Recombinant bone morphogenetic protein-2 and preparation method thereof

Abstract

The present disclosure discloses a nucleic acid molecule including SEQ ID No. 1. The nucleic acid molecule encodes for a recombinant bone morphogenetic protein-2 (rBMP-2) inside a mammalian cell.

Description

[0001] RECOMBINANT BONE MORPHOGENETIC PROTEIN-2 AND PREPARATION METHOD THEREOF

[0002] FIELD OF INVENTION

[0003] [1] The present disclosure relates to a nucleic acid molecule encoding for a recombinant protein. More particularly, the present disclosure relates to nucleic acid molecule encoding for a recombinant bone morphogenetic protein-2.

[0004] BACKGROUND OF INVENTION

[0005] [2] Recombinant bone morphogenetic protein-2 (or BMP-2) is a multi-functional growth factor belonging to the transforming growth factor-beta (or TGF- ) superfamily of proteins. Expression of BMP-2 is critical for both embryonic development and adult tissue maintenance, though its physiological expression levels change. The BMP-2 is essential for developmental processes, for example, bone formation, cardio-genesis, neurogenesis, osteogenesis, etc. The BMP-2 is involved primarily at the time of embryo development as well as bone remodeling and homeostasis in adulthood. In other words, BMP-2 is essential for major developmental processes like heart and bone formation in the fetus, and it continues to be expressed into adulthood to induce ossification (bone formation), maintain bone and cartilage, and regulate other adult physiological processes. The concentration of BMP-2 in humans varies considerably depending on physiological or clinical conditions.

[0006] [3] As a key regulator of bone growth and repair, its presence is tightly controlled within the body. For example, endogenous production of BMP-2 is typically detected at low levels, averaging around 164.6 ± 12.39 pg / mL and is localized around specific tissues. This makes large-scale extraction and purification of BMP-2 from natural tissue impractical and inefficient. Obtaining substantial amounts of BMP-2 demands a large volume of raw materials and typically results in low and inconsistent protein yields.

[0007] [4] Furthermore, downstream purification of BMP-2 would be difficult and costly because the much amount of BMP-2 will be lost due to very low production yield.

[0008] [5] Conventionally, BMP-2 is produced in media supplemented with serum. Thus, purified BMP-2 that is free of albumin, is very hard to procure and is very expensive.

[0009] [6] Thus, there arises a need for a method to produce a recombinant BMP-2 that overcomes the drawbacks associated with the conventionally available BMP-2 and methods to prepare thereof. SUMMARY OF INVENTION

[0010] [7] Particular embodiments of the present disclosure are described herein below with reference to the accompanying drawings, however, it is to be understood that the disclosed embodiments are mere examples of the disclosure, which may be embodied in various forms. Well-known functions or constructions are not described in detail to avoid obscuring the present disclosure in unnecessary detail. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present disclosure in virtually any appropriately detailed structure.

[0011] [8] In an exemplary embodiment, the present disclosure relates to a nucleic acid molecule including SEQ ID No. 1. The nucleic acid molecule encodes a recombinant bone morphogenetic protein-2 (rBMP-2) inside a mammalian cell.

[0012] [9] In another exemplary embodiment, the present disclosure relates to a construct including a vector having a promoter and a nucleic acid molecule disposed downstream of the promoter. The nucleic acid molecule including SEQ ID No. 1 and encodes for a recombinant bone morphogenetic protein-2 (rBMP-2) inside a mammalian cell.

[0013]

[0010] In yet another exemplary embodiment, the present disclosure relates to a single cell colony including a plurality of mammalian cells including the construct as described above.

[0014] BRIEF DESCRIPTION OF DRAWINGS

[0015]

[0011] The summary above, as well as the following detailed description of illustrative embodiments, is better understood when read in conjunction with the apportioned drawings. For the purpose of illustrating the present disclosure, exemplary constructions of the disclosure are shown in the drawings. However, the disclosure is not limited to specific methods and instrumentality disclosed herein. Moreover, those in the art will understand that the drawings are not to scale.

[0016]

[0012] Fig. la depicts a nucleic acid molecule 110, according to an embodiment of the present disclosure.

[0017]

[0013] Fig. lb depicts a vector 130, according to an embodiment of the present disclosure.

[0014] Fig. lc depicts a construct 150, according to an embodiment of the present disclosure.

[0018]

[0015] Fig. 2 depicts a method 200 to prepare a mammalian cell capable of producing recombinant BMP-2 (rBMP-2), according to an embodiment of the present disclosure.

[0019]

[0016] Fig. 3 depicts a method 300 to prepare a rBMP-2 from the single-cell colony, according to an embodiment of the present disclosure.

[0020]

[0017] Figs. 4-9 depict experimental data of rBMP-2 protein, according to an embodiment of the present disclosure.

[0021] DETAILED DESCRIPTION OF THE DRAWINGS

[0022]

[0018] Prior to describing the invention in detail, definitions of certain words or phrases used throughout this patent document will be defined: the terms "include" and "comprise", as well as derivatives thereof, mean inclusion without limitation; the term "or" is inclusive, meaning and / or; the phrases "coupled with" and "associated therewith", as well as derivatives thereof, may mean to include, be included within, interconnect with, contain, be contained within, connect to or with, couple to or with, be communicable with, cooperate with, interleave, juxtapose, be proximate to, be bound to or with, have a property of, or the like. Definitions of certain words and phrases are provided throughout this patent document, and those of ordinary skill in the art will understand that such definitions apply in many, if not most, instances to prior as well as future uses of such defined words and phrases.

[0023]

[0019] Reference throughout this specification to "one embodiment," "an embodiment," or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases "in one embodiment," "in an embodiment," and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment, but mean "one or more but not all embodiments" unless expressly specified otherwise. The terms "including," "comprising," "having," and variations thereof mean "including but not limited to" unless expressly specified otherwise. An enumerated listing of items does not imply that any or all of the items are mutually exclusive and / or mutually inclusive, unless expressly specified otherwise. The terms "a," "an," and "the" also refer to "one or more" unless expressly specified otherwise.

[0024]

[0020] Although the operations of exemplary embodiments of the disclosed method may be described in a particular, sequential order for convenient presentation, it should be understood that the disclosed embodiments can encompass an order of operations other than the particular, sequential order disclosed. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Further, descriptions and disclosures provided in association with one particular embodiment are not limited to that embodiment, and may be applied to any embodiment disclosed herein. Moreover, for the sake of simplicity, the attached figures may not show the various ways in which the disclosed system, method, and apparatus can be used in combination with other systems, methods, and apparatuses.

[0025]

[0021] Furthermore, the described features, advantages, and characteristics of the embodiments may be combined in any suitable manner. One skilled in the relevant art will recognize that the embodiments may be practiced without one or more of the specific features or advantages of a particular embodiment. In other instances, additional features and advantages may be recognized in certain embodiments that may not be present in all embodiments. These features and advantages of the embodiments will become more fully apparent from the following description and apportioned claims, or may be learned by the practice of embodiments as set forth hereinafter.

[0026]

[0022] The present disclosure relates to a nucleic acid molecule encoded by SEQ ID No. 1. In an embodiment, the nucleic acid molecule encodes a recombinant bone morphogenetic protein-2 (rBMP-2) inside a mammalian cell. The nucleic acid molecule is transcribed to at least one messenger (mRNA) that is then translated to the rBMP-2.

[0027]

[0023] The rBMP-2 of the present disclosure may be used for development and / or regeneration of bone and other human tissues. It may be used as a standard for various stem cell assays. It can be used to prepare bone graft generation. It can be added in demineralize bone matrix and applied to a wounded area for faster healing. It can accelerate healing of bone fractures when used with synthetic bone graft material.

[0028]

[0024] In an embodiment, the rBMP-2 is produced by a single cell colony of a plurality of mammalian cells. Each mammalian cell includes the nucleic acid molecule and is capable of producing rBMP-2. The use of mammalian cell in producing rBMP-2 eliminates the requirement of human-derived fluids or tissues and thereby overcomes variability observed in age-related decline of endogenous BMP-2 levels.

[0025] Further, the present disclosure relates to a method of preparation of a recombinant bone morphogenetic protein-2 (rBMP-2). In an embodiment, the single cell colony is suspended in a serum free media that has no albumin adulteration. Thus, the method enables stringent control over purity of the rBMP-2 obtained and ensures consistent yield of rBMP-2 in large scale production batches. The present disclosure provides high yield of rBMP-2, for example, more than 10 mg / L. In an embodiment, the method of preparing rBMP-2 provides a 48.2 mg of rBMP-2 per liter of spent media before purification, and 30.9 mg of rBMP-2 per liter of spent media after purification (>64% yield). In this manner, the present disclosure provides a reliable and commercially viable source of rBMP-2 that meets clinical standards for safety, quality, and economic feasibility.

[0029]

[0026] Due to its purity, a 5-10 pg of rBMP-2 per dose would be sufficient for the desired effect thereby, reducing financial burden on the user. Moreover, the method of preparing rBMP-2 is inherently scalable, supporting production at laboratory, pre-clinical, and industrial volumes depending upon the requirement. The method allows for consistent protein yield in large scale production batches.

[0030]

[0027] Now referring to figures, Figs, la-lc depict a nucleic acid molecule 110, a vector 130 and a recombinant construct 150 (hereinafter, referred to as construct 150), respectively, according to an embodiment of the present disclosure. In an embodiment, the nucleic acid molecule 110 (as shown in Fig. la) is encoded at least by SEQ ID No. 1. The nucleic acid molecule 110 is transcribed to at least one messenger (mRNA) that is then translated to the recombinant bone morphogenetic protein-2 (hereinafter, referred to as rBMP-2). The rBMP- 2 protein is encoded by SEQ ID No. 2. The sequence of a plurality of amino acid residues in rBMP-2 (i.e., SEQ ID No. 2) is identical to that of the wild type BMP-2 protein, thus, ensuring minimal to none adverse effect arising from use of rBMP-2 of the present disclosure.

