and yield of the protein of interest. The method involves the preparation of an expression vector construct capable of expressing the fusion protein or polypeptide, wherein the expression vector encoding a protein / gene of interest is incorporated into the vector backbone and further transfected into suitable host cells, preferably mammalian cells, resulting in higher expression with desired quality of protein. It is also an object of the present invention to develop an expression vector construct that ensures expression of high yield and desired quality of protein of interest, specifically Glucagon-like peptide-l / IgG4 fusion protein.

[0012] SUMMARY OF THE INVENTION

[0013] An embodiment of the present invention provides a method for high expression of recombinant fusion protein of interest, using an expression vector construct.

[0014] In an embodiment, the invention provides a stable expression vector construct having suitable optimized elements ensuring efficient transcription, translation, long term cell viability that enhances genomic integration.

[0015] The present invention provides method of high expression of recombinant protein of interest through an expression vector construct capable of expressing protein or polypeptide wherein the expression vector encoding a gene of interest is incorporated into vector backbone and further transfected into suitable host cell, preferably mammalian cells resulting in higher expression and yield with desired quality of protein.

[0016] In an embodiment, the invention provides a method for expressing recombinant fusion protein of interest comprising: i. Providing a suitable expression vector construct comprising; a. a hybrid promoter or a functional variant; b. a secretory signal peptide system operably linked to a recombinant fusion protein of interest; c. a gene sequence encoding the protein of interest; d. a dual selection system having antibiotic resistance and glutamine synthetase (GS) markers; e. an origin of replication; and f. a termination sequence comprising an SV40 polyadenylation sequence or a functional variant thereof; ii. transfecting the expression vector construct into a mammalian host cell to form a clone capable of expressing the protein of interest; iii. culturing the mammalian host cell comprising the expression vector construct, through cell culture methods selected from fed-batch mode, perfusion culture for at least more than 12 Days, to up to 16 Days in a suitable medium; wherein the cell culture method has first pH and second pH, wherein the second pH is at least two log lower than first pH; wherein the cell culture method maintains a temperature selected from about 32°C to about 36 °C; wherein the culture method maintains osmolality at about 300 Osm / kg to 470 Osm / kg; wherein the cell culture method maintains residual glucose less than 5gm / L; wherein the cell culture method maintains glutamine ImM to 6mM; wherein the cell culture method maintains glutamate ImM to 7mM; wherein the cell culture method maintains cell viability at least 40 million cells / ml to 50 million cells / ml; wherein the cell culture method reduces the oxidation rate to form a oxidized species of the protein of interest by at least 30% from Day 10 till harvest; iv. harvesting and purifying the recombinant fusion protein; wherein the expression vector construct directs the expression of recombinant fusion protein of interest in the mammalian host cell at yield of at least or more than 1 gm / L, to 3 gm / L; wherein the harvested recombinant fusion protein of interest is GLPl-IgG4 fusion protein comprising at least less than about 25% basic variants, at least less than about 30% acidic variants, less than about 2% high molecular weight aggregates, and predominantly higher monomer of GLPl-IgG4 fusion protein.

[0017] In one embodiment, method of claim 1, wherein the expression vector construct is transfected into a microbial host cell; selecting and isolating expression vector construct from the microbial host cell.

[0018] In one embodiment, wherein mammalian host cell capable to express protein of interest is selected and isolated by applying a dual selection system.

[0019] In one embodiment, the cell culture method is performed through fed- batch mode.

[0020] In one embodiment, the cell culture method is performed through fed- batch mode for at least 16 Days.

[0021] In an embodiment, the mammalian host cell is cultured through fed batch mode for at least 12 Days.

[0022] In one embodiment the titre of the protein of interest is about 1 gm / L to about 8gm / L, about Igm / L to about 5 gm / L; about Igm / L to about 4gm / L, about Igm / L to about 3 gm / L.

[0023] In an embodiment the titer of protein of interest is determined at the harvest level using protein A HPLC. In an embodiment, the cell culture is performed at a temperature of about 31°C, about 32°C, about 33°C, about 34°C, about 35 °C, about 36 °C, and about 37°C. In one embodiment, the positive clone in the microbial host cell is selected applying a suitable glutamine free media at temperature of about 37°C. The cell culture is performed at a temperature of about 34°C.

[0024] In an embodiment, the cell culture maintains first and second pH, where the second pH is about two log lower than the first pH. The cell culture maintains first pH selected from 7.0 to 7.5. The cell culture maintains second pH selected from 6.5 to 6.8.

[0025] In an embodiment, the cell culture maintains the cell density at about 40 million cells / mL to about 50 million cells / mL. In one embodiment cell culture maintains the cell density at least about 45 million cells / mL. In one embodiment, the cell culture has the initial seeding density of about 0.5 million cells / mL, about 1.0 million cells / mL, about 1.5 million cells / mL, about 2.0 million cells / mL cultured through fed-batch mode.

[0026] In an embodiment, the cell culture is subjected to a feed regimen continuously or periodically. In some embodiment, the cell culture is supplemented with a feed medium periodically from Day 3, Day 5, Day 7, Day 9, Day 11, and Day 13. In one embodiment, the cell culture is subjected to a feed regimen periodically, wherein glucose is maintained at 4gm / L. The feeding of feed medium is performed such that; glutamine is maintained below at about ImM to about 6mM, glutamate is maintained below at about ImM about 7mM.

[0027] In an embodiment, the cell culture method reduces the oxidation impurities by at least 33%. The cell culture method reduces the oxidation impurities between Day 12 and Day 16. The cell viability is maintained at least 85% during the process.

[0028] In an embodiment the oxidation impurities is determined at the neutralized protein A (NPEL) level using techniques selected from Mass spectrometry and RP-HPLC.

[0029] In an embodiment, the cell culture has a dissolved oxygen controlled at about 50% saturation through a cascaded system employing an air overlay and a variable air sparging rate from about 0.004 vvm to about 0.018 vvm.

[0030] In an embodiment, the cell viability of the culture for Day 12 is at least 90%. In one embodiment, the cell viability decreases by at least 5% between Day 12 and Day 16.

[0031] In an embodiment, the recovered recombinant fusion protein of interest is GLPl-IgG4 fusion protein. In an embodiment the recovered recombinant fusion protein of interest is characterized at harvest by suitable technique known in the art. The GLPl-IgG4 fusion protein comprises wherein basic variants about 20% or below, 18% or below, 16% or below; 14% or below; 12% or below; 10% or below; 8% or below, and 5% or below. Acidic variants is about 24% or below, 22% or below, 20% or below, 18% or below, 16% or below; 14% or below; 12% or below; 10% or below; 8% or below and 5% or below. In an embodiment the basic variant and acidic variant is measured by anion exchange (AEX) HPLC.

[0032] In an embodiment, the purification comprises the techniques selected from filtration, affinity chromatography, and virus inactivation and neutralization.

[0033] In an embodiment, the invention provides a vector construct for an expression of Glucagon-like peptide- 1-Fc fusion protein with titer 2 gm / L or above with desired quality having targeted levels of glycans selected from fucosylated glycans, galactosylated glycans, high mannose glycans, and / or sialylated glycans. In an embodiment, the titer of Glucagon-like peptide- 1-Fc fusion protein is determined at the harvest level using protein A HPLC. In an embodiment, the glycans of GLP1- IgG4 fusion protein is determined at the NPEL using HILIC UPLC.

[0034] In an embodiment, the hybrid promoter of the expression vector construct is selected from simian vacuolating virus 40 (SV40), or cytomegalovirus (CMV), or cytomegalovirus major immediate early enhancer (CMV / MIE), or CMV- MIA (melanoma inhibitory activity promoter), or musclespecific creatine kinase (MIA) promoter, or elongation factor (EF)-l, or lactase LAC4, pPolh, or trp, Z.PL, A0X1, GALI, or GAL10, or nmtl, or nmt42, or nmt81, or glyceraldehyde- 3 -phosphate dehydrogenase (GAP), and combinations thereof.

[0035] In one embodiment, the expression vector construct comprises more than one hybrid promoter.

[0036] In some embodiments, the hybrid promoter of the expression vector construct is CMV / MIE hybrid promoter or CMVZEF1 hybrid promoter or SV40 promoter.

[0037] In one embodiment, the secretory signal peptide sequence of the expression vector construct is selected from but not limited to Homo sapiens albumin, or alpha Lactalbumin, or immunoglobulin light chain (partial Homo sapiens).

[0038] In an embodiment, the secretory signal peptide system of the expression vector construct comprises amino acid sequences selected from SEQ ID No: 6, or SEQ ID No: 7, or SEQ ID No: 8 or any other sequence with at least 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% or similar functional properties.

[0039] In one embodiment, the secretory signal peptide system of the expression vector construct translates amino acid exhibited as SYGN013 having SEQ ID NO : 9, SYGN014 having SEQ ID NO: 10, SYGN015 having SEQ ID NO: 11; wherein the secretory signal peptide system of the expression vector construct translates amino acid at least 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97% or 98%, or 99% identical to SEQ ID 9, or SEQ ID 10, and SEQ ID 11.

[0040] In an embodiment, selection system of the expression vector construct comprises at least two antibiotic resistance markers or a functional variant, and a glutamine synthetase marker or a functional variant.

[0041] In one embodiment, the antibiotic resistance marker of the expression vector construct is selected from but not limited to puromycin, or kanamycin, hygromycin, or geneticin, or neomycin, and combination thereof.

[0042] In one embodiment, the glutamine synthetase marker of the expression vector construct is glutamine synthetase (GS) gene sequence or functional variant.

[0043] In an embodiment, the origin of replication of the expression vector construct is microbial origin of replication is pBR322 with pMBl origin.

[0044] In an embodiment, the expression vector construct further comprises one or more cloning sites having a nucleotide sequence encoding a translation initiator codon, a secretory signal peptide system and a gene of interest.

[0045] In an embodiment, the nucleotide sequence of the expression vector construct encoding the translation initiator codon, secretory signal peptide system and gene of interest is selected from SEQ ID No: 2, or SEQ ID No: 3, or SEQ ID No: 4 or any other sequence with at least 90% or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97% or 98%, or 99% or similar functional properties.

[0046] In an embodiment, one or more cloning site of the expression vector construct comprises restriction sites selected from Sbfl, Nhel, Pad, Avril, BamHl, Xbal, Notl, and Spel amongst others. In one embodiment, one or more cloning site of the expression vector construct comprises restriction sites selected form combination of Sbfl and Pad, or Nhel and Avril, or Xbal and Spel, or BamHl and Notl.

[0047] In another embodiment, the expression vector construct comprises a Kozak sequence having sequence SEQ ID NO: 12, positioned upstream of the initiation codon.

[0048] In another embodiment, the expression vector construct comprises: a) hybrid promoter selected from CMV / MIE, or CMVZEF1; b) one or more cloning sites comprising nucleotide sequence encoding translation initiator codon, signal peptide, and gene of interest; c) a secretory signal peptide system selected from Homo sapiens albumin, or alpha Lactalbumin, or immunoglobulin light chain (partial Homo sapiens),' d) a protein of interest; e) an antibiotic selection marker selected from puromycin, or kanamycin, or hygromycin, or geneticin, or neomycin; f) a glutamine selection marker; g) an origin of replication; h) a simian virus 40 polyadenylation sequence; wherein the protein of interest is depicted as amino acid sequence set forth as SEQ ID No: 1 or any other sequence with at least 90% or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97% or 98%, or 99% or nucleotide sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or any other sequence with at least 90% or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97% or 98%, or 99%; wherein gene of interest is operably linked to signal peptide selected from SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8 or any other sequence with at least 90% or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97% or 98%, or 99%; wherein the expression vector construct expressed in the host cell provides an expression and / or titer of recombinant fusion protein greater than at least 2 gm / L.

[0049] In an embodiment, the expression vector construct exhibits the sequence as set forth in SEQ ID No: 5. In some embodiments the expression vector construct comprises a sequence having at least 99%, 98%, 97%, 96%, or 95%, identity to the sequence of SEQ ID NO:5.

[0050] In an embodiment, the recombinant fusion protein of interest is Glucagon-like peptide- 1.

[0051] In an embodiment, the Glucagon -like peptide- 1 is set forth as amino acid sequence SEQ ID NO: 1; or Glucagon-like peptide-1 comprises the amino acid sequence at least 90% or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97% or 98%, or 99% identical to sequence SEQ ID NO: 1 . In one embodiment, the Glucagon-like peptide-1 comprises the nucleotide sequence as set forth in SEQ ID NO: 2, or SEQ ID NO 3, or SEQ ID 4; or Glucagon-like peptide-1 comprises the nucleotide sequence at least 90% or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97% or 98%, or 99% identical to sequence SEQ ID NO: 2, or SEQ ID NO 3, or SEQ ID 4.

[0052] In an embodiment, the microbial host cell is E.coli.

[0053] In an embodiment, the host cell is mammalian host cell, Chinese hamster ovary (CHO) cell selected from CHO DG44, CHO-S, CHO-K1, or CHO DUKX-B11. In an embodiment, the operating conditions for host cell culture are optimized and adjusted for each stage.

[0054] In some embodiments, the selection pressure is applied using glutamine-free media (like BalanCD CHO Growth A) and antibiotics to isolate a stable, high-expressing clone.

[0055] In some embodiments, the selected clone is subjected to fed batch production in a bioreactor with initial volume of 3.6L and seed density about 1.0 million cells / mL and is grown under tightly controlled conditions for a total batch duration of 16 days. The temperature is selected from 31°C, 32°C, 33°C,34°C, 35°C,36°C,and 37°C preferably 34°C and pH is maintained at 7±01.

[0056] In one embodiment, dissolved oxygen (D.O.) is held at 50% through a cascaded system of air overlay, air sparging (increasing from 0.004 vvm to 0.018 vvm), and controlled oxygen flow. Agitation is also increased from 250 rpm (0 day) to 350 rpm after day 10 to improve mixing and oxygen transfer.

[0057] In one embodiment, nutrient levels in the fed-batch condition are sustained through a feed regime, using Cell Boost 7a and 7b along with supplements like LC and managed glucose additions, ensuring a stable environment for high-level protein expression.

[0058] In an embodiment, the cell culture is performed in a 2L bioreactor, 5L bioreactor, 20L bioreactor, 50L bioreactor, 100L bioreactor, 500L bioreactor and larger scale bioreactors.

[0059] In an embodiment, high molecular weight impurities (HMWs) in recombinant fusion protein are less than about 2%, 1.5%, 1%, 0.5%, 0.1%, 0.05%. In an embodiment, the HMWs of a recombinant fusion protein is determined at the NPEL using techniques selected from size exclusion chromatography.

[0060] In an embodiment, the glycan profile of recombinant fusion protein comprises afucosylated glycans less than about 2%, and total afucosylated glycans less than about 5%. In an embodiment, the afucosylated glycans is determined at the NPEL using techniques selected from HILIC UPLC.

[0061] In an embodiment, the glycan profile of recombinant fusion protein comprises total galactosylation less than about 23%. In an embodiment, the galactosylation is determined at the NPEL using techniques selected from HILIC UPLC.

[0062] In an embodiment, the glycan profile of recombinant fusion protein comprises total sialylation less than about 2%. In an embodiment, the sialyation is determined at the NPEL using techniques selected from HILIC UPLC. In an embodiment the expression vector construct provides an expression of protein of interest more than Ig / L, more than 1 ,5g / L, more than 2g / L, more than 2.2g / L, more than 2.5g / L, and more than 3g / L. In an embodiment the titer of protein of interest is determined at the harvest level using protein A HPLC.

[0063] In an embodiment, the recombinant fusion protein is dulaglutide. In one embodiment the method to produce dulaglutide comprises steps of: i. providing an expression vector construct having: a) a MIA / CMV promoter; b) a cloning site encoding translation initiator codon, a secretory signal peptide system and a gene of interest; c) a SV40 polyadenylation sequence; d) a CMVZEF 1 promoter; e) one or more cloning site encoding translation initiator codon, a secretory signal peptide system and a gene of interest; f) a SV40 polyadenylation sequence; g) a Puromycin selection marker; h) a PBR322 origin of replication; i) a Kanamycin selection marker; j) a Glutamine Selection Marker; k) a SV40 polyadenylation sequence; ii. transfecting the recombinant expression vector construct into a microbial host cell; iii. culturing the mammalian host cell comprising the expression vector construct, through fed batch mode; iv. harvesting and purifying dulaglutide; wherein dulaglutide has purity more than 95% and reduced amount of high molecular weight (HMW) impurities of about 2% or below; wherein the purity and HMW impurity is determined by HP-SEC.

[0064] In an embodiment, a composition comprising a mixture of GLPl-IgG4 fusion protein of interest is disclosed. The composition comprising: a. a GLPl-IgG4 fusion protein having an amino acid sequence as set forth in SEQ ID NO: 1; and b. a GLPl-IgG4 fusion protein having an amino acid sequence as set forth in SEQ ID NO: 13, wherein SEQ ID NO 1 is predominantly higher than SEQ ID NO 13; wherein the SEQ ID NO 1 is dulaglutide.

[0065] In one embodiment, the composition is set forth as sequence SEQ ID NO 13 present less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%. The composition where the SEQ ID NO 13 lacks at least two amino acids at N terminal in comparison to SEQ ID NO 1. The two amino acids are histidine and glycine. In an embodiment the N-terminal lacking two amino acids in sequence ID no 13 is determined by techniques selected by AEX HPLC and Mass spectrometry.

[0066] In one embodiment the composition comprising a mixture of GLPl-IgG4 fusion protein of interest, comprises: a. a GLPl-IgG4 fusion protein having an amino acid sequence as set forth in SEQ ID NO: 1; and b. a GLPl-IgG4 fusion protein having an amino acid sequence as set forth in SEQ ID NO: 14, wherein SEQ ID NO 1 is predominantly higher than SEQ ID NO 14; wherein the SEQ ID NO 1 is dulaglutide.

[0067] In one embodiment, the composition is set forth as sequence SEQ ID NO 14 present less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%. The composition where the SEQ ID NO 14 lacks at least two amino acids at N terminal in comparison to SEQ ID NO 1, as determined by AEX HPLC, mass spectrometry. The four amino acids are histidine, glycine, glutamic acid, and glycine.

[0068] In another embodiment, the expressed protein of interest is for the treatment of metabolic disorder selected from type-2 diabetes, or obesity, or metabolic syndrome.

[0069] BRIEF DESCRIPTION OF THE FIGURES

[0070] Figure 1: pKSB2.0 vector map

[0071] Figure 2: Characterization of pKSHB2.0_SYGN013 (CL#1 & CL#2)

[0072] Figure 3: Characterization of pKSHB2.0_SYGN014 (CL#2) & pKSHB2.0_SYGN015 L#l)

[0073] BRIEF DESCRIPTION OF THE ACCOMPANYING SEQUENCE LISTINGS

[0074] SEQ ID NO. 1 represents amino acid sequence of GLPl-IgG4 fusion protein (Dulaglutide) SEQ ID NO. 2 represents nucleotide sequence for GLPl-IgG4 fusion protein (Dulaglutide)

[0075] SEQ ID NO. 3 represents nucleotide sequence for GLPl-IgG4 fusion protein (Dulaglutide)

[0076] SEQ ID NO. 4 represent nucleotide sequence for GLPl-IgG4 fusion protein (Dulaglutide)

[0077] SEQ ID NO. 5 represents the recombinant vector construct for expressing GLPl-IgG4 fusion protein (Dulaglutide)

[0078] SEQ ID NO. 6 represents signal peptide sequence for translation of GLPl-IgG4 fusion protein (Dulaglutide) SEQ ID NO. 7 represents signal peptide sequence for translation of GLPl-IgG4 fusion protein (Dulaglutide) SEQ ID NO. 8 represents signal peptide sequence for translation of GLPl-IgG4 fusion protein (Dulaglutide) SEQ ID NO. 9 represents translated amino acid sequence of GLPl-IgG4 fusion protein (Dulaglutide)

[0079] SEQ ID NO. 10 represents translated amino acid sequence of GLPl-IgG4 fusion protein (Dulaglutide)

[0080] SEQ ID NO. 11 represents translated amino acid sequence of GLPl-IgG4 fusion protein (Dulaglutide)

[0081] SEQ ID NO. 12 represents Kozak sequence

[0082] SEQ ID NO. 13 represents amino acid sequence of GLPl-IgG4 fusion protein (Dulaglutide)

[0083] SEQ ID NO. 14 represents amino acid sequence of GLPl-IgG4 fusion protein (Dulaglutide)

[0084] DETAILED DESCRIPTION OF INVENTION

[0085] As used herein, the following terms shall have the meanings as indicated below unless otherwise specified or required by context:

[0086] The term “comprises” or “comprising” is used in the present description, it does not exclude other elements or steps. For purpose of the present invention, the term “consisting of is considered to be an optional embodiment, of the term “comprising of . If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which optionally consists only of these embodiments.

[0087] As used throughout the specification and in the appended claims, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise.

[0088] The term “about”, as used herein, is intended to refer to ranges of approximately 10-20% greater than or less than the referenced value. In certain circumstances, one of skill in the art will recognize that, due to the nature of the referenced value, the term “about” can mean more or less than a 10- 20% deviation from that value.

[0089] The term “host cell” or “cell line” refers to mammalian cells i.e., Chinese hamster ovary (CHO) cells cultured in suitable medium to express protein of interest. In an embodiment, the Chinese hamster ovary cell being utilized to produce polypeptide directly into the cell culture.

[0090] The term “gene of interest” or “GOI” or “protein of interest”, “recombinant fusion protein”, used herein refers to nucleotide sequences of Glucagon-like peptide-1 fusion protein in expression construct. GOI can be obtained from a variety of sources, including source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.

[0091] The term “ hybrid promoter” in the present invention refers to a sequence of nucleic acid that allows efficient, high-level expression of recombinant protein. The hybrid promoters can be selected from simian vacuolating virus 40 (SV40), cytomegalovirus (CMV), Cytomegalovirus major immediate early enhancer (CMV / MTE), CMV- MIA (melanoma inhibitory activity promoter), muscle-specific creatine kinase (MIA) promoter, elongation factor (EF)-l, lactase LAC4, pPolh, trp, Z.PL, A0X1, GALI, GAL 10, nmtl, nmt42, nmt81 and glyceraldehyde- 3 - phosphate dehydrogenase (GAP).

[0092] The term "expression vector construct" or "expression construct" or “expression vector” are interchangeable and refers to the nucleotide sequences of the invention containing the nucleotide sequences to be expressed. The restriction sites between the 5 'and 3' ends of the sequences allows insertion, removal of sequences in a vector.

[0093] The term “transfection” refers to introduction of expression vector into the host cell such as mammalian cells (Chinese hamster ovary), E. coli (DH5a) to produce or express protein of interest. The transfection methods known in the art for example calcium phosphate co-precipitation, liposome transfection and electroporation. The glutamine Synthetase (GS) gene incorporated into the vector construct that allows selection of transfected CHO-S™ cells using Methionine Sulfoximine (MSX).

[0094] The term “Leader sequence” or “Secretion signal sequence” or “Signal peptide” refers to a sequence of amino acids which can be enzymatically or chemically removed to produce the desired polypeptide of interest. The secretion signal sequence generally present at the N-terminal region of a larger polypeptide functioning to initiate association of that polypeptide with the cell membrane compartments like endoplasmic reticulum and secretion of polypeptide through the plasma membrane. The nucleotide sequence of signal peptide incorporated into the vector along with protein of interest.

[0095] The term “Digestion” or “Restriction” of DNA refers to the catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA (“sequence-specific endonucleases'). The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors, and other requirements were used as would be known to one of ordinary skill in the art. In an embodiment, the linearization of isolated plasmid using restriction endonuclease selected from EcoRI, EcoRII, BamHI, Hindlll, TaqI, Notl, HinFI, Sau3 Al, PvuII*, Smal*, Haelll*, Hgal, Alul*, EcoRV*, EcoP15I, Rsal, Kpnl, Pstl, BstBI , Mfel, BciVI, Xhol, PacI, BstEII, SacI, Sall, Seal, SacI, Tthllll, EagI, Sadi, Spel, SphI, Stul*, Bglll, Xbal, Nrul, Sbfl, BamHI, Notl and Pad. In an embodiment, the vedor construd comprises multiple doning site to insert the gene of interest for the expression of protein MCS 1 (Sbfl or Nhel and Pad or Avril), MCS 2 (BamHI or Xbal and Notl or Spel). The restriction enzymes linearize or cut at spedfic site through which gene of interest can be released and checked for expression.

[0096] The term “culture medium” or “suitable medium” or “cell culture medium” or “cell culture” refers to medium for culturing mammalian cells (such as CHO DUKX-B11, CHO S, CHO KI, CHO DG44) capable to produce in protein of interest in the bioreactor or fermenter. The culture medium solution or media contains nutrients for growth, propagation, expansion and maintenance of the cells.

[0097] The term “feed” or “feed solution” or “feed medium” refers to addition of ingredients or substances to maintain and promote growth of cells in bioreactor. In an embodiment, the feed supplemented to culture medium after inoculation comprises one or more nutrients, basal media, carbohydrate source, etc. Typical ingredients that are used in cell culture media include amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins and the like. Other ingredients that promote or maintain cultivation of cells ex vivo can be selected by those of skill in the art within the scope of the invention, and in accordance with the particular need.

[0098] The terms “fed batch cell culture” and “fed batch culture” refers to a cell culture wherein the cells, preferably mammalian (chinese hamster ovary), and culture media are supplied to the culturing vessel initially and additional culture nutrients are fed, continuously or in discrete increments, to the culture during culturing, with or without periodic cell and / or product harvest before termination of culture. A “fed batch method,” refers to a method by which a fed batch cell culture is supplied with additional nutrients. A fed batch method may comprise adding supplemental media as per determined feeding schedule. The term “perfusion batch culture” or “continuous cell culture” as used herein refers to a process that uses a method to keep cells in a bioreactor while continuously exchanging culture medium. Fresh medium replenishes nutrients and carbon sources, while cellular waste and medium depleted of nutrients are removed. This exchange of medium is commonly expressed as the number of operating vessel volumes per day (WD). The term “continuous large-scale 10 fermenter” or “perfusion-batch cell culture” or “continuous cell culture” as used herein are interchangeable.

[0099] The term “expanding” as used herein refers to culturing one or more cells in vitro for the purpose of obtaining a larger number of cells in the culture.

[0100] The term "Cell Density" refers to the number of cells in a given volume of culture medium. In an embodiment, “viable cell density” or “VCD” refers to the number of live cells in a given volume of culture medium.

[0101] The term “Titer” or “Yield” or “expression of protein” as used herein, refers to the total amount of protein produced by a cell culture in given amount of cell culture volume.

[0102] The term high yield and desired quality refer to high yielding expression vector construct that provides large quantity of protein (gm / L) along with high purity, acceptable range of glycans, functional potency, with less impurities or degraded products.