[0031]

[0028] The nucleic acid molecule 110 may be a single stranded DNA (ssDNA) or a double stranded DNA (dsDNA). In an embodiment, the nucleic acid molecule 110 is a double stranded, complementary DNA (cDNA) that is chemically synthesized.

[0032]

[0029] The nucleic acid molecule 110 includes a plurality of codons. The nucleic acid molecule 110, as encoded by the SEQ ID No. 1, includes one-hundred fifteen codons. Each codon includes three nucleotides and encodes for an amino acid residue of the rBMP-2. In an embodiment, the plurality of codons within the nucleic acid molecule 110 are optimized to enhance transcriptional and translational efficiency in a suitable host cell (explained later). Optimization of the plurality of codons improves overall yield of the rBMP-2 protein.

[0033]

[0030] In an embodiment, out of one-hundred fifteen codons present in wild type gene of BMP-2, a predefined number of codons (each codon corresponding to a specific position) are optimized. In an exemplary embodiment, sixty-seven codons out of the one-hundred fifteen codons of SEQ ID NO. 1 are optimized. The sixty-seven optimized codons of SEQ ID No. 1 compared to the corresponding codon in the wild type gene of BMP-2 are tabulated below in Table 1:

[0034] Table 1: Optimized codon of SEQ ID No. 1

[0031] Fig. lc depicts the construct 150, according to an embodiment of the present disclosure. In an embodiment, the construct 150 includes the nucleic acid molecule 110 ligated to the vector 130. The construct (150) has the vector 130 and the nucleic acid molecule 110. In an embodiment, the construct 150 is substantially circular shaped polynucleotide molecule. The construct 150 facilitates production of the rBMP-2 protein by expressing the nucleic acid molecule 110 in the suitable host cell.

[0035]

[0032] In an embodiment, the vector 130 (as shown in Fig. lb) includes at least one first origin of replication region (hereinafter referred to as first ori 131), at least one first promoter region (or promoter) 133, one or more signaling region 135, one or more selectable markers 137, a plurality of restriction sites 139 (i.e., pre-defined nucleotide sequences that are recognized by restriction enzymes), etc. In an exemplary embodiment, the vector 130 is encoded by SEQ ID No. 3.

[0036]

[0033] The first ori 131 helps the vector 130 to replicate inside a host cell. In an embodiment, the first ori 131 of the vector 130 includes a pUC origin. Additionally, or optionally, the vector 130 includes a second ori 131a for single-stranded DNA replication. In an embodiment, the second ori 131a includes fl origin.

[0037]

[0034] The first promoter 133 may have a binding affinity to at least one RNA polymerase enzyme of the host cell. The first promoter 133 is provided upstream to one or more sequences (including the signaling region 135 and the nucleic acid molecule 110). In other words, the nucleic acid molecule 110 is disposed downstream of the first promoter 133. The construct 150 facilitates production of the rBMP-2 protein by expressing the nucleic acid molecule 110 via the first promoter 133. The first promoter 133 helps initiate the transcription of the one or more sequences. The at least one RNA polymerase enzyme helps to produce mRNA molecules of the one or more sequences provided downstream of the first promoter 133 via transcription. The first promoter 133 may be one of CMV (encoded by SEQ ID No. 4), EFla (encoded by SEQ ID No. 5), etc. In an embodiment, the first promoter 133 is CMV.

[0038]

[0035] The signaling region 135 is disposed downstream of the first promoter 133. Specifically, the signaling region 135 is provided between the first promoter 133 and the nucleic acid molecule 110. The signaling region 135 (for example, Gaussia luciferaseor Glue cDNA) is encoded by SEQ ID No. 6. The signaling region 135 is transcribed into an mRNA molecule and is then translated into a signal peptide (encoded by SEQ ID No. 7) coupled to an N-terminal of the rBMP-2 protein (encoded by SEQ ID No. 2) expressed by the nucleic acid molecule 110. The signal peptide helps to guide the rBMP-2 protein out of the host cell, and into a media (explained later). This facilitates cheap and easy purification of rBMP-2.

[0039]

[0036] The selectable markers 137 may include at least one resistant genes (hereinafter, referred to as resistance genes), for example, ampicillin, kanamycin, gentamycin, neomycin, zeocin, etc. resistant genes. In an exemplary embodiment, as shown in Figs, lb-lc, the vector 130 includes ampicillin resistant gene (hereinafter referred to as AmpR) (encoded by SEQ ID No. 8) and zeocin resistant gene (hereinafter referred to as ZeoR) (encoded by SEQ ID No. 9) as selectable markers 137. The resistance gene(s) of the selectable marker 137 may be disposed downstream of a respective second promoter region (not shown) that facilitates the expression of the corresponding resistance gene(s). For example, ampicillin resistance gene (encoded by SEQ ID No. 8) and zeocin resistance gene (encoded by SEQ ID No. 9) are disposed downstream of AmpR promoter, and ZeoR promoter, respectively. The AmpR promoter and ZeoR promoter facilitate expression of the ampicillin resistance gene (encoded by SEQ ID No. 8) and the zeocin resistance gene (encoded by SEQ ID No. 9), respectively. Each of the selectable markers 137 help the host cell having the vector 130 to resist the effects of the corresponding antibiotic.

[0040]

[0037] The plurality of restriction sites 139 may include Ndel, Pmel, BamHI, Xbal, Hindu, EcoRV, EcoRI, Pstl, Xhol, etc. The nucleic acid molecule 110 is inserted into at least one restriction site 139 of the plurality of restriction sites 139 of vector 130. In an embodiment, the nucleic acid molecule 110 is inserted to the vector 130 using two restriction sites namely, Ndel and Xhol.

[0041]

[0038] Additionally, or optionally, the nucleic acid molecule 110 may be flanked by one or more segments of additional nucleotides (hereinafter, segments). The segments are provided in at least one of upstream of 5' end or downstream of 3' end of the nucleic acid molecule 110. In an embodiment, a first segment (encoded by sequence "GCTAGC") is provided at the 5' end positioned upstream of the initiation codon (i.e., ATG, 1stcodon) of the nucleic acid molecule 110, while a second segment (encoded by sequence "CTCGAG") is positioned downstream of the 3' end of the nucleic acid molecule 110. The nucleic acid molecule 110 flanked by the segments is referenced by SEQ ID No. 10. The segments facilitate cloning of the nucleic acid molecule 110 to the vector 130 at a restriction site selected from the plurality of restriction sites 139. In an embodiment, the first segment and the second segment include a respective recognition site for the corresponding restriction enzyme, such as, Ndel and XhoL Depending upon the restriction enzyme, the restriction sites selected from the plurality of restriction sites 139 of the vector 130, and cloning strategy, the sequence of the segments of the nucleic acid molecule 110 may be changed and the same is within the scope of the teachings of the present disclosure.

[0042]

[0039] Additionally, or optionally, the vector 130 is provided with one or more orientation markers 130a which helps to ensure correct orientation of the nucleic acid molecule 110 with respect to the vector 130 and / or the first promoter 133. In an exemplary embodiment, the orientation markers 130a include a T7 promoter region (encoded by SEQ ID NO. 11) disposed upstream of the nucleic acid molecule 110 and a T7 terminator region (not shown) disposed downstream of the nucleic acid molecule 110. In an embodiment, a primer having specific binding affinity to the T7 promoter region is encoded by SEQ ID No. 12. The orientation markers 130a in conjunction with a sequencing technique help to determine and ensure the orientation of the nucleic acid molecule 110 relative to the first promoter 133 of the vector 130. In an embodiment, the nucleic acid molecule 110 is inserted downstream of the first promoter 133 and the signaling region 135.

[0043]

[0040] The construct 150 is configured for expression of the nucleic acid molecule 110 via the first promoter 133 in the suitable host cell. In an embodiment, the suitable host cell is a mammalian cell. The mammalian cell is selected from at least one of a Human Embryonic Kidney T (HEK293T) cell, a Chinese hamster ovary (CHO) cell, an AD293 cell, a Human Embryonic Kidney (HEK293) cell, etc. The mammalian cell includes the construct 150 and allows transcription and translation of the nucleic acid molecule 110 to produce rBMP-2 protein. The mammalian cell, thus serves as an expression system embodying the construct 150 and exhibiting the functional capability to synthesize rBMP-2 encoded by the nucleic acid molecule 110. In an embodiment, a plurality of mammalian cell (each mammalian cell harboring the construct 150) forms a single cell colony. In other words, the single colony includes the plurality of mammalian cells, each mammalian cell includes the construct 150. The single cell colony is capable of maintaining the construct 150 and continuously expressing rBMP-2 in a predefined culture media under predefined culture conditions. The single cell colony (constituting plurality of mammalian cells) serves as a defined and controllable source for obtaining the rBMP-2 with high purity and yield.

[0044]

[0041] The plurality of mammalian cells of the single cell colony is incubated at a predefined temperature for a predefined time period in a humidified atmosphere. In an embodiment, the plurality of mammalian cells of the single cell colony is suspended in a serum-free media (SFM) and is incubated at a predefined temperature for 37°C in a 5% CO2for approximately 15 days. This allows each mammalian cell to maintain stable expression of the nucleic acid molecule 110 and produce the corresponding rBMP-2 consistently. The plurality of mammalian cells produces a pre-defined amount of rBMP-2 by transcribing and then translating the nucleic acid molecule 110.