[0103] “Bioreactor” herein is referred as vessel, equipment or a system to support the growth and development of microorganisms or cell lines under aseptic condition. The bioreactor body can be made of materials selected from Stainless steel, glass or polymers like polystyrene, polycarbonate, Polyethylene terephthalate, PETG, LDPE, PVA, PVC, PVA or PP.

[0104] “Collected protein mixture” is the mixture obtained at the end of the cell culture process, the cellculture is harvested, and cell separation is carried out by centrifugation or filtration, the resultant cell free harvest comprising the protein of interest and one or more impurities for example Host cell proteins and Low molecular weight species; is called the collected protein mixture.

[0105] “Purified protein mixture” herein refers to the solution comprising the protein of interest and reduced one or more impurities as compared to the collected protein mixture.

[0106] “Separation matrix” herein refers to a solid support material that holds a stationary phase and is used to separate a mixture selected based on molecular characteristics and interaction type use mechanisms of the desired molecule to be separated from the mixture from ion exchange, surface adsorption, partition, and size exclusion. “Host cell proteins” or “HCP” here refers to the proteins other than the protein of interest generated by host organisms or cell lines pertaining to cellular metabolism during the production of the protein of interest considered as process-related protein impurities.

[0107] “Product quality” is the identity, purity, potency, stability, and safety of the protein of interest determined by the predefined product quality attributes for example glycan profile, charge variants profile, size variants profile, etc. which impacts the degree to which a drug substance or product meets its intended use and fulfils its inherent properties.

[0108] The term “protein mixture” herein refers to the protein of interest essentially consisting of protein of interest and one or more impurities. A “protein of interest” is a “Physiologically active protein” capable to bind with target or receptor to induce therapeutic effect. In an embodiment the physiologically active protein capable to bind with GLP-1 receptors on the beta cells. In an embodiment the physiologically active protein comprising human GLP-1 receptor agonist. The protein of interest is a fusion protein that consists of two identical, disulfide-linked chains, each containing an N-terminal GLP-1 analog sequence covalently linked to the Fc portion of a modified human immunoglobulin G4 (IgG4) heavy chain by a small peptide linker. The GLP-1 analog portion of dulaglutide is 90% homologous to native human GLP-1 (7-37). In an embodiment the protein of interest is GLPl-Fc fusion protein more particularly GLPl-IgG4 fusion protein. In an embodiment the protein of interest is GLP-l-Fc fusion protein. In an embodiment the protein of interest is dulaglutide. Wherein the physiologically active protein has a molecular weight of below 150 KDa, below lOOKDa, 63 KDa, 60KDa. In certain embodiment the Physiologically active protein is conjugated with polyethylene glycol (PEG) to form pegylated protein which further increases efficiency of the Physiologically active protein.

[0109] The term “inoculation” as used herein refers to the addition of cells to starting medium to begin the culture.

[0110] The term “basal medium” and “basal media” as used herein refers to starting medium to which cells are added to begin the culture.

[0111] The term “liquid culture” as used herein refers to cells (for example, bacteria, plant, insect, yeast, or animal cells) grown on supports, or growing suspended in a liquid nutrient medium.

[0112] The term “seed culture” as used herein refers to a cell culture grown in order to be used to inoculate larger volumes of culture medium. The seed culture can be used to inoculate larger volumes of media in order to expand the number of cells growing in the culture (for example, cells grown in suspension).

[0113] The term “culturing” as used herein refers to growing one or more cells in vitro under defined or controlled conditions. Examples of culturing conditions which can be defined include temperature, gas mixture, time, and medium formulation.

[0114] The term “cell culture” or “culture medium” or “production phase” as used herein, refer to culturing of mammalian cells (such as CHO DUKX-B11, CHO S, CHO KI, CHO DG44) capable to product in antibody in bioreactor or fermenter wherein bioreactor comprising a solution or media containing nutrients for growth, propagation, expansion and maintenance of the cells.

[0115] The term “growth phase” as used herein of the cell culture as used herein refers to the period of exponential cell growth (for example, the log phase) where cells are primarily dividing rapidly. During this phase, the rate of increase in the density of viable cells is higher than at any other time point.

[0116] The term “production phase” of the cell culture as used herein refers to the period of time during which cell growth is stationary or is maintained at a near constant level. The density of viable cells remains approximately constant over a given period of time. Logarithmic cell growth has terminated, and protein production is the primary activity during the production phase. The medium at this time is generally supplemented to support continued protein production and to achieve the desired glycoprotein product.

[0117] The term “cell culture condition” as used herein refers to the conditions suitable for improving cell biomass and protein titer. In an embodiment, cell culture conditions are selected from basal media, feed media, feeding including constant and exponential, temperature, pH, duration of culture, dissolved oxygen, CO2.

[0118] The term “cell density” as used herein refers to the number of cells present in a given volume of medium. In an embodiment, the present method provides a cell density at least 30 million. In an embodiment, the present method provides a cell density at least 40 million to 50 million. The mammalian cell culture keeps growing up to the desired days selected from day 12 or day 13 or day 14, or day 15, or day 16, and maintains a product quality and titer in specific conditions of cell culture as mentioned in the present disclosure. The present invention successfully developed and designed the expression vector and upstream process conditions which is suitable for the production of recombinant fusion protein, specifically GLPl-IgG4 fusion protein or dulaglutide. The present disclosure provides improved product quality and reduces undesired impurities selected from HMWs, LMWs, acidic variants, basic variants, oxidized impurities, truncated fragments or protein.

[0119] The term “expression cassette” herein refer to Genetic material comprising clone or vector comprising nucleotide sequence capable to encode protein of interest, promoter sequence and one or more nucleotide sequence encoding elements selected from enhancer, terminator, selection marker, proximal promoters, distal enhancers, silencers, insulators / boundary elements, and locus control regions. The clone or vector is transfected into mammalian host cell thereby formed the expression cassette which is capable to express the protein. In an embodiment, the heavy chain and light chain is expressed together in the same vector. In another embodiment, the heavy chain and light chain is expressed separately in two vectors.

[0120] The term “expression vector” or “expression vector construct” or “vector” are interchangeable herein and refers to the nucleotide sequences of the invention containing the nucleotide sequences to be expressed. The restriction sites between the 5 'and 3' ends of the sequences allows insertion, removal of sequences in a vector. The expression vector construct includes nucleotide sequence, transcriptional and translational control sequences, such as a promoter and / or termination sequences, operably linked to the nucleotide sequence and allowing expression in a host cell. In an embodiment, the expression vector comprises Kozak sequence before initiation codon.

[0121] The term “promoter” or “promoting sequence” or “promotor sequence” are interchangeable herein refers to a region of DNA upstream of a gene where relevant proteins (such as RNA polymerase and transcription factors) bind to initiate transcription of that gene. The resulting transcription produces an RNA molecule (such as mRNA). Herein the promoters are selected from but not limited to Simion Virus Early promoter also known as P SV40E, and Simion Virus late promoter also known as P SV40L. The hybrid promoters selected from simian vacuolating virus 40 (SV40), cytomegalovirus (CMV), Cytomegalovirus major immediate early enhancer (CMV / MIE), CMV- MIA (melanoma inhibitory activity promoter), muscle-specific creatine kinase (MIA) promoter, elongation factor (EF)-l, lactase LAC4, pPolh, trp, Z.PL, A0X1, GALI, GAL10, nmtl, nmt42, nmt81 and glyceraldehyde- 3 -phosphate dehydrogenase (GAP). In an embodiment, the promoter used in construct is CMV / MIE hybrid promoter (MIE functions as an enhancer to increase the expression of CMV). In an embodiment, the “promoter sequence” used in the present invention comprising a sequence of nucleic acid required to turn a gene 30 on or off.

[0122] The term “Enhancer” or “Enhancer sequence” are interchangeable herein and refer to regulatory DNA sequences that, when bound by specific proteins called transcription factors, enhance the transcription of an associated gene. Herein the enhancers is Simion Virus enhancer also known as E SV40 Enhancer.

[0123] The term “Kozak Sequence” or “Scanning sequence” are interchangeable herein and refers to is a nucleic acid motif that initiates protein translation in eukaryotic mRNA. A start codon is located within the Kozak sequences where the assembly of ribosomes begins.

[0124] Plasmid DNA vector comprises a multiple cloning site, an RNA promoter sequence, a selection marker, such as an antibiotic resistance factor (selected from Puromycin, Kanamycin, Hygromycin, G418 (Geneticin), and Neomycin), and a sequence suitable for multiplication of the vector, such as an origin of replication. In an embodiment, the plasmid backbone is pBR322 with pMBl origin of replication (ori) in the context of the present invention.

[0125] The term “multiple cloning site” or “MCS” are interchangeable here and herein refers to a small DNA segment in a plasmid that contains multiple restriction enzyme cut sites. These sites are used to introduce foreign DNA into a plasmid, which is a common feature in engineered plasmids. There are two MCS sequences used in the present invention selected from MCS-I and / or MCS-II.

[0126] The term “chromatin opening element”, or “ubiquitous chromatin opening element” (UCOE), is a DNA element that helps to open chromatin and promote gene expression. Herein the chromatin opening element is "3.0 KB UCOE".

[0127] The term “polyadenylation” refers to a process that adds a chain of adenosine nucleotides, known as a poly(A) tail, to the 3' end of RNA transcripts. This process is catalysed by a family of enzymes called poly(A) polymerases. “Polyadenylation sequence” or “poly(A) signal”, is a conserved motif that is required for the process of polyadenylation. Herein the “Polyadenylation sequence” is a bidirectional sequence namely “pA_SV40”.

[0128] The term “origin of replication” or “origins of bidirectional replication” (OBR) is a specific DNA sequence where DNA replication begins on a chromosome, plasmid, or virus. Herein the origin of replication is Simon Virus Origin of Replication also known as SV40 origin of replication (“Ori_SV40”), and pUC origin of replication (“Ori_pUC”).

[0129] The term “transfection” refers to introduction of expression vector into the host cell such as mammalian cells to produce or express protein of interest. The transfection methods known in the art for example calcium phosphate co-precipitation, liposome transfection and electroporation. The term “clone” comprises the expression vector construct, protein of interest in a host cell.

[0130] “Glutamine Synthetase” gene plays a role in transfection as a selection marker in cell line development. The GS gene encodes a protein that catalyses the conversion of glutamate and ammonia into glutamine, which is important for cell growth and proliferation. The GS gene can be used as a selection marker to generate stable clones with higher antibody productivity. The glutamine Synthetase (GS) gene incorporated into the vector construct that allows selection of transfected CHO-S™ cells using Methionine Sulfoximine (MSX).

[0131] The term “Digestion” or “Restriction” of DNA refers to the catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA (“sequence-specific endonucleases'). The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors, and other requirements were used as would be known to one of ordinary skill in the art. In an embodiment, the linearization of isolated plasmid using restriction endonuclease selected from EcoRI, EcoRII, BamHI, Hindlll, TaqI, Notl, HinFI, Sau3 Al, PvuII*, Smal*, Haelll*, Hgal, Alul*, EcoRV*, EcoP15I, Rsal, Kpnl, Pstl, BstBI , Mfel, BciVI, Xhol, PacI, BstEII, SacI, Sall, Seal, SacI, Tthllll, EagI, Sadi, Spel, SphI, Stul*, Bglll, Xbal, Nrul, Sbfl, BamHI, Notl and Pad. In an embodiment, the vedor construd comprises multiple doning site to insert the gene of interest for the expression of protein MCS 1 (Sbfl or Nhel and Pad or Avril), MCS 2 (BamHI or Xbal and Notl or Spel). The restriction enzymes linearize or cut at spedfic site through which gene of interest can be released and checked for expression.

[0132] The term “Secretory signal sequence” or “Signal peptide” or “Leader sequence” are interchangeable here and refers to regions located between the promoter and coding region of a gene. They regulate gene expression at the transcription or translation level. In eukaryotes, leader sequences can range from a few nucleotides to over 1000 nucleotides. Secretory signal sequences are sequences of amino acids which can be enzymatically or chemically removed to produce the desired polypeptide of interest. The secretion signal sequence generally present at the N-terminal region of a larger polypeptide functioning to initiate association of that polypeptide with the cell membrane compartments like endoplasmic reticulum and secretion of polypeptide through the plasma membrane. The nucleotide sequence of signal peptide incorporated into the vector along with gene of interest. Herein the Secretory signal sequences are selected from Immunoglobulin Kappa Secretory chain, and Albumin secretory signal chain. The term “terminator” or “terminating sequence” herein refers to the termination sequence, which follows the promoter and coding region, and is the last region of the gene. During transcription the termination sequence signals to the RNA polymerase molecule that it has reached the end of the gene and should stop transcribing. If this region were not present, RNA polymerase would continue down the chromosome to more of the genes.

[0133] The term “Digestion” or “Restriction” of DNA are interchangeable here and refers to the catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA (“sequence-specific endonu cieases'). The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors, and other requirements were used as would be known to one of ordinary skill in the art. In an embodiment in the present invention the vector construct comprises of Multiple cloning site to aid the insertion of Gene of interest, the vector construct is commenced through PCR wherein, the purified DNA fragments are digested using suitable restriction enzymes. Hence restriction enzymes linearize or cut at specific site through which gene of interest can be released and checked for expression. Herein the restriction enzymes used to cut DNA fragments at MCS I are selected from but not limited to BamHl, Pmll, Sbfl, Fsel, and Nael. The restriction enzymes used to cut DNA fragments at MCS II are selected from but not limited to Pmel, Swal, and BstBI.

[0134] The term “electroporation” herein refers to a process of using an electric pulse to introduce DNA into cells by creating temporary pores in the cell membrane, allowing for efficient gene delivery in both in vivo and in vitro settings, particularly useful in neuroscience research for manipulating gene expression in specific brain regions.

[0135] The term “mammalian cell” herein refers to a cell which is highly organised, has dynamic structure that compartmentalises its many functions into organelles such as the nucleus, Golgi, and endoplasmic reticulum. The nucleus retains the genetic material for cell maintenance and replication, whereby efficient signal dependent targeting of cellular proteins into or out of the nucleus. Herein mammalian cell refers to the host cells which can grow in culture and expressing a desired recombinant product protein. In an embodiment the mammalian cell is CHO cell.

[0136] The term “protein expression” herein involves making an RNA copy of the DNA code, a process called Transcription and translating information carried by nucleic acids to give the sequences of amino acids that make up proteins.

[0137] The term “Glycan” used herein refers to sugar residues such as glucose, N-acetylglucosamine, N- acetyl neuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, 2'- fluororibose, 2'-deoxyribose, phosphomannose, 6'-sulfo N-acetylglucosamine etc present in glycoprotein or fusion protein. The term "High Mannose" used herein refers to the sugar mannose present in the fusion protein molecule includes at least 3 mannose sugar residues and that terminates in a mannose on a nonreducing end of the glycan.

[0138] The term “Terminal sialylation” used herein refers to the addition of sialic acid residues to the terminal ends of glycan (sugar) chains that are attached to proteins or lipids. During the glycosylation process, a form of post-translational modification, and it plays a crucial role in various biological functions.

[0139] The term "Galactosylation" or "used herein refers to the protein molecule containing or adding sugar galactose on the N-linked site of the protein molecule. The term "N-Glycan" used herein refers to the method that determines the total individual sugar molecule present in the fusion protein.

[0140] The term “Sialylated glycan” used herein refers to a glycan that includes at least 1 sialic acid. In an embodiment, a sialylated glycan includes at least 1, 2, 3, or 4 sialic acids such as monosialylated glycan (e.g., a branched glycan monosialylated on an al-3 arm of the branched glycan (e.g., with a NeuAc-a2,6-Gal terminal linkage)), and / or a disialylated glycan (e.g., a branched glycan sialylated on both an al-3 arm and an al -6 arm of the branched glycan).

[0141] The term “Galactosylated glycan” used herein refers to a glycan that includes at least 1 galactose sugar residue. In an embodiment, a galactosylated glycan is a Gl, G2, GIF, G2F, Al, and / or A2 glycan. In an embodiment, a galactosylated glycan is a galactose-alpha-l-3-galactose-containing glycan. In an embodiment, a galactosylated glycan is a tri-antennary glycan or a tetra-antennary glycan.

[0142] The term “Mature glycan” used herein refers to a glycan that includes at least one sugar residue in addition to a core pentasaccharide structure and does not include more than three mannose sugar residues. In an embodiment, a mature glycan includes a core pentasaccharide structure and at least one N-acetylglucosamine.

[0143] The term used “high molecular weight” or “HMW” is product-related impurities that contribute to the size heterogeneity of antibody products. The formation of HMW species within a therapeutic antibody drug product as a result of protein aggregation can potentially compromise both drug efficacy and safety (e.g., eliciting unwanted immunogenic response). HMW is considered critical quality attribute that are routinely monitored during drug development and as part of release testing of purified drug product during manufacturing. In certain embodiment the HMW relates to aggregates. In an embodiment the neutralized protein A step refers to one step purification wherein the harvest obtained from cell culture method like fed-batch or perfusion is purified by protein A affinity chromatography wherein the purified protein elute of protein A affinity chromatography is further incubated at acidic pH for virus inactivation for at least 40 minutes and thereafter shift the pH to 6.5 to 7.0 for neutralization.

[0144] Neutralized Protein A Eluate (NPEL):As used herein, the term “Neutralized Protein A Eluate (NPEL)” refers to the solution obtained after elution of an antibody or Fc-fusion protein from a Protein A chromatography resin under acidic conditions, followed by adjustment of the eluate to a near-neutral pH (about 6.0-8.0) using a suitable neutralizing agent. The NPEL represents a process intermediate that maintains antibody stability and is suitable for subsequent downstream operations.

[0145] The invention is further described hereinafter with reference to its general method steps, which exemplify but do not limit the scope of the invention.

[0146] An embodiment of the invention discloses a method for high expression of recombinant fusion protein of interest, using an expression vector construct.

[0147] In an embodiment, a method for expressing recombinant fusion protein of interest is described. The method comprises: i. Providing a suitable expression vector construct comprising; a. a hybrid promoter or a functional variant; b. a secretory signal peptide system operably linked to a recombinant fusion protein of interest; c. a gene sequence encoding the protein of interest; d. a dual selection system having antibiotic resistance and glutamine synthetase (GS) markers; e. an origin of replication; and f. a termination sequence comprising an SV40 polyadenylation sequence or a functional variant thereof; ii. transfecting the expression vector construct into a mammalian host cell to form a clone capable of expressing the protein of interest; iii. culturing the mammalian host cell comprising the expression vector construct, through cell culture methods selected from fed-batch mode, perfusion culture for at least more than 12 Days, to up to 16 Days in a suitable medium; wherein the cell culture method has first pH and second pH, wherein the second pH is at least two log lower than first pH; wherein the cell culture method maintains a temperature selected from about 32°C to about 36 °C; wherein the culture method maintains osmolality at about 300 Osm / kg to 470 Osm / kg; wherein the cell culture method maintains residual glucose less than 5gm / L; wherein the cell culture method maintains glutamine ImM to 6mM; wherein the cell culture method maintains glutamate ImM to 7mM; wherein the cell culture method maintains cell viability at least 40 million cells / ml to 50 million cells / ml; wherein the cell culture method reduces the oxidation rate to form a oxidized species of the protein of interest by at least 30% from Day 10 till harvest; iv. harvesting and purifying the recombinant fusion protein; wherein the expression vector construct directs the expression of recombinant fusion protein of interest in the mammalian host cell at yield of at least or more than 1 gm / L, to 3 gm / L; wherein the harvested recombinant fusion protein of interest is GLPl-IgG4 fusion protein comprising at least less than about 25% basic variants, at least less than about 30% acidic variants, less than about 2% high molecular weight aggregates, and predominantly higher monomer of GLPl-IgG4 fusion protein.

[0148] In an embodiment, wherein the expression vector construct is transfected into a microbial host cell; selecting and isolating expression vector construct from the microbial host cell.

[0149] In an embodiment, wherein mammalian host cell capable to express protein of interest is selected and isolated by applying a dual selection system.

[0150] In an embodiment, the monomer percentage purity of expressed recombinant fusion protein is more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% and more than 99%.

[0151] In an embodiment, the hybrid promoter of the expression vector construct is selected from simian vacuolating virus 40 (SV40), or cytomegalovirus (CMV), or cytomegalovirus major immediate early enhancer (CMV / MIE), or CMV- MIA (melanoma inhibitory activity promoter), or musclespecific creatine kinase (MIA) promoter, or elongation factor (EF)-l, or lactase LAC4, pPolh, or trp, Z.PL, A0X1, GALI, or GAL10, or nmtl, or nmt42, or nmt81, or glyceraldehyde- 3 -phosphate dehydrogenase (GAP), and combinations thereof. The “promoter sequence” used in the present invention comprises a sequence of nucleic acid required to turn a gene on or off. The expression vector construct includes nucleotide sequence, transcriptional and translational control sequences, such as a promoter and / or termination sequences, operably linked to the nucleotide sequence, allowing expression in a host cell.

[0152] In one embodiment, the expression vector construct comprises more than one hybrid promoter.

[0153] In some embodiments, the hybrid promoter of the expression vector construct is CMV / MIE hybrid promoter or CMVZEF1 hybrid promoter or SV40 promoter.

[0154] In one embodiment, the secretory signal peptide sequence of the expression vector construct is selected from but not limited to Homo sapiens albumin, or alpha Lactalbumin, or immunoglobulin light chain (partial Homo sapiens).

[0155] In an embodiment, the secretory signal peptide system of the expression vector construct comprises amino acid sequences selected from SEQ ID No: 6, or SEQ ID No: 7, or SEQ ID No: 8 or any other sequence with at least 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% or similar functional properties.

[0156] In one embodiment, the secretory signal peptide system of the expression vector construct translates amino acid exhibited as SYGN013 having SEQ ID NO : 9, SYGN014 having SEQ ID NO: 10, SYGN015 having SEQ ID NO: 11; wherein the secretory signal peptide system of the expression vector construct translates amino acid at least 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97% or 98%, or 99% identical to SEQ ID 9, or SEQ ID 10, and SEQ ID 11.

[0157] In some embodiment, the selection system of the expression vector construct comprises one antibiotic resistance marker or a functional variant and a glutamine synthetase marker or a functional variant.

[0158] In one embodiment, the antibiotic resistance marker of the expression vector construct is selected from but not limited to puromycin, or kanamycin, hygromycin, or geneticin, or neomycin, and combination thereof.

[0159] In one embodiment, the glutamine synthetase marker of the expression vector construct is glutamine synthetase (GS) gene sequence or functional variant.

[0160] In an embodiment, the plasmid DNA vector comprises multiple cloning site, an RNA promoter sequence, a selection system, such as an antibiotic resistance factor (selected from kanamycin, hygromycin, G418 (geneticin), or neomycin), and a sequence suitable for multiplication of the vector, such as an origin of replication. The plasmid DNA vector is the origin of replication for the present expression vector construct. The origin of replication is the microbial origin of replication pBR322 with pMBl origin. In an embodiment, the expression vector construct further comprises one or more cloning sites having a nucleotide sequence encoding a translation initiator codon, a secretory signal peptide system and a gene of interest.

[0161] In an embodiment, the nucleotide sequence of the expression vector construct encoding the translation initiator codon, secretory signal peptide system and gene of interest is selected from SEQ ID No: 2, or SEQ ID No: 3, or SEQ ID No: 4 or any other sequence with at least 90% or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97% or 98%, or 99% or similar functional properties.

[0162] In an embodiment, one or more cloning site of the expression vector construct comprises restriction sites selected from Sbfl, Nhel, Pad, Avril, BamHl, Xbal, Notl, and Spel amongst others. In one embodiment, one or more cloning site of the expression vector construct comprises restriction sites selected form combination of Sbfl and Pad, or Nhel and Avril, or Xbal and Spel, or BamHl and Notl.

[0163] In an embodiment the one or more cloning site comprises first cloning site and the second cloning site In one embodiment, the nucleotide sequences encoding the light chain are incorporated in the first multiple cloning site (MCS-1) and the nucleotide sequences encoding the heavy chain are incorporated in the second multiple cloning site (MCS-2).

[0164] In an embodiment, the first cloning site of the expression vector construct comprises restriction sites for Sbfl or Nhel and Pad or Avril or Sbfl and Pad, or Nhel and Avril.

[0165] In an embodiment, the second cloning site of the expression vector construct comprises restriction sites for BamHl or Xbal and Notl or Spel or Xbal and Spel, or BamHl and Notl.

[0166] In another embodiment, the expression vector construct comprises a Kozak sequence having sequence SEQ ID NO: 12, upstream of the initiation codon.

[0167] In some embodiment, the expression vector construct comprises: a) More than one hybrid promoter or a functional variant thereof; b) Secretary signal peptide system; c) More than one cloning site; d) A coding sequence for Gene / Protein of interest and; e) More than one selection marker.

[0168] In some embodiment, the expression vector construct comprises: a) Two hybrid promoters; b) Secretary signal peptide system; c) Two cloning sites; d) Coding sequence for protein / gene of interest; e) Two selection markers; and f) Other regulatory elements.

[0169] In some embodiment, the expression vector construct comprises: a) CMV / MIA, CMV / EF1, SV40 Promoter or a functional variant thereof, b) Glutamine Selection Marker or a functional variant thereof, c) Antibiotic selection Marker or a functional variant thereof, and d) Polyadenylation Sequence or a functional variant thereof.

[0170] In some embodiment, the expression vector construct comprises: a) MIA / CMV promoter; b) First cloning site and gene of interest; c) SV40 polyadenylation sequence; d) CMV / EF1 promoter; e) Second, cloning site and gene of interest; f) SV40 polyadenylation sequence; g) Puromycin selection marker; h) PBR322 origin of replication; i) Kanamycin selection marker; j) Glutamine Selection Marker k) SV40 polyadenylation sequence.

[0171] In some embodiment, the expression vector construct comprises: a) MIA / CMV promoter; b) First cloning site and gene of interest; c) SV40 polyadenylation sequence; d) Puromycin selection marker; e) PBR322 origin of replication; f) Kanamycin Selection Marker; g) Glutamine Selection Marker h) SV40 polyadenylation sequence. In another embodiment, the expression vector construct comprises: a) hybrid promoter selected from CMV / MIE, or CMVZEF1; b) a first and a second cloning site comprising nucleotide sequence encoding translation initiator codon, signal peptide, and gene of interest; c) a secretory signal peptide system selected from Homo sapiens albumin, or alpha Lactalbumin, or immunoglobulin light chain (partial Homo sapiens),' d) a protein of interest; e) an antibiotic selection marker selected from puromycin, or kanamycin, or hygromycin, or geneticin, or neomycin; f) a glutamine selection marker; g) an origin of replication; h) a simian virus 40 polyadenylation sequence; wherein the protein of interest is depicted as amino acid sequence set forth as SEQ ID No: 1 or any other sequence with at least 90% or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97% or 98%, or 99% or nucleotide sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or any other sequence with at least 90% or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97% or 98%, or 99%; wherein gene of interest is operably linked to signal peptide selected from SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8 or any other sequence with at least 90% or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97% or 98%, or 99%; wherein the expression vector construct expressed in the host cell provides an expression and / or titer of recombinant fusion protein greater than at least 2 gm / L.