[0045]

[0042] The serum free medium (SFM) includes a carbon source, a plurality of inorganic salts, a plurality of amino acids, a plurality of vitamins, a plurality of trace elements, a plurality of growth factors, etc.

[0046]

[0043] A predefined amount of carbon source in the SFM ranges from 5 mM to 25 mM, the plurality of inorganic salts ranges from 0.1 mM to 50 mM, the plurality of amino acids ranges from 0.01 mM to 10 mM, the plurality of vitamins ranges from 0.001 mM to 1 mM, the plurality of trace elements ranges from 0.1 pM to 1 pM, the plurality of growth factors ranges from 0.1 pM to 1 mM, etc.

[0047]

[0044] The plurality of inorganic salts in serum free medium is selected from a group of sodium chloride (NaCI), potassium chloride (KCI), calcium chloride (CaCl2), magnesium sulfate (MgS04) and sodium bicarbonate (NaHC03), etc., or a combination thereof.

[0048]

[0045] The plurality of amino acids in the serum-free medium is selected from a group of L- Glutamine, L-Arginine, L-Leucine, L-Lysine, L-Phenylalanine, L-Tyrosine, L-Methionine, L- Histidine, L-Valine, Glycine, etc., or a combination thereof.

[0049]

[0046] The plurality of vitamins may be selected from a group of folic acid, thiamine HCI (vitamin Bl), riboflavin (vitamin B2), nicotinamide (vitamin B3), pyridoxine HCI (vitamin B6), biotin, pantothenic acid, etc., or a combination thereof.

[0050]

[0047] The plurality of trace elements may be selected from a group of zinc sulfate (ZnS04), ferrous sulphate (FeS04) / ferric nitrate (Fe(NO3)3, copper sulphate (CuS04), manganese (II) sulphate (MnS04), etc., or a combination thereof.

[0051]

[0048] The plurality of growth factors may be selected from a group of at least one recombinant growth factors (e.g., Epidermal Growth Factor (EGF), basic fibroblast growth factor (bFGF), etc.), at least one antioxidant (e.g., glutathione, ascorbate, etc.).

[0052]

[0049] In an embodiment, the serum free medium (SFM) includes D-glucose (as carbon source), a predefined amount of sodium chloride (NaCI), a predefined amount of potassium chloride (KCI), a predefined amount of calcium chloride (CaCl2), a predefined amount of magnesium sulfate (MgS04) and a predefined amount of (NaHCO3), a predefined amount of L-Glutamine, a predefined amount of L-Arginine, a predefined amount of L-Leucine, a predefined amount of L-Lysine, a predefined amount of L-Phenylalanine, a predefined amount of L-Tyrosine, a predefined amount of L-Methionine, a predefined amount of L- Histidine, a predefined amount of L-Valine, a predefined amount of Glycine, a predefined amount of folic acid, a predefined amount of thiamine HCI (vitamin Bl), a predefined amount of riboflavin (vitamin B2), a predefined amount of nicotinamide (vitamin B3), a predefined amount of pyridoxine HCI (vitamin B6), a predefined amount of biotin, a predefined amount of pantothenic acid, a predefined amount of zinc sulfate (ZnS04), a predefined amount of ferrous sulphate (FeS04) / ferric nitrate (Fe(NO3)3, a predefined amount of copper sulphate (CuS04), a predefined amount of manganese(ll) sulphate (MnS04), a predefined amount of one or more recombinant growth factors (e.g., Epidermal Growth Factor (EGF), basic fibroblast growth factor (bFGF), etc.), a predefined amount of one or more antioxidants (e.g., glutathione, ascorbate, etc.), etc.

[0053]

[0050] The predefined amount of sodium chloride (NaCI), the predefined amount of potassium chloride (KCI), the predefined amount of calcium chloride (CaCl2), the predefined amount of magnesium sulfate (MgS04) and the predefined amount of (NaHC03) may range from 120 mM to 150 mM, from 4 mM to 6 mM, from 1 mM to 2 mM, from 0.5 mM to 1 mM, and from 10 mM to 30 mM, respectively.

[0054]

[0051] The predefined amount of L-Glutamine, L-Arginine, L-Leucine, L-Lysine, L- Phenylalanine, L-Tyrosine, L-Methionine, L-Histidine, L-Valine and Glycine may range from 2 mM to 4 mM, from 0.4 mM to 1 mM, from 0.4 mM to 0.8 mM, from 0.1 mM to 0.8 mM, from 0.1 mM to 0.3 mM, from 0.05 mM to 0.2 mM, from 0.05 mM to 0.2 mM, from 0.02 mM to 0.1 mM, from 0.03 mM to 0.6 mM, and from 0.2 mM to 0.4 mM, respectively.

[0055]

[0052] The predefined amount of folic acid, thiamine HCI (vitamin Bl), riboflavin (vitamin B2), nicotinamide (vitamin B3), pyridoxine HCI (vitamin B6), biotin, pantothenic acid may range from 1 pM to 10 pM, from 0.5 pM to 50 pM, from 0.01 pM to 0.2 pM, from 0.02 mM to 0.2 mM, from 0.003 mM to 0.05 mM, from 0.01 pM to 0.1 pM, and from 0.002 mM to 0.1 mM, respectively.

[0056]

[0053] The predefined amount of zinc sulfate (ZnS04), ferrous sulphate (FeS04) / ferric nitrate (Fe(NO3)3, copper sulphate (CuS04), manganese (II) sulphate (MnS04) may range from 0.5 pM to 10 pM, from 0.2 pM to 20 pM, from 0.05 pM to 1 pM, from 0.05 pM to 1 pM, etc.

[0057]

[0054] The predefined amount of one or more recombinant growth factors, one or more lipids (e.g., cholesterol, fatty acids, phospholipids, Pluronic® F68, etc.) and one or more antioxidants may range from 1 ng / mL to 20 ng / mL, from 0.01 pM to 100 pM, and from 10 pM to 100 pM, respectively.

[0058]

[0055] Alternately, the plurality of mammalian cells may be suspended in a media supplemented with a predefined amount of serum under the same culture conditions (including, a predefined temperature for a predefined time period in a humidified atmosphere). The serum supplemented medium includes all constituents of the serum-free media along with a predefined amount of serum. In an embodiment, the serum supplemented media includes the SFM supplemented with 10% Fetal Bovine Serum (FBS).

[0059]

[0056] Fig. 2 depicts an exemplary method 200 of preparing a mammalian cell capable of expressing the nucleic acid molecule 110 and producing rBMP-2.

[0060]

[0057] The method 200 commences at step 201, by obtaining the nucleic acid molecule 110 and the vector 130.

[0061]

[0058] At an optional step 201a, if the vector 130 has a circular shaped structure, the vector 130 is digested to obtain a linearized structure of the vector 130. The vector 130 may be digested using one or two restriction enzymes based on a respective restriction site 139 of the vector 130. The restriction digestion enzyme is selected from at least one of Hindi II, Xhol, Pstl, Ndel, Pmel, BamHI, EcoRV, EcoRI, Xbal, etc. The restriction enzyme is configured to introduce double-stranded breaks and cut the vector 130 at the restriction sites selected from the plurality of restriction sites 139, thereby obtaining the linearized structure of the vector 130. Depending upon the restriction enzyme used for linearization of the vector 130, the linearized structure of the vector 130 defines sticky overhang or blunt ends at each of the two (free) ends of the vector 130. In an exemplary embodiment, the vector 130 is linearized to define two sticky overhangs using Ndel restriction enzyme and Xhol restriction enzyme. Using two different restriction enzymes prevents the vector 130 to self-circularize (and self-ligate).

[0062]

[0059] At an optional step 201b, the nucleic acid molecule 110 encoded by SEQ ID No. 10 may be digested using the same restriction enzyme(s) with which the vector 130 is digested in step 101a. Upon digesting the nucleic acid molecule 110 with the restriction enzyme, each segment of the one or more segments of the nucleic acid molecule 110 creates sticky overhang at the respective ends of the nucleic acid molecule 110 that complements the sticky overhangs of the vector 130. This ensures that the two free ends of the nucleic acid molecule 110 have affinity to bind with respective free ends of the vector 130. In an embodiment, two different restriction enzymes are used for digesting the vector 130 and the nucleic acid molecule 110, thus ensuring appropriate orientation of the nucleic acid molecule 110 with respect to the vector 130. The appropriate orientation of the nucleic acid molecule 110 corresponds to positioning the initiation codon (or 1stcodon) of the nucleic acid molecule 110 adjacent to the first promoter 133 (and the signaling region 135).

[0063]

[0060] Additionally, or optionally, prior to digesting the nucleic acid molecule 110, the nucleic acid molecule 110 may be amplified to increase their number and prepare a large- scale stock. In an embodiment, the nucleic acid molecule 110 is ligated to a plasmid (e.g., pUC57 plasmid encoded by SEQ ID No. 13) via a blunt-end EcoRV restriction site 139 and transformed into chemically competent E. coli DH5a cells. Thereafter, the plasmids are extracted and then restriction digested as detailed above. Although the method 200 is described with natural replication of the plasmids, the plasmids (or portions thereof) may be synthetically amplified using technique such as polymerase chain reaction (PCR), etc. The same is within the scope of the teachings of the present disclosure.

[0064]

[0061] At step 203, the construct 150 is obtained by ligating the nucleic acid molecule 110 (obtained from step 201 or 201b) to the vector 130 (obtained from step 201 or 201a) using a DNA ligase enzyme. In an embodiment, the nucleic acid molecule 110 is ligated downstream of the first promoter 133 and the signaling region 135 using T4 DNA ligase enzyme.

[0065]

[0062] At step 205, a plurality of mammalian cells is obtained and expanded. The mammalian cells may be any one of Human Embryonic Kidney T (HEK293T) cells, Chinese hamster ovary (CHO) cells, AD293 cells, Human Embryonic Kidney (HEK293) cells, etc. Additionally, or optionally, the plurality of mammalian cells may be derived from cryopreserved cell stocks maintained under controlled storage conditions. At an optional step, the plurality of mammalian cells is revived from cryopreservation and expanded inside a flask incubated at 37 °C in a humidified incubator with 5% CO2.

[0066]

[0063] At step 207, the constructs 150 (obtained from step 203) are transfected into the plurality of mammalian cells (obtained from step 205) to obtain a plurality of clones capable of producing rBMP-2 from the nucleic acid molecule 110. In an embodiment, each clone includes a mammalian cell harboring at least one construct 150, and is capable of expressing the nucleic acid molecule 110 and producing rBPM-2.

[0067]

[0064] In an exemplary embodiment, the constructs 150 are transfected into HEK293T cells via a transfection reagent, for example, Effectene. Post transfection, the clones (i.e., mammalian cells having the construct 150) is selected based on the selectable marker 137, for example, zeocin at 250 pg / mL. This helps in eliminating non-transformed mammalian cells (i.e., mammalian cells not harboring the construct 150). Post-selection, a single cell colony of a plurality of mammalian cells (having the construct 150) is obtained from the clones. In an embodiment, the mammalian cells in the single cell colony are genetically identical and collectively capable of sustained expression of the nucleic acid molecule 110 encoding rBMP-2.

[0068]

[0065] The singe colony (obtained from step 207) is cryopreserved in a vial at a pre-defined temperature (for example, -140°C). In an embodiment, the single cell colony obtained yield about 6.43 mg / L of rBMP-2 in routine lab scale conditions.

[0069]

[0066] Fig. 3 depicts a method 300 to prepare rBMP2 from the single cell colony obtained from method 200.

[0070]

[0067] The method 300 commences at step 301, by culturing the single cell colony in a predefined nutrient medium. In an exemplary embodiment, the single cell colony is cultured in a media optionally including at least one antimycotic agent or the like.

[0071]

[0068] In an exemplary embodiment, the single cell colony is cultured in Dulbecco's Modified Eagle Medium (DMEM) complete medium supplemented with 10% fetal bovine serum (FBS). Other functionally equivalent nutrient mediums are also within the scope of the teachings of the present disclosure.

[0072]

[0069] In an exemplary embodiment, the DMEM complete medium includes 25mM of D- glucose, 4 mM of L-glutamine, ImM of sodium pyruvate, 6.4g / L of sodium chloride (NaCI), 400mg / L of potassium chloride (KCI), 200mg / L calcium chloride (CaC ), 96.67 mg / L of magnesium sulfate (MgSOzi), sodium bicarbonate (NaHCOs), 15mg / L of phenol red, amino acids, and vitamins. In an embodiment, the amino acids include 84 mg / L of L-Arginine HCI, 63 mg / L of L-Cystine-2HCI, 584 mg / L of L-Glutamine, 30 mg / L of Glycine, 42 mg / L of L- Histidine-HCI, 105 mg / L of L-lsoleucine, 105 mg / L of L-Leucine, 146 mg / L of L-Lysine-HCI, 30 mg / L L-Methionine, 66 mg / L of L-Phenylalanine, 95 mg / L of L-Threonine, 16 mg / L of L- Tryptophan, 103 mg / L of L-Tyrosine-2Na-2H2O, and 94 mg / L of L-Valine. In an embodiment, the vitamins include 4 mg / L of Choline chloride, 4 mg / L of Folic acid, 7.2 mg / L of Inositol, 4 mg / L of Niacinamide, 4 mg / L of D-Pantothenic acid, 4 mg / L of Pyridoxine-HCI, 0.4 mg / L of Riboflavin, 4 mg / L of Thiamine-HCI, and 1.36 pg / L of Vitamin B12.

[0073]

[0070] The single cell colony is cultured in a predetermined temperature, and at a predefined CO2. In an exemplary embodiment, the single cell colony is cultured at 37 °C in a humidified incubator with 5% CO2.

[0074]

[0071] At step 303, a pre-defined number of clones (or cells) from the single cell colony are seeded into shake flasks containing serum-free media and incubated for a pre-defined time period to produce a spent media. The number of clones (or cells) seeded ranges from 0.15 x 106cells / mL to 0.2 x 106cells / mL.

[0075]

[0072] The serum free medium (SFM) includes a carbon source, a plurality of inorganic salts, a plurality of amino acids, a plurality of vitamins, a plurality of trace elements, a plurality of growth factors, etc.

[0076]

[0073] A predefined amount of carbon source in the SFM ranges from 5 mM to 25 mM, the plurality of inorganic salts ranges from O.lmM to 50 mM, the plurality of amino acids ranges from 0.01 mM to 10 mM, the plurality of vitamins ranges from O.OlmM to ImM, the plurality of trace elements ranges from 0.1 pM to 1 pM, the plurality of growth factors ranges from 0.1 pM to 1 pM, etc.

[0077]

[0074] The plurality of inorganic salts in serum free medium is selected from a group of sodium chloride (NaCI), potassium chloride (KCI), calcium chloride (CaCl2), magnesium sulfate (MgS04) and sodium bicarbonate (NaHC03), etc., or a combination thereof.

[0078]

[0075] The plurality of amino acids in the serum-free medium is selected from a group of L- Glutamine, L-Arginine, L-Leucine, L-Lysine, L-Phenylalanine, L-Tyrosine, L-Methionine, L- Histidine, L-Valine, Glycine, etc., or a combination thereof.

[0079]

[0076] The plurality of vitamins may be selected from a group of folic acid, thiamine HCI (vitamin Bl), riboflavin (vitamin B2), nicotinamide (vitamin B3), pyridoxine HCI (vitamin B6), biotin, pantothenic acid, etc., or a combination thereof.

[0080]

[0077] The plurality of trace elements may be selected from a group of zinc sulfate (ZnS04), ferrous sulphate (FeS04) / ferric nitrate (Fe(NO3)3, copper sulphate (CuS04), manganese (II) sulphate (MnS04), etc., or a combination thereof.

[0081]

[0078] The plurality of growth factors may be selected from a group of at least one recombinant growth factors (e.g., Epidermal Growth Factor (EGF), basic fibroblast growth factor (bFGF), etc.), at least one antioxidant (e.g., glutathione, ascorbate, etc.), a predefined amount of Dexamethasone (Dxn), and a predefined amount of L-alanyl-L-glutamine dipeptide in a predefined amount of inorganic salt (such as, sodium chloride (NaCI)).

[0082]

[0079] In an embodiment, the serum free medium (SFM) includes D-glucose, a predefined amount of sodium chloride (NaCI), a predefined amount of potassium chloride (KCI), a predefined amount of calcium chloride (CaCl2), a predefined amount of magnesium sulfate (MgS04) and a predefined amount of (NaHCO3), a predefined amount of L-Glutamine, a predefined amount of L-Arginine, a predefined amount of L-Leucine, a predefined amount of L-Lysine, a predefined amount of L-Phenylalanine, a predefined amount of L-Tyrosine, a predefined amount of L-Methionine, a predefined amount of L-Histidine, a predefined amount of L-Valine, a predefined amount of Glycine, a predefined amount of folic acid, a predefined amount of thiamine HCI (vitamin Bl), a predefined amount of riboflavin (vitamin B2), a predefined amount of nicotinamide (vitamin B3), a predefined amount of pyridoxine HCI (vitamin B6), a predefined amount of biotin, a predefined amount of pantothenic acid, a predefined amount of zinc sulfate (ZnS04), a predefined amount of ferrous sulphate (FeS04) / ferric nitrate (Fe(NO3)3, a predefined amount of copper sulphate (CuS04), a predefined amount of manganese(ll) sulphate (MnS04), a predefined amount of one or more recombinant growth factors (e.g., Epidermal Growth Factor (EGF), basic fibroblast growth factor (bFGF), etc.), a predefined amount of one or more antioxidants (e.g., glutathione, ascorbate, etc.), etc.

[0083]

[0080] The predefined amount of sodium chloride (NaCI), the predefined amount of potassium chloride (KCI), the predefined amount of calcium chloride (CaCl2), the predefined amount of magnesium sulfate (MgS04) and the predefined amount of (NaHC03) may range from 120 mM to 150 mM, from 4 mM to 6 mM, from 1 mM to 2 mM, from 0.5 mM to 1 mM, and from 10 mM to 30 mM, respectively.

[0081] The predefined amount of L-Glutamine, L-Arginine, L-Leucine, L-Lysine, L- Phenylalanine, L-Tyrosine, L-Methionine, L-Histidine, L-Valine and Glycine may range from 2 mM to 4 mM, from 0.4 mM to 1 mM, from 0.4 mM to 0.8 mM, from 0.1 mM to 0.8 mM, from 0.1 mM to 0.3 mM, from 0.05 mM to 0.2 mM, from 0.05 mM to 0.2 mM, from 0.02 mM to 0.1 mM, from 0.03 mM to 0.6 mM, and from 0.2 mM to 0.4 mM, respectively.

[0084]

[0082] The predefined amount of folic acid, thiamine HCI (vitamin Bl), riboflavin (vitamin B2), nicotinamide (vitamin B3), pyridoxine HCI (vitamin B6), biotin, pantothenic acid may range from 1 pM to 10 pM, from 0.5 pM to 50 pM, from 0.01 pM to 0.2 pM, from 0.02 mM to 0.2 mM, from 0.003 mM to 0.05 mM, from 0.01 pM to 0.1 pM, and from 0.002 mM to 0.1 mM, respectively.