[0172] In an embodiment, the expression vector construct comprises: a) a hybrid promoter; b) a signal peptide linked to protein of interest wherein the signal peptide is set forth as SEQ ID NO: 6 or functional variant; c) a coding sequence for protein of interest; d) a termination sequence; wherein the coding sequence of protein of interest is set forth in SEQ ID NO: 2 wherein the protein of interest is recombinant protein comprising the amino acid sequence SEQ ID NO: 1 or functional variant. In an embodiment, the expression vector construct comprises: a) a hybrid promoter; b) a signal peptide linked to protein of interest wherein the signal peptide is set forth as SEQ ID NO:7 or functional variant; c) a coding sequence for protein of interest; d) termination sequence; wherein the coding sequence of protein of interest is set forth in SEQ ID NO: 3 wherein the protein of interest is recombinant protein comprising the amino acid sequence SEQ ID NO: 1 or functional variant.

[0173] In an embodiment, an expression vector construct comprises: a) a hybrid promoter; b) a signal peptide is linked to protein of interest wherein the signal peptide is set forth as SEQ ID NO: 8 or functional variant; c) a coding sequence for protein of interest; d) a termination sequence; wherein the coding sequence of protein of interest is set forth in SEQ ID NON wherein the protein of interest is recombinant protein comprising the amino acid sequence SEQ ID NO: 1 or functional variant.

[0174] In an embodiment, the expression vector construct comprises: a) a hybrid promoter; b) a signal peptide; c) a coding sequence for protein of interest; d) a termination sequence; wherein recombinant protein is selected from antibody and / or fusion protein; wherein the signal peptide nucleotide sequence is selected from SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8 or any other functional variant; wherein the expression vector construct provides a high expression and / or yield of antibody or fusion protein more than 1.5 gm / L, wherein the yield is determined at harvest level through protein A HPLC. In an embodiment, the nucleotide sequence encoding expression vector construct SEQ ID No: 5, wherein the expression vector construct further comprises nucleotide sequence selected from SEQ ID No: 2, or SEQ ID No: 3, or SEQ ID No: 4 , or any other sequence with at least 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97% or 98%, or 99% or similar functional properties functional variant thereof to express the protein of interest.

[0175] In one embodiment, the method for expressing the recombinant fusion protein comprises: a) Providing the recombinant expression vector construct; b) Introducing the recombinant expression vector construct into a microbial host cell; c) Selecting and isolating recombinant expression vector construct from positive clone; d) Isolating the recombinant expression construct form the positive clone; e) Transfecting the recombinant expression construct into mammalian host cells to form a clone capable of expressing the protein of interest; f) Selecting the clone, applying a dual selection system for selection of clone and isolating the clone; g) Culturing the mammalian host cell comprising the expression vector construct, through cell culture methods selected from fed-batch mode, perfusion culture, under optimized culture conditions; h) Harvesting and purifying recombinant fusion protein.

[0176] In one embodiment, the cell culture method is performed through fed- batch mode.

[0177] In one embodiment, the cell culture method is performed through fed- batch mode for at least 16 Days.

[0178] In an embodiment, the mammalian host cell is cultured through fed batch mode for at least 12 Days with a titre of at least 1.5 gm / L. The mammalian host cell is cultured through fed batch mode for at least 16 Days with a titre of at least 2.5 gm / L. In an embodiment the titer is determined at harvest level through protein A HPLC.

[0179] In an embodiment, the cell culture is performed at a temperature of about 31°C, about 32°C, about 33°C, about 34°C, about 35 °C, about 36 °C, and about 37°C. In one embodiment, the positive clone in the microbial host cell is selected applying a suitable glutamine free media at temperature of about 37°C. The cell culture is performed at a temperature of about 34°C.

[0180] In an embodiment, the cell culture maintains first and second pH where the second pH is about two log lower than the first pH. In some embodiment the first pH at about day 0 to day 3 is selected from 7.0 to 7.5 and second pH at day 4 to day 14 is selected from 6.5 to 6.8. In certain embodiment the second pH at day 4 to day 16 is selected from 6.5 to 6.8.

[0181] In an embodiment, the cell culture maintains the cell density at about 30 million cells / mL. In an embodiment, the cell culture maintains the cell density at about 40 million cells / mL to about 50 million cells / mL. In one embodiment, the cell culture has the initial seeding density of about 0.5 million cells / mL, about 1.0 million cells / mL, about 1.5 million cells / mL, about 2.0 million cells / mL cultured through fed-batch mode.

[0182] In an embodiment, the cell culture is subjected to a feed regimen continuously or periodically. In some embodiment, the cell culture is supplemented with a feed medium periodically from Day 3, Day 5, Day 7, Day 9, Day 11, Day 13 and Dayl5. In one embodiment, the cell culture is subjected to a feed regimen periodically, where the glucose concentration of the culture medium is maintained at 4gm / L.

[0183] In an embodiment, the feeding is started on or before day 5 of the culture; on or before day 4 of the culture; on or before day 3 of the culture; on or before day 2 of the culture; on or before day 1 of the culture; preferably on day 3 of the culture.

[0184] In an embodiment, feeding is performed periodically from day 3.

[0185] In an embodiment, feeding is performed by supplementing feed media at about 2% (v / v) to about 200% (v / v) periodically from day 3.

[0186] In an embodiment, feeding is performed by supplementing feed media at about 30% (v / v) to about 200% (v / v) periodically from day 3.

[0187] In an embodiment, feeding is performed by supplementing feed media at about 30% (v / v) on day 3, about 100% (v / v) on day 5, about 120% (v / v) on day 7, about 150% (v / v) on day 9, and about 200% (v / v) from day 11.

[0188] In an embodiment, feeding is performed by supplementing feed media at about 2% (v / v) to about 10% (v / v) periodically from day 3, preferably about 3% (v / v) periodically.

[0189] In an embodiment, feed media can be a mixture of one or more than one cell culture media. In an embodiment, the one or more cell culture media can be mixed before feeding or can be fed separately without mixing. In an embodiment, feeding comprises supplementation of feed media and optionally simultaneous removal of spent media.

[0190] In an embodiment, the cell culture media contains buffers, salts, carbohydrates, amino acids, vitamins, hormones, growth factors, buffers, antibiotics, lipids and trace essential elements various culture media are commercially available and can be used: RPMI- 1640 Medium, RPMI-1641 Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Eagle, F- 12K Medium, Ham's F12 Medium, Iscove's Modified Dulbecco's Medium, CD FortiCHO , CD Forti CHO +1 x HT, McCoy's 5 A Medium, Leibovitz's L-15 Medium, and serum- free media such as EX-CELL™ 300 Series (JRH Biosciences, Lenexa, Kansas).

[0191] In an embodiment, the cell culturing in CHO cells starts from Day 0, Day 1, Day 2, Day 3, Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, Day 11, Day 12, Day 13, Day 14, Day 15 and Day 16. In an embodiment, the culture duration is about 5 Days to 16 Days.

[0192] In an embodiment, the feeding of feed medium is performed such that; glutamine is maintained at about ImM to 6mM, glutamate is maintained at about ImM to 7mM, and osmolality is maintained at about 300 Osm / kg to 470 Osm / kg.

[0193] In one embodiment, the feeding of feed medium is performed such that; glutamine is maintained at about 2.5mM to about 5.5mM, glutamate is maintained at about 2.5mM to about 6mM, and osmolality is maintained at about 350 Osm / kg to 450 Osm / kg. In one embodiment, the feeding of feed medium is performed such that; glutamine is maintained below at about 5.5mM, glutamate is maintained below at about 6mM.

[0194] In some embodiments, the selection pressure is applied using glutamine-free media (like BalanCD CHO Growth A) and antibiotics to isolate a stable, high-expressing clone.

[0195] In some embodiments, the selected clone is subjected to fed batch production in a bioreactor with initial volume of 3.6L and seed density about 1.0 million cells / mL and is grown under tightly controlled conditions for a total batch duration of 16 days. The temperature is shifted from 37 °C to 31 °C, preferably 34 °C and pH is maintained at 7±1.

[0196] In an embodiment, the cell culture method reduces the oxidation impurities by at least 33%. The cell culture method reduces the oxidation impurities between Day 12 and Day 16. The cell viability is maintained at least 85% during the process.

[0197] In an embodiment, the cell culture has a dissolved oxygen controlled at about 50% saturation through a cascaded system employing an air overlay and a variable air sparging rate from about 0.004 vvm to about 0.018 vvm. Agitation is also increased from 250 rpm (0 day) to 350 rpm after day 10 to improve mixing and oxygen transfer.

[0198] In one embodiment, the nutrient levels in the fed-batch condition are sustained through a feed regime, using Cell Boost 7a and 7b along with supplements like LC and managed glucose additions, ensuring a stable environment for high-level protein expression.

[0199] In an embodiment, harvesting is performed at the end of culturing.

[0200] In an embodiment, the harvesting is performed by filtration and / or by centrifugation.

[0201] In an embodiment, filtration and / or centrifugation is performed continuously or in batches.

[0202] In an embodiment, harvesting is performed continuously during culturing and at the end of culturing.

[0203] In an embodiment, continuous harvesting is started on day 14 of culturing.

[0204] In an embodiment, harvesting is performed such that the host cell is retained in the bioreactor.

[0205] In an embodiment, purification is performed by continuous filtration.

[0206] In an embodiment, the continuous filtration is selected from tangential flow filtration and alternating tangential flow filtration.

[0207] In another embodiment, the expression vector construct comprises: a) hybrid promoter selected from CMV / MIE, or CMV / EF1; b) one or more cloning sites comprising nucleotide sequence encoding translation initiator codon, signal peptide, and gene of interest; c) a secretory signal peptide system selected from Homo sapiens albumin, or alpha Lactalbumin, or immunoglobulin light chain (partial Homo sapiens),' d) a protein of interest; e) an antibiotic selection marker selected from puromycin, or kanamycin, or hygromycin, or geneticin, or neomycin; f) a glutamine selection marker; g) an origin of replication; h) a simian virus 40 polyadenylation sequence; wherein the protein of interest is depicted as amino acid sequence set forth as SEQ ID No: 1 or any other sequence with at least 90% or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97% or 98%, or 99% or nucleotide sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or any other sequence with at least 90% or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97% or 98%, or 99%; wherein gene of interest is operably linked to signal peptide selected from SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8 or any other sequence with at least 90% or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97% or 98%, or 99%; wherein the expression vector construct expressed in the mammalian host cell through the cell culture method, provides an expression and / or titer of recombinant fusion protein greater than at least 2.5 gm / L measured by protein A HPLC; wherein the cell culture method is Fed-batch mode.

[0208] In an embodiment, the expression vector construct exhibits the sequence as set forth in SEQ ID No: 5. In some embodiments the expression vector construct comprises a sequence having at least 99%, 98%, 97%, 96%, or 95%, identity to the sequence of SEQ ID NO:5.

[0209] In an embodiment, the method for expressing recombinant fusion protein of interest comprising: i. Providing an expression vector construct having; a. a hybrid promoter or a functional variant; b. a secretory signal peptide system operably linked to recombinant fusion protein of interest; c. a coding sequence for the protein of interest; d. a selection system using antibiotic resistance and glutamine synthetase (GS) markers; e. an origin of replication; and f. a termination sequence comprising an SV40 polyadenylation sequence or a functional variant thereof; ii. transfecting the expression vector construct into E-col , iii. selecting and isolating the expression vector expressed in E-colv, iv. transfecting the isolated expression vector in a host mammalian host cells; v. selecting a clone expressing the high yield or titer with desired quality of protein maintaining suitable culture conditions vi. harvesting and purifying the protein of interest.

[0210] In an embodiment, the protein of interest is cultured or grown in suitable host cells selected from bacteria, mammalian, yeast amongst others. In an embodiment, the microbial host cell is E-coli. In an embodiment, the host cell is mammalian host cell, Chinese hamster ovary (CHO) cell selected from CHO DG44, CHO-S, CHO-K1, or CHO DUKX-B11. In an embodiment, the initiation of cell culture takes place at small scale and extends at larger scale say from shake flask level to 2L bioreactor, to 5L bioreactor, to 20L bioreactor, to 50L bioreactor, to 100L bioreactor, to 500L bioreactor and larger scale bioreactors.

[0211] In certain embodiments, an expression vector construct encoding a nucleotide sequence results in high yield and / or high levels of protein production.

[0212] In an embodiment, wherein the recovered recombinant protein of interest is fusion protein.

[0213] In an embodiment, the fusion protein is Glucagon-like peptide-1 fusion protein. In an embodiment, the fusion protein is Glucagon-like peptide-1 -IgG4 fusion protein In one embodiment the fusion protein is dulaglutide.

[0214] In an embodiment, the Glucagon -like peptide-1 fusion protein exhibits sequence coverage of at least 98% as determined by mass spectrometry.

[0215] In an embodiment, the Glucagon-like peptide-1 fusion protein comprises the amino acid sequence SEQ ID NO: 1 or any other sequence with at least 90% or 92% or 94% or 96% or 98% similar functional properties.

[0216] In certain embodiment the HMW relates to aggregates of dimer, trimer or tetramer of GLP-1 Fc fusion protein. In an embodiment, the Glucagon-like peptide-1 fusion protein composition comprises reduced amount of high molecular weight (HMW) impurities selected from about 15% or below, about 14% or below, about 13% or below, about 12% or below, about 11% or below, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5% or below HMW, determined SEC HPLC.

[0217] In an embodiment, the Glucagon-like peptide-1 fusion protein comprises HMW (%) less than 2 %, less than 1.7 %, less than 1.5 %, less than 1.4 %, less than 1.3 %, less than 1.2 %, less than 1 %, less than 0.5% , less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%.