[0085]

[0083] The predefined amount of zinc sulfate (ZnS04), ferrous sulphate (FeS04) / ferric nitrate (Fe(NO3)3, copper sulphate (CuS04), manganese (II) sulphate (MnS04) may range from 0.5 pM to 10 pM, from 0.2 pM to 20 pM, from 0.05 pM to 1 pM, from 0.05 pM to 1 pM, etc.

[0086]

[0084] The predefined amount of one or more recombinant growth factors, one or more lipids (e.g., cholesterol, fatty acids, phospholipids, Pluronic® F68, etc.), one or more antioxidants may range from 1 ng / mL to 20 ng / mL, from 0.01 pM_to 100 pM and from 10 pM to 100 pM, respectively. Further, in an embodiment, the serum-free media is supplemented with 10 mL / L of 2Mm of L-alanyl-L-glutamine dipeptide in 0.0085% NaCI and 1 mg / L of dexamethasone.

[0087]

[0085] In an embodiment, the serum-free media includes Hybridoma- serum free medium (Hybridoma-SFM™), lg / L Cell Boost 6™ (CB6) supplement, lmg / L Dexamethasone (Dxn), and IX GlutaMAX™. The IX GlutaMAX™ includes 10 mL / L of 2mM of L-alanyl-L-glutamine dipeptide (in 0.0085% sodium chloride, NaCI). Alternatively, instead of GlutaMAX™, the serum-free media may include L-glutamine.

[0088]

[0086] In an embodiment, the combination of hybridoma-serum free medium (Hybridoma- SFM™) and the cell boost 6™ supplement includes 10 mM of D-glucose, 120 mM of sodium chloride (NaCI), 5mM mM potassium chloride (KCI), 1.8 mM of calcium chloride (CaCl2), 0.5 mM of magnesium sulfate (MgS04) and 10 mM of (NaHC03), 4 mM mM of L-Glutamine, 1 mM of L-Arginine, 0.4 mM of L-Leucine, 0.1 mM of L-Lysine, 0.1 mM of L-Phenylalanine, 0.1 mM of L-Tyrosine, 0.05 mM of L-Methionine, 0.05 mM of L-Histidine, 0.03 mM of L-Valine, 0.25 mM of Glycine, 0.002 mM of folic acid, 0. 5 pM of thiamine HCI (vitamin Bl), 0.2 pM riboflavin (vitamin B2), 0.02 mM of nicotinamide (vitamin B3), 3 pM of pyridoxine HCI (vitamin B6), 0.1 pM of biotin, 0.002 mM of pantothenic acid, 0. 5 pM of zinc sulfate (ZnS04), 0.2 pM of ferrous sulphate (FeS04) / ferric nitrate (Fe(NO3)3, 0. 05 pM of copper sulphate (CuS04), 0.05 pM of manganese(ll) sulphate (MnS04), 0.1 pM of basic fibroblast growth factor (bFGF), 0.01 mM of glutathione, 0.01 pM of Pluronic® F68,etc.

[0089]

[0087] In an embodiment, the shake flasks were incubated at 37°C in a 5% CO2shaker incubator for about 15 days, with addition of fresh Hybridoma-SFM™ (lOOmL / L) on day 6. The clones produce a pre-defined amount of rBMP-2 by transcribing and then translating the nucleic acid molecule 110.

[0090]

[0088] Although the step 303 is described with the example of shake flask, the clones (or cells) may be incubated in bench-scale reactors, wave bioreactors, etc. and the same is within the scope of the teachings of the present disclosure.

[0091]

[0089] The rBMP-2 produced by the clones (or cells) is ejected out of the cell and into the serum-free media, hereon after called the spent media. In an embodiment, the 48.2 mg of rBMP-2 per liter of spent media is obtained.

[0092]

[0090] At step 305, the rBMP-2 produced by the clones (or cells) at step 303 is purified from the spent media. One or more purification techniques may be employed to increase the amount of rBMP-2 protein and reduce the amount of remaining (total) protein in the spent media. The purification techniques include, but are not limited to, ultrafiltration, dialysis, affinity chromatography, column chromatography, or a combination thereof. In an exemplary embodiment, the rBMP-2 is purified by subjecting the spent media to dialysis, ultrafiltration, heparin affinity chromatography, dialysis, carboxymethyl cellulose column chromatography, and dialysis. In an embodiment, 30.9 mg of rBMP-2 per liter of spent media is obtained after purification (i.e., >64% yield). Other functionally equivalent techniques to purify the rBMP-2 are within the scope of the teachings of the present disclosure.

[0093]

[0091] The rBMP-2 disclosed above will now be described with the help of the following examples.

[0094]

[0092] Example 1: Preparation of the construct 150 of the present disclosure

[0095]

[0093] The nucleic acid molecule 110 encoded by SEQ ID No. 10 was chemically synthesized.

[0096] The nucleic acid molecule 110 was inserted into the pUC57 vector encoded by SEQ ID No. 13 (procured from GenScript, USA) (having an ampicillin resistance marker for bacterial selection) via a blunt-end EcoRV site to obtain a recombinant pUC57-BMP2 plasmid. To prepare a large-scale stock of the recombinant pUC57-BMP2 plasmid, 100 pg of plasmid DNA was transformed into chemically competent E. coli DH5a cells. Transformed cells were plated on Luria Bertani agar (procured from HiMedia, India) supplemented with 100 pg / mL of ampicillin. Individual colonies were then inoculated into 4.5 mL of Lurai Bertani broth (procured from HiMedia, India) containing the same antibiotic concentration and cultured at 37 °C with shaking at 230 RPM for approximately 16 hours. Following incubation, cells were harvested by centrifugation at 8000 RPM in 1.5 mL microcentrifuge tubes. Plasmid DNA was extracted using a commercial miniprep kit (procured from Sigma, USA) according to the manufacturer's instructions and eluted in an elution buffer. The purified plasmid DNA was stored at -20 °C.

[0097]

[0094] The recombinant pUC57-BMP2 plasmid and the vector 130 (pCMVMD, encoded by SEQ ID No. 3) were individually digested using Ndel and Xhol restriction enzymes. This enabled to clone the Gaussia luciferase (Glue) signaling region 135 upstream and in frame with the open reading frames (ORFs) of the nucleic acid molecule 110. This facilitated proper expression and secretion of the rBMP-2 protein into the culture medium. The digested nucleic acid molecule 110 and the vector 130 was ligated using T4 DNA ligase to obtain the construct 150.

[0098]

[0095] Example 2: Characterization of the construct 150 of the present disclosure

[0099]

[0096] The construct 150 obtained from example 1 above was characterized by digesting the construct 150 with Ndel and Xhol restriction enzymes. The digested construct 150 (well 2 and 3) was subjected to gel electrophoresis. As shown in Fig. 4, the digested construct 150 was loaded in well 2 and 3, against a lkb DNA ladder loaded in well 1. The image of agarose gel confirmed the approximate size of the nucleic acid molecule 110 (356 bp) and the vector 130 (5071 bp), as shown in Fig. 4.

[0100]

[0097] Example 3: Method to prepare the single cell clone of HEK293T cells using the construct 150 obtained from Example 1 above

[0101]

[0098] A cryopreserved vial of HEK293T cells (frozen in 90% fetal bovine serum and 10% dimethyl sulfoxide) was retrieved from liquid nitrogen storage and rapidly thawed in a 37 °C water bath for 1-2 minutes. The cells from the vial were transferred into a sterile tube and was suspended in 5-10 mL of pre-warmed Dulbecco's Modified Eagle Medium (DMEM) complete medium (procured from HiMedia, India) supplemented with 10% fetal bovine serum (FBS). Composition of the DMEM complete medium used was 25mM of D-glucose, 4 mM of L-glutamine, ImM of sodium pyruvate, 6.4g / L of sodium chloride (NaCI), 400mg / L of potassium chloride (KCI), 200mg / L calcium chloride (CaC ), 96.67 mg / L of magnesium sulfate (MgSC ), sodium bicarbonate (NaHCCh), 15mg / L of phenol red, amino acids, and vitamins. In an embodiment, the amino acids include 84 mg / L of L-Arginine HCI, 63 mg / L of L- Cystine-2HCI, 584 mg / L of L-Glutamine, 30 mg / L of Glycine, 42 mg / L of L-Histidine-HCI, 105 mg / L of L-lsoleucine, 105 mg / L of L-Leucine, 146 mg / L of L-Lysine-HCI, 30 mg / L L-Methionine, 66 mg / L of L-Phenylalanine, 95 mg / L of L-Threonine, 16 mg / L of L-Tryptophan, 103 mg / L of L- Tyrosine-2Na-2H2O, and 94 mg / L of L-Valine. In an embodiment, the vitamins include 4 mg / L of Choline chloride, 4 mg / L of Folic acid, 7.2 mg / L of Inositol, 4 mg / L of Niacinamide, 4 mg / L of D-Pantothenic acid, 4 mg / L of Pyridoxine-HCI, 0.4 mg / L of Riboflavin, 4 mg / L of Thiamine-HCI, and 1.36 pg / L of Vitamin B12.

[0102]

[0099] The suspension was centrifuged at 1000 rpm for 5 minutes at room temperature (~25 °C) using a tabletop centrifuge. After centrifugation, the supernatant was removed, and the pellet was detached from the wall of the tube by tapping the tube. Thereafter, the cells were then resuspended in 5 mL of fresh DMEM complete medium (supplemented with 10% FBS) and was transferred into a T25 culture flask. The flask was incubated at 37 °C in a humidified incubator with 5% CO2. The culture was maintained until the cells reached full confluency, refreshing with fresh DMEM complete medium as required.

[0103]

[0100] To remove residual medium, cells were gently washed before adding 1 mL of 0.05% trypsin. The flask was incubated at 37 °C in a humidified atmosphere containing 5% CO2for 5 minutes to promote cell detachment. Once detached, 2 mL of complete DMEM medium was added to neutralize the trypsin activity.

[0104]

[0101] A portion of the resulting cell suspension was transferred to a sterile conical tube and centrifuged at 1000 rpm for 5 minutes at room temperature (~25 °C). After discarding the supernatant, the cell pellet was gently resuspended by tapping and reconstituted in 5 mL of fresh complete DMEM medium supplemented with 10% FBS. Cells were then seeded into a 96-well tissue culture plate at a density of approximately 0.4 million cells per well in 2 mL of 10% DMEM complete medium. The following day, zeocin was added to each row of wells at increasing concentrations: 0 pg / mL in the first row, followed by 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, and 1000 pg / mL in the subsequent rows. Media in each well was replaced every 2-3 days with fresh medium containing the corresponding Zeocin concentration. Cell viability and morphological changes were monitored daily for up to 7 days. After 7 days of treatment, complete cell death was observed at 250 pg / mL Zeocin, identifying it as the minimum effective concentration for selective elimination of non- resistant cells. This concentration was therefore chosen for subsequent antibiotic selection experiments.

[0105]

[0102] The remaining cell suspension were maintained in the T25 culture flask for the transfection. For this, the spent medium in the T25 culture flask was aspirated, and 1 mL of 0.05% trypsin was added to detach adherent cells. The flask was incubated at 37 °C in a humidified incubator with 5% CO2for approximately 5 minutes to allow complete detachment. Thereafter, 2 mL of DMEM complete medium supplemented with 10% FBS was added to neutralize the enzymatic activity of trypsin. The resulting cell suspension was transferred into a sterile tube and centrifuged at 1000 rpm for 5 minutes at room temperature (~25 °C). After centrifugation, the supernatant was discarded, and the cell pellet was gently resuspended in 5 mL of fresh complete medium. 0.3 million HEK293T cells were seeded into individual wells of a 6-well culture plate, each containing 2 mL of complete DMEM. The plate was incubated overnight (approximately, 16 hours) at 37 °C in a 5% CO2incubator to allow cells to adhere and reach approximately 80% confluency, which is optimal for transfection.

[0106]

[0103] For transfection of the construct 150 (obtained from Example 1 above) into the HEK293T cells, a transfection complex was prepared. For this, a mixture of 1 pg of the construct 150 (diluted in 0.1X TrisEDTA buffer) with 150 pL of EC condensation buffer and 8 pL of enhancer reagent was prepared (using Effectene kit, procured from Qiagen). The mixture was vortexed and pipetted to ensure proper mixing and incubated at room temperature (approximately, 25 °C) for 5 minutes. Following this incubation, 25 pL of Effectene transfection reagent was added to the mixture, and was incubated for an additional 10 minutes at room temperature to allow the formation of the transfection complex. After 10 minutes, the resulting transfection complex was then diluted in 1 mL of DMEM complete medium (containing 10% FBS).

[0107]

[0104] Meanwhile, the old culture medium from each well was carefully aspirated, and 1 mL of fresh DMEM complete medium (containing 10% FBS) was added along the side of each well without disturbing the cell monolayer. The transfection complex (diluted in the DMEM complete media) is added dropwise to the cells to all the rows of the wells expect for one well (which was the non-transfected control wells). The non-transfected control wells were subjected to the same handling but without the addition of transfection reagents. The cells were then incubated for 24 hours at 37 °C in a humidified 5% CO2incubator.

[0108]

[0105] After 24 hours, transfected cells were dissociated using a cell dissociation buffer and seeded into a sterile T75 flask containing 15 mL of fresh DMEM complete medium supplemented with 10% FBS. Upon confirmation of cell detachment, antibiotic selection was initiated with Zeocin at a final concentration of 250 pg / mL.

[0109]

[0106] Cells were maintained in selection medium for 3 to 4 weeks. During this period, resistant colonies began to form. These parental polyclonal populations were cryopreserved for future use. To isolate monoclonal, stably transfected lines expressing rBMP2, single-cell clones was subsequently isolated using limiting dilution method in 96-well plates.

[0110]

[0107] Single cell clones were isolated using the limiting dilution technique. Prior to initiating the cloning procedure, cells from the parental polyclonal population were evaluated for viability and active proliferation to confirm their suitability for cloning. Cells grown in a T75 culture flask were harvested, and a single-cell suspension was prepared by resuspension. A precise cell count was performed using a hemocytometer to determine the appropriate dilution factor. Based on this count, the cell suspension was diluted in a cloning medium (procured from HiMedia, India) to achieve a final concentration of 300 cells in 40 mL. To ensure even distribution, 100 pL of the suspension was dispensed into each well of four 96- well tissue culture plates. The plates were then incubated at 37 °C in a humidified 5% CO2incubator for 10 to 15 days. During incubation, colony formation was regularly monitored under an inverted microscope to identify colonies derived from single cell.

[0111]

[0108] Wells containing clearly defined single-cell-derived colonies were allowed to proliferate until the colony covered roughly one-third of the well surface. Selected monoclonal colonies were gradually expanded into 24-well plates using DMEM complete medium supplemented with 10% FBS. The monoclonal colonies were screened for rBMP2 expression using an ELISA kit (procured from Thermo Scientific, USA). The single cell colony giving the highest rBMP2 yield (about 6.43 mg / L at routine lab scale conditions) was selected and cryopreserved.

[0109] Example 4: Expansion of the single cell clone obtained from Example 3 above in T175

[0112] Flask

[0113]

[0110] A cryopreserved vial of the single cell clone was thawed and revived in T25 culture flasks containing DMEM complete medium supplemented with 10% serum. Upon reaching approximately 80% confluency, the cells were treated with 1 ml of 0.05% trypsin and incubated at 37°C for 5 minutes to detach the cells from the walls of the flask. The trypsin activity was neutralized with DMEM complete medium having 10% FBS. The cells were collected by centrifugation at 1000 rpm for 5 minutes at room temperature (25°C). The resulting cell pellet was gently resuspended in 5 ml fresh 10% DMEM and transferred to a T175 culture flask containing 25 ml of DMEM having 10% FBS. Cultures were incubated at 37°C with 5% CO2until ready for adaptation or further scale-up.

[0114]

[0111] Once confluent, the cells were trypsinized, counted using a hemocytometer, and seeded at 0.15-0.2 million cells / ml into 100 mL shake flasks containing 30 mL of Hybridoma- serum free medium (Hybridoma-SFM™) (procured form Gibco™) supplemented with 1 g / L Cell Boost 6™ (CB6) (procured from Cytiva, USA), 1 mg / L Dexamethasone (Dxn), and 10 mL / L of 2mM of L-alanyl-L-glutamine dipeptide (in 0.0085% NaCI). The cell boost 6™ (CB6) added includes lipids, amino acids, vitamins, and growth factors. The combination of hybridoma- serum free medium (Hybridoma-SFM™) and the cell boost 6™ supplement includes 10 mM of D-glucose, 120 mM of sodium chloride (NaCI), 5 mM potassium chloride (KCI), 1.8 mM of calcium chloride (CaCl2), 0.5 mM of magnesium sulfate (MgS04) and 10 mM of (NaHC03), 4 mM of L-Glutamine, 1 mM of L-Arginine, 0.4 mM of L-Leucine, 0.1 mM of L-Lysine, 0.1 mM of L-Phenylalanine, 0.1 mM of L-Tyrosine, 0.05 mM of L-Methionine, 0.05 mM of L-Histidine, 0.03 mM of L-Valine, 0.25 mM of Glycine, 0.0002 mM of folic acid, 0.5 pM of thiamine HCI (vitamin Bl), 0.2 pM riboflavin (vitamin B2), 0.02 mM of nicotinamide (vitamin B3), 0.3 pM of pyridoxine HCI (vitamin B6), 0. 1 pM of biotin, 0.002 mM of pantothenic acid, 0.5 mM of zinc sulfate (ZnS04), 0.2 pM of ferrous sulphate (FeS04) / ferric nitrate (Fe(NO3)3, 0.05 pM of copper sulphate (CuS04), 0.05 pM of manganese(ll) sulphate (MnS04), 0. 1 pM of basic fibroblast growth factor (bFGF), 0.01 mM of glutathione, 0.01 pM of Pluronic® F68, etc. Cultures were incubated at 37°C in a 5% CO2shaker incubator for 6 days. On day 6, fresh Hybridoma-SFM™ (100 ml per liter) was added, and incubation continued for an additional 9 days under the same conditions. Cultures were harvested on day 15 based on cell viability. The culture was centrifuged at 10000 relative centrifugal force (ref) for 10 minutes at 4°C using centrifuge. The pellet obtained, containing the cells, was discarded. The spent media in the harvested cultures had the rBMP-2 protein. At the time of harvest, cell viability dropped to 70%, compared to the typical >95% observed under standard culture conditions.

[0115]

[0112] Example 5: Purification of the rBMP-2 protein from the spent media obtained from example 4

[0116]

[0113] 1 Liter of the spent media was dialyzed using the equilibration buffer. The equilibration buffer was specific for the heparin column. The buffer includes 25 mM Tris, 50 mM NaCI, 10% glycerol, 1 mM EDTA, and 1 mM PMSF, with a pH of 7.0 ± 0.2. 1 liter of the spent media was dialyzed against 20 liters of this buffer. During the dialysis, the buffer was changed four times at three-hour intervals.