[0218] In an embodiment, high molecular weight impurities in recombinant fusion protein are less than about 2%, 1.5%, 1%, 0.5%, 0.1%, 0.05%.

[0219] In an embodiment, the glycan profile of recombinant fusion protein comprises afucosylated glycans less than about 2%, and total afucosylated glycans less than about 5%, determined by HILIC UPLC.

[0220] In an embodiment, the glycan profile of recombinant fusion protein comprises total galactosylation less than about 23%, determined by HILIC UPLC. In an embodiment, the glycan profile of recombinant fusion protein comprises total sialylation less than about 2%, determined by HILIC UPLC.

[0221] In an embodiment the expression vector construct provides an expression more than Ig / L, more than 1.5g / L, more than 2g / L, more than 2.2g / L, more than 2.5g / L, more than 3g / L.

[0222] In an embodiment, the protein expression provides a yield of more than 1 gm / L, more than 1.1 gm / L, more than 1.2 gm / L, more than 1.3 gm / L, more than 1.4 gm / L, more than 1.5 gm / L, more than 1.6 gm / L, more than 1.7 gm / L, more than 1.8 gm / L, more than 1.9 gm / L, more than 2 gm / L, more than 2.1 gm / L, more than 2.2 gm / L, more than 2.3 gm / L, more than 2.4 gm / L, more than 2.5 gm / L, more than 2.6 gm / L, more than 2.7 gm / L, more than 2.8 gm / L more than 2.9 gm / L, more than 3 gm / L, more than 3.1 gm / L, more than 3.2 g / L, more than 3.3 gm / L, more than 3.4 gm / L, more than 3.5 gm / L, more than 3.6 gm / L, more than 3.7 gm / L, more than 3.8 gm / L, more than 3.9 gm / L, more than 4 gm / L, more than 4.1 gm / L, more than 4.2 gm / L, more than 4.3 gm / L, more than 4.4 gm / L, more than 4.5 gm / L, more than 4.6 gm / L, more than 4.7 gm / L, more than 4.8 gm / L, more than 4.9 gm / L, more than 5 gm / L.

[0223] The term “Low molecular weight” or “LMWs” used herein refers to species which is a protein backbone-truncated fragments & considered as product-related impurities that contribute to the size heterogeneity of fusion protein or other biotherapeutic products. LMW species often have low or substantially reduced activity relative to the monomeric form of the fusion protein and can lead to immunogenicity or potentially impact pharmacokinetic properties in vivo. In an embodiment, the Glucagon-like peptide- 1 fusion protein composition comprises reduced amount of low molecular weight (LMW) impurities selected from about 15% or below, about 14% or below, about 13% or below, about 12% or below, about 11% or below, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5% or below LMW.

[0224] The term “substantially pure fusion protein” used herein includes an GLP-1 Fc fusion protein / dulaglutide that is substantially free of impurity selected from product or process related impurity. In certain embodiment fusion protein is free of acidic variant, basic variant, low molecular weight and high molecular weight impurities or aggregates, substantially pure fusion protein has purity more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%.

[0225] The term “Acceptable range of glycans” or “Target value of glycans” used herein refers to a value of one or more glycans selected from fucosylated glycans, galactosylated glycans, high mannose glycans, and sialylated glycans is in acceptable range or fall within the range of reference value. In an embodiment, the reference value is obtained from approved therapeutic glycoprotein with about 95% to 100% identical amino acids. The reference value expressed in percentages (%). As an example, the acceptable range of glycan from about 0.22% to about 0.71%

[0226] In an embodiment, an acceptable value or target value is a level of one or more particular glycans, such as high mannose glycans (e.g., HM3, HM4, HM5, HM6, HM7, HM8, HM9, or combinations), fucosylated glycans (e.g., GOF, GIF, G2F, or combinations), galactosylated glycans (e.g., Gl, G2, GIF, G2F, Al, A2 or combinations) and / or sialylated glycans (e.g., monosialylated, disialylated, or combinations), in a reference glycoprotein product or described in a specification or master batch record for a pharmaceutical product.

[0227] In one embodiment, a composition comprising dulaglutide and one or more basic variants wherein the composition comprises one or more basic variant selected from below 20% or less as measured by anion exchange high performance chromatography analysis; wherein the composition has dulaglutide purity more than 80%; wherein the basic variants are measured in the composition obtained from neutralized protein A step.

[0228] In one embodiment, a composition comprising dulaglutide and one or more basic variants wherein the composition comprising one or more basic variants selected from below 18% or less, about 15% or less, about 12% or less, about 9% or less, about 7% or less, about 5% or less, as measured by anion exchange high performance liquid chromatography analysis; wherein the composition has dulaglutide purity more than 80%, more than about 83%, about 85%, more than about 90%. more than about 95% and more than 98%.

[0229] In one embodiment, a composition comprising dulaglutide and one or more acidic variants wherein the composition comprising one or more acidic variants selected from below 25% or less as measured by anion exchange high performance liquid chromatography analysis; wherein the composition has dulaglutide purity more than 80%; wherein the acidic variants are measured in the composition obtained from neutralized protein A step.

[0230] In one embodiment, a composition comprising dulaglutide and one or more acidic variants wherein the composition comprising one or more acidic variants selected from below 18% or less, about 15% or less, about 12% or less, about 9% or less, about 7% or less, about 5% or less, as measured by anion exchange high performance liquid chromatography analysis; wherein the composition has dulaglutide purity more than 80%, more than about 83%, about 85%, more than about 90%. more than about 95% and more than 98%; wherein the acidic variants are measured in the composition obtained from neutralized protein A step. In one embodiment, a composition comprising dulaglutide and one or more HMWs wherein the composition comprising one or more HMWs selected from below 2% or less, about 1.5% or less, about 1% or less, about 0.5% or less, about 0.4% or less, and about 0.2% as measured by SEC- HPLC; wherein the composition has dulaglutide purity more than 80%, more than about 83%, about 85%, more than about 90%. more than about 95% and more than 98%; wherein the HMWs are measured in the composition obtained from neutralized protein A step.

[0231] In one embodiment, a composition comprising dulaglutide and one or more LMWs wherein the composition comprising one or more LMWs selected from below 15% or less, about 12% or less, about 10% or less, about 7% or less, about 5% or less, about 3% or less, about 1% or less, about 0.8% or less, about 0.5% or less, about 0.4% or less, and about 0.2% as measured by SEC-HPLC; wherein the composition has dulaglutide purity more than 80%, more than about 83%, about 85%, more than about 90%. more than about 95% and more than 98%; wherein the LMWs are measured in the composition obtained from neutralized protein A step.

[0232] In one embodiment, a composition comprising dulaglutide and one or more glycan wherein the composition comprising one or more afucosylated glycans selected from below about 3% or less, about 2% or less, about 1% or less, about 0.8% or less, about 0.5% or less, about 0.4% or less, and about 0.2% as measured by HILIC UPLC; one or more galactosylation less than about 23%, less than about 21%, less than about 19%, less than about 17%, less than about 15%, less than about 13%, less than about 11%, less than about 9% less than about 7% less than about 5% less than about 3%, less than about 1%; wherein the composition sialylation less than about 2% about 1% or less, about 0.8% or less, about 0.5% or less, about 0.4% or less, and about 0.2% as measured by HILIC UPLC; wherein the composition has dulaglutide purity more than 80%, more than about 83%, about 85%, more than about 90%. more than about 95% and more than 98%.

[0233] In one embodiment, a composition comprising dulaglutide and one or more reduced oxidation impurities wherein the composition comprising one or more reduced oxidation impurities selected from below 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 1% or less, about 0.8% or less, about 0.5% or less, about 0.4% or less, and about 0.2% as measured by RP-HPLC; wherein the composition has dulaglutide purity more than 80%, more than about 83%, about 85%, more than about 90%. more than about 95% and more than 98%; wherein the oxidation impurities are measured in the composition obtained from neutralized protein A step.

[0234] In another embodiment, the method to produce dulaglutide comprises steps of: i. providing an expression vector construct having: a. a MIA / CMV promoter; b. a cloning site encoding translation initiator codon, a secretory signal peptide system and a gene of interest; c. a SV40 polyadenylation sequence; d. a CMVZEF 1 promoter; e. one or more cloning site encoding translation initiator codon, a secretory signal peptide system and a gene of interest; f. a SV40 polyadenylation sequence; g. a Puromycin selection marker; h. a PBR322 origin of replication; i. a Kanamycin selection marker; j . a Glutamine Selection Marker; k. a SV40 polyadenylation sequence; ii. introducing the recombinant expression vector construct into a microbial host cell; iii. selecting and isolating recombinant expression vector construct from positive clone; iv. isolating the recombinant expression construct form microbial host cell; v. transfecting the recombinant expression construct into a mammalian host cell to form a clone capable of expressing dulaglutide; vi. selecting the clone, applying dual selection system for selection of clone and isolating the clone; vii. culturing the mammalian host cell comprising the expression vector construct, through fed batch mode; viii. harvesting and purifying dulaglutide; wherein dulaglutide has purity more than 95% and reduced amount of high molecular weight (BMW) impurities of about 2% or below.

[0235] In another embodiment, a composition of GLPl-IgG4 fusion protein is disclosed. The GLPl-IgG4 fusion protein is dulaglutide as set forth in SEQ ID NO 1. The composition comprises a mixture of GLPl-IgG4 fusion protein of interest: a. a GLPl-IgG4 fusion protein having an amino acid sequence as set forth in SEQ ID NO: 1; and b. a GLPl-IgG4 fusion protein having an amino acid sequence as set forth in SEQ ID NO:13, wherein SEQ ID NO 1 is predominantly higher than SEQ ID NO 13.

[0236] In one embodiment of the composition, the SEQ ID NO 13 is present less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.4%, less than about 0.3%, and less than about 0.2%. The composition of SEQ ID NO 13 lacks at least two amino acids at N terminal in comparison to SEQ ID NO 1, where the two amino acids are in order histidine and glycine.

[0237] In one embodiment, the composition of GLPl-IgG4 fusion protein, dulaglutide comprises: a. a GLPl-IgG4 fusion protein having an amino acid sequence as set forth in SEQ ID NO:1; and b. a GLPl-IgG4 fusion protein having an amino acid sequence as set forth in SEQ ID NO: 14, wherein SEQ ID NO 1 is predominantly higher than SEQ ID NO 14.

[0238] In one embodiment of the composition, the SEQ ID NO 14 is present less than 6% less than 5%, less than 4%, less than 3%, less than 2%, less than about 1%, less than about 0.5%, less than about 0.4%, less than about 0.3%, and less than about 0.2%. The composition of SEQ ID NO 14 lacks at least four amino acids at N terminal in comparison to SEQ ID NO 1, where the four amino acids are in order histidine, glycine, glutamic acid, and glycine.

[0239] In some embodiment, the GLPl-IgG4 fusion protein exhibit a sequence coverage of at least 98% as determined by mass spectrometry.

[0240] In one embodiment, a composition comprising dulaglutide, one or more reduced oxidation impurities, one or more truncated sequences selected from N-2, N-4; wherein the composition comprising one or more reduced oxidation impurities selected from below 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 1% or less, about 0.8% or less, about 0.5% or less, about 0.4% or less, and about 0.2% as measured by RP-HPLC; wherein the composition has dulaglutide purity more than 80%, more than about 83%, about 85%, more than about 90%. more than about 95% and more than 98%; wherein the oxidation impurities are measured in the composition obtained from neutralized protein A step. In another embodiment, the expressed protein of interest is for the treatment of metabolic disorder selected from type-2 diabetes, or obesity, or metabolic syndrome.

[0241] Example 1: Designing and Chemical Synthesis of Mammalian Expression Vector & Gene of Interest (GOI)

[0242] The constructs have been designed to get the expression of target protein in high amount. The designing for the vector & insert is as follows: a) The amino acid sequence of dulaglutide (SEQ ID NO: 1) was obtained from US7452966B2 and subsequently reverse-translated into a nucleotide sequence, which was codon- optimized according to CHO amino acid codon preferences. By incorporating a Kozak Sequence, a secretory signal sequence, stop codons and restriction consequences, the complete gene sequence of interest is selected from SEQ ID NO: 2 SYGN013 (MP2), SEQ ID NO: 3 SYGN014 (MP48) & SEQ ID NO: 4 SYGN015 (MP60) were designed and chemically synthesized. b) The sequences of signal sequence and gene of interest are illustrated in Figure 1, which were utilized for cloning in mammalian expression vector pKSB2.0 (~11.28 bp). c) The pKSB2.0 vector comprises the elements listed in Table 1 and Figure 1 and sequence (SEQ ID NO: 5) in Figure 2.

[0243] Table 1: Elements of Mammalian Expression Vector pKSB2.0 Example 2: Designing and Chemical Synthesis of Mammalian Expression Vector & Gene of Interest (GOI) a) The chemically synthesized GOI (SYGN013, 14 and 15) was transformed into E.coli DH5a and isolated the single colony for inoculation followed by plasmid DNA isolation. b) The isolated plasmid DNA of SYGN013 was digested with cloning enzyme Sbfl - PacI and checked release of GOI (~ 825 bp) on 0.8 % agarose gel. Similarly, mammalian expression vector (pKSB2.0) was digested with Sbfl-PacI and the desired fragment of approx.11.28 kb was excised out from gel. In case of gene of interest SYGN014 & SYGN015, the cloning sites (Sbfl-PacI) was incorporated by PCR using primers, performed to create the related sites of restriction digestion followed by restriction digestion using restriction enzymes Sbfl-PacI. Specially 40 cycles of PCR amplification were carried out using 100 picomoles of gene specific oligonucleotide primers in a volume of 50 pL containing 2 x Fusion master mix. Each PCR amplification cycle consisted of incubation at 98 °C, for 30 sec (denaturation), 70 °C. for 45 sec (annealing) and 70 °C for 1 min (extension). Amplified product of the PCR reaction was resolved on a 1% agarose gel. The desired fragment of approx. 825 base pairs size was excised out from the gel and purified using Qiagen Gel Extraction Kit. The purified DNA fragments was ligated into pKSHB2.0. The composition of digestion reaction is mentioned in Table 2 & 3.