[0117]

[0114] After dialysis, the spent media was sequentially filtered through 0.45 pm and 0.22 pm filters to ensure clarity and sterility. The filtered media was then concentrated by ultrafiltration to approximately 200 ml. The concentrated media was loaded on the heparin column.

[0118]

[0115] 50 ml of heparin gel was laid into a column. The resin was first washed with 5 column volumes of 2 M NaCI at a flow rate of 4 ml / min, followed by 5 column volumes of 100 mM NaCI at the same flow rate. Thereafter, the heparin gel was equilibrated with 10 column volumes of equilibration buffer (25 mM Tris, 50 mM NaCI, 10% glycerol, 1 mM EDTA, 1 mM PMSF, pH 7.0 ± 0.2) at 4 ml / min. Filtered lysate (filtered through a 2 pm filter) was then loaded onto the column at a flow rate of 2.5-3 ml / min using a peristaltic pump.

[0119]

[0116] The column was washed sequentially with 7 column volumes (~200 ml) of equilibration buffer at 3 ml / min, followed by 7 column volumes of equilibration buffer containing 100 mM NaCI at 4 ml / min. Additional washes were performed with 8 column volumes (~240 ml) of equilibration buffer containing 150 mM NaCI and then 8 column volumes of equilibration buffer containing 300 mM NaCI, both at 4 ml / min. The resin was further washed with 5 column volumes (150 ml) of buffer containing 500 mM NaCI and then 5 column volumes (150 ml) of buffer containing 750 mM NaCI.

[0120]

[0117] Thereafter, the eluent is collected in a tube. For elution, a sterile 15 ml centrifuge tube was prepared by adding 0.5 ml of 0.2 pm filtered 50% trehalose solution. BMP-2 was eluted with buffer containing 1.5 M NaCI, collecting 5 ml fractions with 5 column volumes directly into tubes containing 5% trehalose, and stored at 2-8°C. Elution fractions with absorbance at 280 nm greater than 0.05 were pooled, as shown in Fig. 5a.

[0121]

[0118] Thereafter, the eluent was dialyzed. For this, a dialysis bag of 15cm length and with a 5 kDa molecular weight cutoff was placed in a beaker containing ten times the volume of intermediate dialysis buffer (25 mM sodium phosphate, pH 6.4 ± 0.2, 10% glycerol, 1 mM PMSF, 1 mM EDTA). The dialysis buffer was stirred gently using a magnetic stirrer. Dialysis was performed overnight at 4°C with four buffer changes. After dialysis, the sample was carefully collected from the bag into a sterile Nalgene bottle, the volume was measured and labeled, and finally, 5% trehalose was added from the stock solution to the dialyzed material.

[0122]

[0119] After dialysis, the dialyzed solute is loaded onto a cationic exchange chromatographic column. 10 mL of carboxymethyl (CM) cellulose resin was laid in a column. The column was then equilibrated for 30 minutes with equilibration buffer composed of 25 mM sodium phosphate (pH 6.4 ± 0.2), 10% glycerol, 1 mM EDTA, and 1 mM PMSF.

[0123]

[0120] The dialyzed solute is loaded onto the CM cellulose at a flow rate of 3 ml / min using a peristaltic pump. The flow-through was collected and passed through the column again at the same flow rate. The column was washed with 10 column volumes (100 ml) of equilibration buffer, followed by 10 column volumes of buffer containing 50 mM NaCI, both at 3 ml / min. The column was loaded and washed for 3 to 5 hours.

[0124]

[0121] After drying the tubing, buffer containing 750 mM NaCI and 5% trehalose was passed to the column height, and the column was incubated for 15 minutes. The elution fraction of 2 ml each was collected using the same 750 mM NaCI and 5% trehalose equilibration buffer. Fractions with absorbance at 280 nm greater than 0.1 were pooled, as shown in Fig. 5b. The column regeneration process followed subsequently.

[0125]

[0122] Post-second ion exchange chromatography, the eluent was dialyzed. For this, a dialysis bag of a length of 15 cm with a 14 kDa molecular weight cutoff was thoroughly washed with distilled water. The dialysis bag was placed into a beaker containing ten times the volume of final dialysis buffer (25 mM sodium phosphate, pH 7.0 ± 0.2, 100 mM NaCI), and gently stirred using a magnetic stirrer. The eluent was dialyzed for 16 hours at 4°C in a cold room, with four buffer changes.

[0126]

[0123] After dialysis, the solutes were collected into a sterile Nalgene bottle. The final volume of the dialyzed sample containing the rBMP-2 was measured and filtered through 0.2 p - PES sterile syringe filter in sterile falcon tube under aseptic conditions.

[0127] T1

[0124] Example 6: SDS-PAGE analysis of rBMP-2 obtained from example 5 above

[0128]

[0125] The rBMP-2 was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gel containing 15% polyacrylamide was prepared to evaluate the purification profile and determine the purity of rBMP-2. The analysis was performed under reducing conditions in the presence of SDS to ensure complete denaturation of the protein for accurate molecular weight estimation. The gel revealed a single prominent band at approximately 13 kDa (as shown in Fig. 6), consistent with the expected molecular weight of BMP-2. The absence of additional bands indicated high purity, with rBMP2 estimated to be >95% pure.

[0129]

[0126] Example 7: Western blot analysis of rBMP-2 obtained from example 5 above

[0130]

[0127] Western blot analysis was performed to confirm the identity of the purified recombinant BMP-2 (rBMP2) band observed in SDS-PAGE. A monoclonal anti-BMP-2 mouse antibody (procured from Thermo Scientific, USA) was used as the primary probe to specifically detect rBMP-2. A pre-stained protein marker was included for molecular weight reference. The blot revealed a distinct and specific band at approximately 13 kDa (as shown in Fig. 6a), confirming the presence of BMP-2 and validating the results obtained from SDS- PAGE.

[0131]

[0128] Example 8: Dot blot analysis of rBMP-2 obtained from example 5 above

[0132]

[0129] Dot blot analysis was carried out to confirm the presence of purified rBMP2 in its native conformation. A monoclonal anti-BMP-2 mouse antibody (procured from Thermo Scientific, USA) was used as the primary probe for specific detection of BMP-2. An unrelated protein served as a negative control. The blot showed a distinct and specific signal at the marked position (as shown in Fig. 7), confirming the presence of rBMP-2 in its native form.

[0133]

[0130] Example 9: Antigen activity analysis of rBMP-2 obtained from example 5 above by ELISA:

[0134]

[0131] Protein quantitation of rBMP2 was performed by measuring absorbance at 280 nm (A28O), using an extinction coefficient of 1.44. The antigenic activity of rBMP2 was evaluated using a commercial ELISA kit (procured from Thermo Scientific, USA). The antigenic activity values obtained from the ELISA assay were comparable to those determined by Azso measurements, indicating consistency between the immunological and spectrophotometric quantitation methods. A total of 48.2 mg of rBMP2 per liter of spent medium was recovered. After purification, 30.9 mg of rBMP-2 protein per liter of spent medium was obtained per liter of the spent medium, corresponding to an overall yield of 64.10% and 43-fold increase in purity (Table 2).

[0135] Table 2: Purification profile of rBMP2 purified from HEK293T cells a: Total protein (TP) content was estimated by Bradford Protein Assay using bovine serum albumin as a standard protein. b: Total rBMP-2 content of samples / fractions was measured by BMP2 specific ELISA assay.

[0136]

[0132] Example 10: Study of immunocytochemistry (ICC) of HEK293T cells obtained from example 3 above

[0137]

[0133] ICC was performed on rBMP2-expressing HEK293T cells (clones) to confirm the expression and cellular localization of rBMP2 protein. A monoclonal anti-BMP2 antibody (procured from Thermo Scientific, USA) was used as the primary probe for BMP2 detection.

[0138]

[0134] On the Day 1: 35 mm Petri dish containing a sterilized glass coverslip was pre-washed with 70% ethanol and air-dried and was prepared for cell culture. Approximately 100-200 pL of rBMP2-expressing HEK293T cells, corresponding to ~1 x 105cells, were seeded onto the coverslip. The dish was then incubated at 37 °C in a CO2incubator to allow the cells to attach to the surface. After confirming cell adherence, 2 mL of DMEM complete medium was gently added to the dish, and the cells were incubated overnight.

[0139]

[0135] On Day 2: The DMEM complete medium was removed, and the cells were washed three times with lx phosphate buffer saline (PBS) at 5-minute intervals to remove residual medium. Cells were then fixed using 1 mL of 4% paraformaldehyde (prepared in PBS, pH 7.2- 7.4) for 15 minutes at room temperature with gentle shaking. Post-fixation, cells were washed again three times with lx PBS.

[0140]

[0136] Permeabilization was carried out using 0.3% Triton X-100 for 5 minutes, followed by three PBS washes. To prevent non-specific antibody binding, the cells were incubated with 1% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. After blocking, the cells underwent another series of three PBS washes.

[0141]

[0137] Following this, the primary antibody— anti-BMP2 monoclonal antibody (procured from Thermo Scientific, USA) was diluted 1:200 in 1% BSA. Approximately 200 pL of this antibody solution was added to the cells, which were then incubated overnight at 4 °C to allow specific binding.

[0142]

[0138] On Day 3: The primary antibody was removed, and the cells were washed three times with lx PBS at 5-minute intervals. A fluorescence-labeled secondary antibody (procured from Thermo Scientific, USA) (diluted 1:200 in PBS) was then applied, and the cells were incubated for 1 hour in the dark to prevent photobleaching.

[0143]

[0139] After incubation, the cells were washed three more times with PBS. Nuclear staining was performed using 4',6-diamidino-2-phenylindole (DAPI) at a final concentration of either 1.43 pM or 300 nM. Cells were incubated with DAPI for 10 minutes, followed by three final washes with PBS. Fluorescence microscopy was used to visualize and capture images of the stained cells.