[0244] Table 2: Digestion of pKSB2.0

[0245] Table 3: Digestion of SYGN013, SYGN014 & 15 c) Based on the concentration & size of DNA, the molar ratio (1 :3) was calculated & set up ligation reaction as mentioned in Table 4.

[0246] Table-4: Ligation reaction d) The ligated product was transformed into E. coli DH5a host and transformants were scored on the basis of kanamycin resistance. e) Plasmid DNA isolated from few such colonies was analysed for the presence of dulaglutide gene by restriction digestion. Initial screening was done with cloning enzyme Sbfl- PacI and confirmed the release obligated products. f) Further, the positive clones of pKSHB2.0_SYGN013 (CL#1 & CL#2), pKSHB2.0_SYGN014 (CL#2) & pKSHB2.0_SYGN015 (CL#1) were characterized with additional enzymes Sbfl-Xbal, NdeLPacI & Ncol and confirmed the release of expected DNA bands as shown in Figure-3 and 4. g) DNA from positive clone was used for linearization using restriction enzyme Nrul to cut DNA at unique linearization site followed by ethanol precipitation before transfection.

[0247] Example 3: Cloning of Gene of interest in mammalian expression vector pKSB 2.0 and Shortlisting of Mini pools for Single Cell Cloning a) The chemically synthesized GOI (SYGN013, 14 and 15) was transformed into E.coli DH5a and isolated the single colony for inoculation followed by plasmid DNA isolation. b) The positive clones of pKSHB2.0_SYGN013, pKSHB2.0_SYGN014 & pKSHB2.0_SYGN015 were characterized and confirmed the release of expected DNA bands. c) The cell pellet containing cells, resuspended in buffer and mixed with linearized plasmid DNA, creating a suspension for the electroporation. d) Post transfections, the transfected cells were expanded at T-flask scale for antibiotic selection & amplification. e) The data from the sequential amplification of minipools (heterogenous transfectants) was assessed through an analysis of growth profile and protein content analysis using batch mode study. f) The batch mode study was executed at 6-well plate, a significant increment in productivity was observed at different amplification stage. g) Based on the growth profile and productivity data of 60 clones, 3 minipools (MP2, MP48 and MP60) were selected.

[0248] Example 4: Single Cell Cloning and Clone Evaluation

[0249] 1) Single cell cloning:

[0250] Cells from MP2, MP48 and MP60 were diluted in semisolid media enriched with additives(such as glucose) and subsequently dispensed into a 6-well plate, with each well containing 2 mL semisolid media and 200 cells. Same day (day 0), the semisolid plates were scanned using the advanced instrument ClonePix to collect the images of single cell location. Following this, the scanned plates were incubated at 37 °C in a 5 % CO2 incubator for over 14 days to facilitate colony formation. After the incubation period, the grown colonies were scanned, and those exhibiting high intensity were selected based on criteria such as exterior median intensity, colony size, and proximity. The selected high-intensity colonies were then picked and transferred to 96-well plate containing CD Forti CHO +1 x HT media. These plates were incubated at 37 °C with 5% CO2 to promote cell growth, with media changes occurring at 72 hrs intervals. Once the confluency in the wells exceeded 50%, the cells were transferred to 24-well plate, followed by 6-well plates. The confluency of the 6- well plate was monitored, leading to a transfer to T-25 flasks. Cell density in the T-flasks was assessed using cell counting techniques, and a batch mode study was established for screening of clones.

[0251] 2) Clone Screening a) Screening at 6-well plate level (Batch Mode Study)

[0252] The study was conducted using a 6-well plate over a duration of five days. The 6-well plate was set up for a batch mode study with seeding density 0.3 x 106cells per mL. A total of 0.9 x 106cells in 3 mL CD Forti CHO media supplemented with lx HT was maintained in each well for 5 days, at a temperature 37 °C, in a 5 % CO2 incubator. Following the incubation period, cell density & percent viability were assessed, and the supernatant from the sample was utilized for protein content analysis by protein A technique. The batch mode study using single cell clones derived from all three minipools (MP2, MP48 & MP60) and the resulting data were analysed to identify high expressing single cell clones for subsequent expression study at 125 mL shake flask level. More than 70 single cell clones exhibited protein expression level exceeding 150 mg / L within the 5 days batch study in the 6-well plate. b) Clone screening at shake flask level

[0253] Following the analysis of the growth profile & protein yield of clones in the batch study, a total of 19 clones (MP48 / 18 / A2 / 42, MP48 / 18 / A3 / 84, MP48 / 17 / A2 / 4, MP60 / 19 / A1 / 1, MP60 / 20 / A3 / 113,MP60 / 20 / Bl / 169,MP2 / 29 / B3 / 133,MP2 / 29 / B2 / 126,MP2 / 29 / B3 / 140, MP2 / 30 / B3 / 129, MP48 / 18 / A3 / 70, MP48 / 18 / A3 / 79, MP48 / 18 / A2 / 36, MP48 / 18 / A2 / 32, MP60 / 20 / B2 / 214, MP2 / 29 / A3 / 60, MP2 / 30 / B3 / 138, MP2 / 29 / B2 / 117,MP2 / 29 / B2 / 124) have been selected for additional assessment in a fed-batch study conducted at a scale of 125 mL using shake flasks. a) A fed-batch mode expression study was performed using a 125 mL shake flask containing 30 mL of media, a feed supplement was introduced into the flask and physical parameters such as temperature, CO2 concentration and glucose concentration were maintained during the entire process. b) During the handling of shake flasks, the viable cell count, viability, residual glucose, and metabolite profiles, including lactate and ammonia accumulation, were assessed using the advanced Cedex Analyser. Some clones achieved a viable cell density exceeding 30 x 10A6 cells per mL by day 9th, which was sustained until day 13th. Residual glucose levels were maintained below 3 g / L from day 5 through the conclusion of the 14-days process. Notably, clone MP2 / 29 / B2 / 124 demonstrated significant glucose consumption between days 5thand 11thc) On the 14thday, the culture from the flask was subjected to centrifugation to collect the cell pellet, while the supernatant was utilized for protein content analysis employing Protein A analytical tools. Among the 19 clones, nine exhibited protein expression levels exceeding 2 g / L on the 14thday. Further, the clones were evaluated using the amber 250 and 5-L bioreactor systems, resulting in the collection of data pertaining growth profiles, productivity, and protein quality. From the findings obtained with the amber 250, two specific clones, and the data of growth profile, productivity and protein quality were generated. Based on the amber 250 data, two clones MP2 / 29 / B3 / 140, MP48 / 18 / A3 / 70 and MP60 / 20 / B1 / 169, were selected for further evaluation in the 5-L bioreactor batch. 3) Clone Evaluation At 5-L Bioreactor Scale

[0254] The evaluation of both clones, MP2 / 29 / B3 / 140, MP48 / 18 / A3 / 70 and MP60 / 20 / B 1 / 169, was conducted in a 5-L bioreactor to assess their growth profiles, productivity, and protein quality. Clone MP48 / 18 / A3 / 70 exhibited a high viable cell density in the later days of the process, maintaining viability above 90% until the 10th day. Regarding productivity, the clones demonstrated comparable protein content throughout the 14-day duration of the experiment. The assessment of protein quality was conducted by evaluating factors such as monomer, high molecular weight impurities, and terminal sialylation.

[0255] The cel culture harvest is further purified by filtration and affinity chromatography, virus inactivation & neutralization thereby obtaining neutralized protein A eluate, said method of filtration and affinity chromatography, virus inactivation & neutralization are well known in the art.

[0256] Table 5: Protein Quality profile of clones MP2 / 29 / B3 / 140, MP48 / 18 / A3 / 70 and MP60 / 20 / B1 / 169 (NPEL)

[0257] The composition of the fusion protein has specific modifications to enhance the performance and properties of the selected clone or composition, identified as MP48 / 18 / A3 / 70. As detailed in Table 6, the modification involves clipping the HG amino acid at an N-2 position set forth as sequence SEQ ID 13 eliminating histidine and glycine form N terminal of SEQ ID 1 and the HGEG amino group at an N-4 position set forth as SEQ ID 14 eliminating histidine, glycine, glutamic acid, glycine from N terminal of SEQ ID 1, where the clones are obtained from NPEL level. Whereas the truncation for clones MP2 / 29 / B3 / 140, MP48 / 18 / A3 / 70 was identified to be 7.4%

[0258] Table 6 Truncation Modification In certain embodiment, the oxidation impurities in the fusion protein were analysed by RP-HPLC and found to be reduced between Day 12 to Day 16. The oxidation impurities were reduced at least by 30%, 33%, 35%; as shown in table 7.

[0259] Table 7 Oxidation Impurities

[0260] CONCLUSION:

[0261] The development of the cell line to produce Glucagon-like peptide- l / IgG4 fusion protein commenced with the design of genes that included three distinct secretory signals along with the Kozak sequence, which were subsequently cloned into the pKSHB2.0 vector. The CHOK1 (GS- knockout) host cell line underwent transfection, resulting in protein expression levels surpassing 50 PCD in minipools during batch mode experiments. A comprehensive analysis of the protein productivity data led to the selection of minipools for further expression studies in shake flasks. The shake flask experiment was conducted over a period of 14 days, and the harvested samples were utilized for purification and characterization at the minipool stage, which exhibited a heterogeneous population. The analysis identified minipools MP2, MP48, and MP60 for limiting dilution, employing the advanced ClonePix technology. Following initial screenings in 96, 24, and subsequently 6-well plates, a total of 19 clones were evaluated in shake flasks and amber 250. Clones MP2 / 29 / B3 / 140, MP48 / 18 / A3 / 70 and MP60 / 20 / B1 / 169, were selected for testing in 5-L bioreactor to assess their performance on a larger scale. Based on various criteria including growth profiles, productivity, and protein quality of the clones at different stages, MP48 / 18 / A3 / 70 is selected as the final clone for large scale production of Glucagon like peptide -1 fusion protein with desired quality, as the truncation for Clones MP2 / 29 / B3 / 140, MP48 / 18 / A3 / 70 was found to be higher about 7.4% compared to truncation for clone MP48 / 18 / A3 / 70.

[0262] Example 5

[0263] The cell bank vial of the final selected clone is thawed in shake flask 125 (SF125) into the seed media and cell counts are checked. The incubation is continued for 3-5 days during which cells reach the optimum cell density for subculture, cells are subsequently expanded to shake flask 500 (SF500) followed by shake flask 2000 (SF2000) / shake flask 3000 (SF3000) to generate sufficient inoculum for 5L bioreactors. Production perfusion batch bioreactor run is executed at 5L bioreactor using process parameters as mentioned below. The overall batch cycle is kept 30 days and permeate is continuously collected for further DSP purification.

Claims

Claims:

1. A method for expressing a recombinant fusion protein of interest comprising: i. providing a suitable expression vector construct comprising; a. a hybrid promoter or a functional variant; b. a secretory signal peptide system operably linked to a recombinant fusion protein of interest; c. a gene sequence encoding the protein of interest; d. a dual selection system having antibiotic resistance and glutamine synthetase (GS) markers; e. an origin of replication; and f. a termination sequence comprising an SV40 polyadenylation sequence or a functional variant thereof; ii. transfecting the expression vector construct into a mammalian host cell to form a clone capable of expressing the protein of interest; iii. culturing the mammalian host cell comprising the expression vector construct, through cell culture methods selected from fed-batch mode, perfusion culture for at least more than 12 Days, to up to 16 Days in a suitable medium; wherein the cell culture method has first pH and second pH, wherein the second pH is at least two log lower than first pH; wherein the cell culture method maintains a temperature selected from about 32°C to about 36 °C; wherein the culture method maintains osmolality at about 300 Osm / kg to 470 Osm / kg; wherein the cell culture method maintains residual glucose less than 5gm / L; wherein the cell culture method maintains glutamine ImM to 6mM; wherein the cell culture method maintains glutamate ImM to 7mM; wherein the cell culture method maintains cell viability at least 40 million cells / ml to 50 million cells / ml; wherein the cell culture method reduces the oxidation rate to form a oxidized species of the protein of interest by at least 30% from Day 10 till harvest; iv. harvesting and purifying the recombinant fusion protein; wherein the expression vector construct directs the expression of recombinant fusion protein of interest in the mammalian host cell at yield of at least or more than 1 gm / L, to 3 gm / L;wherein the harvested recombinant fusion protein of interest is GLPl-IgG4 fusion protein comprising at least less than about 25% basic variants, at least less than about 30% acidic variants, less than about 2% high molecular weight aggregates, and predominantly higher monomer of GLPl-IgG4 fusion protein.

2. The method of claim 1, wherein the expression vector construct is transfected into a microbial host cell; selecting and isolating expression vector construct from the microbial host cell.

3. The method of claim 1 , wherein mammalian host cell capable to express protein of interest is selected and isolated by applying a dual selection system.

4. The method of claim 1, wherein the mammalian host cell is cultured through fed batch mode for at least 12 Days with a titre of at least 1.5 gm / L.

5. The method of claim 1, wherein the mammalian host cell is cultured through fed batch mode for at least 16 Days with a titre of at least 2.5 gm / L.

6. The method of claim 1, wherein the cell culture is performed at a temperature of about 31°C, about 32°C, about 33°C, about 34°C, about 35 °C, about 36 °C, and about 37°C.

7. The method of claim 1, wherein the cell culture is performed at a temperature of about 34°C.

8. The method of claim 1, wherein the cell culture maintains first pH is selected from 7.0 to 7.5.

9. The method of claim 1, wherein the cell culture maintains second pH is selected from 6.5 to 6.8.

10. The method of claim 1, wherein the cell culture maintains the cell density at least about 45 million cells / mL.

11. The method of claim 1, wherein the cell culture is subjected to a feed regimen continuously or periodically.

12. The method of claim 1, wherein the cell culture method maintains glucose concentration at least 4gm / L.

13. The method of claim 1, wherein the cell culture is supplemented with a feed medium periodically from Day 3, Day 5, Day 7, Day 9, Day 11, and Day 13.

14. The method of claim 1, wherein feeding is performed by supplementing feed media at about 30% (v / v) to about 200% (v / v) periodically from day 3.

15. The method of claim 1, wherein feeding is performed by supplementing feed media at about 30% (v / v) on day 3, about 100% (v / v) on day 5, about 120% (v / v) on day 7, about 150% (v / v) on day 9, and about 200% (v / v) from day 11.

16. The method of claim 1, wherein the feeding of feed medium is performed such that: glutamine is maintained below at about 5.5mM; and glutamate is maintained below at about 6mM.

17. The method of claim 1, wherein the cell culture has a dissolved oxygen controlled at about 50% saturation through a cascaded system employing an air overlay and a variable air sparging rate from about 0.004 vvm to about 0.018 vvm.

18. The method of claim 1, wherein the cell culture method reduces the oxidation impurities by at least 33%.

19. The method of claim 1, wherein the cell culture method reduces the oxidation impurities between Day 12 and Day 16.

20. The method of claim 1, wherein the cell viability is maintained at least 85% during the process.

21. The method of claim 1 , wherein basic variants is about 20% or below, 18% or below, 16% or below; 14% or below; 12% or below; 10% or below; 8% or below.

22. The method of claim 1, wherein acidic variants is about 24% or below, 22% or below, 20% or below, 18% or below, 16% or below; 14% or below; 12% or below; 10% or below; 8% or below.

23. The method of claim 1, wherein the purification comprises the techniques selected from filtration, affinity chromatography, and virus inactivation and neutralization.

24. The method of claim 1, wherein the hybrid promoter of the expression vector construct is selected from simian vacuolating virus 40 (SV40), cytomegalovirus (CMV), cytomegalovirus major immediate early enhancer (CMV / MIE), CMV- MIA (melanoma inhibitory activity promoter), muscle-specific creatine kinase (MIA) promoter, elongation factor (EF)-l, lactase LAC4, pPolh, trp, Z.PL, A0X1, GALI, GAL 10, nmtl, nmt42, nmt81, glyceraldehyde- 3 -phosphate dehydrogenase (GAP), and combinations thereof.

25. The method of claim 24, wherein the hybrid promoter of the expression vector construct is CMV / MIE hybrid promoter or CMVZEF1 hybrid promoter or SV40 promoter.

26. The method of claim 1, wherein the secretory signal peptide sequence of the expression vector construct is selected from Homo sapiens albumin, or alpha Lactalbumin, and immunoglobulin light chain (partial Homo sapiens).

27. The method of claim 1, wherein the secretory signal peptide system of the expression vector construct comprises amino acid sequence selected from SEQ ID No: 6, or SEQ ID No: 7, or SEQ ID No: 8; wherein the secretory signal peptide system of the expression vector construct comprises amino acid sequence at least 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97% or 98%, or 99% identical to a sequence selected from SEQ ID No: 6, or SEQ ID No: 7, or SEQ ID No: 8.

28. The method of claim 1, wherein the secretory signal peptide system of the expression vector construct translates amino acid exhibited as SYGN013 having SEQ ID NO : 9, SYGN014 SEQ ID NO: 10, SYGN015 SEQ ID NO: 11; wherein the secretory signal peptide system of the expression vector construct translates amino acid at least 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97% or 98%, or 99% identical to SEQ ID 9, or SEQ ID 10, and SEQ ID 11.

29. The method of claim 1, wherein the selection system comprises at least two antibiotic resistance marker and a glutamine synthetase marker; wherein the antibiotic resistance marker of the expression vector construct is selected from puromycin, or kanamycin, or hygromycin, or geneticin, or neomycin, and combination thereof; wherein the glutamine synthetase marker of the expression vector construct is glutamine synthetase (GS) gene sequence; wherein the origin of replication of the expression vector construct is microbial origin of replication pBR322 with pMBl origin; wherein the expression vector constructfurther comprises one or more cloning sites having a nucleotide sequence encoding a translation initiator codon, a secretory signal peptide system and a gene of interest; wherein the cloning site of the expression vector construct comprises restriction sites selected from Sbfl, Nhel, Pad and Avril; or wherein the cloning site of the expression vector construct comprises restriction sites Sbfl and Pad, Nhel and Avril; wherein the cloning site of the expression vector construct comprises restriction sites for BamHl, Xbal, Notl and Spel; or wherein the cloning site of the expression vector construct comprises restriction sites Xbal and Spel; wherein the expression vector construct further comprises a Kozak sequence having sequence SEQ ID NO: 12; or wherein the expression vector construct further comprises a Kozak sequence having sequence SEQ ID NO: 12 positioned upstream of the initiation codon.

30. The method of claim 1, wherein the expression vector construct exhibits the sequence as set forth in SEQ ID No: 5.

31. The method of claim 30, wherein the expression vector construct comprises a sequence having at least 99%, 98%, 97%, 96%, 95% identity to the sequence of SEQ ID NO:5.

32. The method of claim 1, wherein the microbial host cell is E.coli.

33. The method of claim 1, wherein the mammalian host cell is Chinese hamster ovary (CHO) cell selected from CHO DG44, CHO-S, CHO-K1, or CHO DUKX-B11.

34. The method of claim 1, wherein high molecular weight impurities of GLPl-IgG4 fusion protein are less than 2 %, less than 1.7 %, less than 1.5 %, less than 1.4 %, less than 1.3 %, less than 1.2 %, less than 1 %, less than 0.5% , less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%.

35. The method of claim 1, wherein the purity of GLPl-IgG4 fusion protein is more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%.

36. The method of claim 1, wherein the GLPl-IgG4 fusion protein is dulaglutide.

37. The method of claim 1, wherein the method to produce dulaglutide comprises steps of: i. providing an expression vector construct comprising: a. a MIA / CMV promoter;b. a cloning site encoding translation initiator codon, a secretory signal peptide system and a gene of interest; c. a SV40 polyadenylation sequence; d. a CMVZEF 1 promoter; e. one or more cloning site encoding translation initiator codon, a secretory signal peptide system and a gene of interest; f. a SV40 polyadenylation sequence; g. a Puromycin selection marker; h. a PBR322 origin of replication; i. a Kanamycin selection marker; j . a Glutamine Selection Marker; k. a SV40 polyadenylation sequence; ii. transfecting the recombinant expression vector construct into a microbial host cell; iii. culturing the mammalian host cell comprising the expression vector construct, through fed batch mode; iv. harvesting and purifying dulaglutide; wherein dulaglutide has purity more than 95% and reduced amount of high molecular weight (BMW) impurities of about 2% or below.

38. A composition comprising a mixture of GLPl-IgG4 fusion protein of interest, the composition comprising: a. a GLPl-IgG4 fusion protein having an amino acid sequence as set forth in SEQ ID NO: 1; and b. a GLPl-IgG4 fusion protein having an amino acid sequence as set forth in SEQ ID NO:13, wherein SEQ ID NO 1 is predominantly higher than SEQ ID NO 13; wherein the SEQ ID NO 1 is dulaglutide.

39. The composition according to claim 38, wherein the SEQ ID NO 13 is present less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%.

40. The composition according to claim 38, wherein the SEQ ID NO 13 is present less than 0.5%.

41. The composition according to claim 38, wherein the SEQ ID NO 13 is present less than 0.4%.

42. The composition according to claim 38, wherein the SEQ ID NO 13 is present less than 0.3%.

43. The composition according to claim 38, wherein the SEQ ID NO 13 is present less than 0.2%.

44. The composition according to claim 38, wherein the SEQ ID NO 13 lacks at least two amino acids at N terminal in comparison to SEQ ID NO 1; wherein the two amino acids are in order histidine and glycine.

45. A composition comprising a mixture of GLPl-IgG4 fusion protein of interest, the composition comprising: a. a GLPl-IgG4 fusion protein having an amino acid sequence as set forth in SEQ ID NO: 1; and b. a GLPl-IgG4 fusion protein having an amino acid sequence as set forth in SEQ ID NO: 14, wherein SEQ ID NO 1 is predominantly higher than SEQ ID NO 14; wherein the SEQ ID NO 1 is dulaglutide.

46. The composition according to claim 45, wherein the SEQ ID NO 14 is present less than 6% less than 5%, less than 4%, less than 3%, less than 2%, less than 1%.

47. The composition according to claim 45, wherein the SEQ ID NO 14 is present less than 0.5%.

48. The composition according to claim 45, wherein the SEQ ID NO 14 is present less than 0.4%.

49. The composition according to claim 45, wherein the SEQ ID NO 14 is present less than 0.3%.

50. The composition according to claim 45, wherein the SEQ ID NO 14 is present less than 0.2%.

51. The composition according to claim 45, wherein the SEQ ID NO 14 lacks at least four amino acids at N terminal in comparison to SEQ ID NO 1; wherein the four amino acids are in an order histidine, glycine, glutamic acid, and glycine.

52. A composition comprising dulaglutide and one or more basic variants wherein the composition comprises one or more basic variant selected from below 20% or less as measured by anion exchange high performance chromatography analysis; wherein the composition has dulaglutide purity more than 80%; wherein the basic variants are measured in the composition obtained from neutralized protein A step.

53. A composition comprising dulaglutide and one or more basic variants wherein the composition comprising one or more basic variants selected from below 18% or less, about 15% or less, about 12% or less, about 9% or less, about 7% or less, about 5% or less, as measured by anion exchange high performance liquid chromatography analysis; wherein the composition has dulaglutide purity more than 80%, more than about 83%, about 85%, more than about 90%. more than about 95% and more than 98%; wherein the basic variants are measured in the composition obtained from neutralized protein A step.

54. A composition comprising dulaglutide and one or more acidic variants wherein the composition comprising one or more acidic variants selected from below 25% or less as measured by anion exchange high performance liquid chromatography analysis; wherein the composition has dulaglutide purity more than 80%; wherein the acidic variants are measured in the composition obtained from neutralized protein A step.

55. A composition comprising dulaglutide and one or more acidic variants wherein the composition comprising one or more acidic variants selected from below 18% or less, about 15% or less, about 12% or less, about 9% or less, about 7% or less, about 5% or less, as measured by anion exchange high performance liquid chromatography analysis; wherein the composition has dulaglutide purity more than 80%, more than about 83%, about 85%, more than about 90%. more than about 95% and more than 98%; wherein the acidic variants are measured in the composition obtained from neutralized protein A step.

56. A composition comprising dulaglutide and one or more HMWs wherein the composition comprising one or more HMWs selected from below 2% or less, about 1.5% or less, about 1% or less, about 0.5% or less, about 0.4% or less, and about 0.2% as measured by SEC- HPLC; wherein the composition has dulaglutide purity more than 80%, more than 83%, about 85%, more than about 90%. more than about 95% and more than about 98%; wherein the HMW are measured in the composition obtained from neutralized protein A step.

57. A composition comprising dulaglutide and one or more LMWs wherein the composition comprising one or more LMWs selected from below 15% or less, about 12% or less, about 10% or less, about 7% or less, about 5% or less, about 3% or less, about 1% or less, about 0.8% or less, about 0.5% or less, about 0.4% or less, and about 0.2% as measured by SEC- HPLC; wherein the composition has dulaglutide purity more than 80%, more than about 83%, about 85%, more than about 90%. more than about 95% and more than 98%; wherein the LMW are measured in the composition obtained from neutralized protein A step.

58. A composition comprising dulaglutide and one or more glycan wherein the composition comprising one or more afucosylated glycans selected from below about 3% or less, about 2% or less, about 1% or less, about 0.8% or less, about 0.5% or less, about 0.4% or less, and about 0.2% as measured by HILIC UPLC; one or more galactosylation less than about 23%, less than about 21%, less than about 19%, less than about 17%, less than about 15%, less than about 13%, less than about 11%, less than about 9% less than about 7% less than about 5% less than about 3%, less than about 1%; wherein the composition sialylation less than about 2% about 1% or less, about 0.8% or less, about 0.5% or less, about 0.4% or less, and about 0.2% as measured by HILIC UPLC; wherein the composition has dulaglutide purity more than 80%, more than about 83%, about 85%, more than about 90%. more than about 95% and more than 98%; wherein the glycans are measured in the composition obtained from neutralized protein A step.

59. A composition comprising dulaglutide and one or more reduced oxidation impurities wherein the composition comprising one or more reduced oxidation impurities selected from below 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 1% or less, about 0.8% or less, about 0.5% or less, about 0.4% or less, and about 0.2% as measured by RP-HPLC; wherein the composition has dulaglutide purity more than 80%, more than about 83%, about 85%, more than about 90%. more than about 95% and more than 98%; wherein the oxidation impurities are measured in the composition obtained from neutralized protein A step.

60. The composition as claimed in any preceding claim is obtained from neutralized protein A step.

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