[0144]

[0140] Immunocytochemistry analysis confirmed the expression of rBMP2 in the transfected HEK293T cells. As shown in Fig. 8a depicts the DAPI-stained nuclei, providing a clear view of cell morphology and nuclear localization. Fig. 8b shows the specific immunofluorescence signal corresponding to rBMP2 expression, detected using the anti-BMP2 primary antibody. Fig. 8c depicts overlapping of the cells from fig. 8a and the cells from fig. 8b, thus indicating the intracellular localization of rBMP2, demonstrating successful expression and specific detection of rBMP2 through immunocytochemical staining.

[0145]

[0141] Example 11: Determination of Endotoxin level in rBMP-2 obtained from example 5 above

[0146]

[0142] The Commercial Chromogenic Endotoxin LAL Kit (procured from Thermo Scientific, USA) was utilized to measure endotoxin levels in the samples of rBMP-2. Initially, a sterile 96-well plate was pre-equilibrated at 37±1°C for 10 minutes. The Endotoxin Standard (ES) was reconstituted by adding 1.5 mL of Endotoxin-Free Water (EFW) to achieve a concentration of 10 EU / mL, followed by vigorous mixing for 15 minutes. From this solution, a 1.0 EU / mL stock was prepared and vortexed thoroughly. Using this stock, a series of endotoxin dilutions were made by adding 20 pL of the stock endotoxin (10 EU / mL) to 180 pL of EFW, resulting in a total volume of 200 pL. Serial dilutions were then carried out to generate concentrations of 0.5 EU / mL, 0.25 EU / mL, 0.1 EU / mL, 0.05 EU / mL, and 0.01 EU / mL, covering a wide range of endotoxin standards. Samples were diluted with endotoxin-free water at ratios of 1:10, 1:100, and 1:1000. The Amebocyte Lysate reagent was reconstituted with 1.7 mL of EFW, gently swirled to avoid foam formation, and used within 5 minutes. To each well, 50 pL of the endotoxin standards, samples, or blanks was added, followed by 50 pL of the Amebocyte Lysate reagent, and mixed gently by tapping the plate. The plate was then covered and incubated for 14 minutes at 37°C. Meanwhile, the Chromogenic Substrate was reconstituted with 3.4 mL of EFW and pre-warmed at 37±1°C for 5 minutes. Subsequently, 100 pL of the warmed substrate was added to each well, mixed gently, and incubated for 6 minutes at 37±1°C. After exactly 6 minutes, 50 pL of stop solution was added to each well, and the plate was gently tapped again. The absorbance was immediately measured at 405 nm to determine the endotoxin levels. Endotoxin level was determined for 3 different batches of rBMP2 (Table 3).

[0147] Table 3: Endotoxin testing result

[0148]

[0143] Endotoxin level of all 3 batches of rBMP-2 showed <0.1 EU / ng of protein and hence suitable for cell culture work.

[0149]

[0144] Example 12: Biological activity assay of rBMP-2

[0150]

[0145] To study the biological activity of the rBMP-2, dose-dependent effect of r BMP-2 on alkaline phosphatase (ALP) production in ATDC-5 mouse chondrogenic cells using p- nitrophenyl phosphate (pNPP) as a chromogenic substrate was assessed.

[0151]

[0146] For this, ATDC-5 cells were cultured in DMEM / F12 medium supplemented with 5% fetal bovine serum (FBS). These cells were seeded into 96-well plates at a density of 1 x 104cells per well. The cells were incubated for 24 hours at 37°C in a humidified atmosphere containing 5% CO2to allow proper attachment. After this initial incubation, the cells were treated with increasing concentrations of rBMP-2) at 0, 10, 50, 100, and 200 ng / ml, prepared in fresh culture medium. The treatment was continued for 72 hours under standard culture conditions. Following the incubation period, cells were gently washed with PBS, and 50-100 pl of lysis buffer was added to each well. The plates were then incubated on ice for 10-15 minutes to ensure complete cell lysis. Lysates were collected and clarified by centrifugation at 12,000 x g for 10 minutes at 4°C.

[0152]

[0147] To assess alkaline phosphatase (ALP) activity, 80-100 pl of ALP assay buffer, 10-20 pl of the cell lysate, and 50 pl of p-nitrophenyl phosphate (pNPP) substrate solution (final concentration ~5 mM) were added to a fresh 96-well plate. The reaction mixtures were incubated at 37°C for 30-60 minutes, protected from light. To stop the reaction, 50 pl of 1 N or 3 N NaOH was added to each well. Absorbance was then measured at 405 nm using a microplate reader. Blank values (reaction mixture without lysate) were subtracted from all readings, and ALP activity was compared across the different concentrations of rBMP-2. The results were expressed as fold change relative to the untreated control or normalized to total protein content, as required, as shown in Fig. 9. The assay result demonstrates the biological activity of rBMP-2. The fold change of the protein increased with the increased concentration of ALP.

[0153]

[0148] The scope of the invention is only limited by the appended patent claims. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used.

Claims

WE CLAIM,1. A nucleic acid molecule (110) comprising SEQ ID No. 1, the nucleic acid molecule (110) encodes a recombinant bone morphogenetic protein-2 (rBMP-2) inside a mammalian cell.

2. A construct (150) comprising: a. a vector (130) having a first promoter (133); and b. a nucleic acid molecule (110) disposed downstream of the promoter (133), the nucleic acid molecule (110) as claimed in claim 1.

3. The construct (150) as claimed in claim 2, wherein the vector (130) includes at least one first origin of replication region (131), and one or more selectable markers (137).

4. The construct (150) as claimed in claim 3, wherein the one or more selectable markers (137) include a resistance gene(s) of one of ampicillin encoded by SEQ. ID. No. 8, or zeocin encoded by SEQ. ID. NO. 9.

5. The construct (150) as claimed in claim 4, wherein the resistance gene(s) is disposed downstream of a corresponding promoter region, including one of a AmpR promoter or a ZeoR promoter.

6. The construct (150) as claimed in claim 2, wherein the vector (130) includes a signaling region (135) encoded by SEQ ID No. 6, the signaling region (135) is disposed between the promoter (133) and the nucleic acid molecule (110).

7. A single cell colony comprising a plurality of mammalian cells, each mammalian cell including the construct (150) as claimed in any of the claims 2-6.

8. The single cell colony as claimed in claim 7, wherein the mammalian cell includes one of Human Embryonic Kidney T (HEK293T) cells, Chinese hamster ovary (CHO) cells, AD293 cells, or Human Embryonic Kidney (HEK293) cells.

9. The single cell colony as claimed in claim 7, wherein the plurality of mammalian cells is suspended in a serum-free media including a carbon source, a plurality of inorganic salts, a plurality of amino acids, a plurality of vitamins, a plurality of trace elements, a plurality of growth factors.

10. The single cell colony as claimed in claim 9, wherein the carbon source includes 5 mMto 25 mM of D-glucose.

11. The single cell colony as claimed in claim 9, wherein the plurality of inorganic salts includes 120 mM to 150 mM of sodium chloride (NaCI), 4 mM to 6 mM of potassium chloride (KCI), 1 mM to 2 mM of calcium chloride (CaCl2), 0.5 mM to 1 mM of magnesium sulfate (MgS04) and 10 mM to 30 mM of sodium bicarbonate (NaHC03).

12. The single cell colony as claimed in claim 9, wherein the plurality of trace elements includes 0.5 pM to 10 pM of zinc sulfate (ZnS04), 0.2 pM to 20 pM of one of ferrous sulphate (FeS04) or ferric nitrate (Fe(NO3)3, 0.05 pM to 1 pM of copper sulphate (CuS04) and 0.05 pM to 1 pM of manganese (II) sulphate (MnS04).

13. The single cell colony as claimed in claim 9, wherein the plurality of amino acids includes 2 mM to 4 mM of L-Glutamine, 0.4 mM to 1 mM of L-Arginine, 0.4 mM to 0.8 mM of L-Leucine, 0.1 mM to 0.8 mM of L-Lysine, 0.1 mM to 0.3 mM of L- Phenylalanine, 0.05 mM to 0.2 mM of L-Tyrosine, 0.05 mM to 0.2 mM of L- Methionine, 0.02 mM to 0.1 mM of L-Histidine, 0.03 mM to 0.6 mM of L-Valine and 0.2 mM to 0.4 mM of Glycine.

14. The single cell colony as claimed in claim 9, wherein the plurality of vitamins includes 1 pM to 10 pM of folic acid, 0.5 pM to 50 pM of thiamine HCI (vitamin Bl), 0.01 pM to 0.2 pM of riboflavin (vitamin B2), 0.02 mM to 0.2 mM of nicotinamide (vitamin B3), 0.003 mM to 0.05 mM pyridoxine HCI (vitamin B6), 0.01 pM to 0.1 pM biotin, and 0.002 mM to 0.1 mM pantothenic acid.

15. The single cell colony as claimed in claim 9, wherein the plurality of growth factors include: a. 1 ng / mL to 20 ng / mL of at least one of Epidermal Growth Factor (EGF), basic fibroblast growth factor (bFGF), or a combination thereof; b. 0.01 to 100 pM of one or more lipids including cholesterol, fatty acids, phospholipids, Pluronic® F68; c. 10 pM to 100 pM of at least one antioxidant including glutathione, ascorbate, or a combination thereof; d. lmg / L Dexamethasone (Dxn); and e. 10 mL / L of 2mM of L-alanyl-L-glutamine dipeptide in 0.0085% NaCI.

16. A method (300) to prepare a recombinant bone morphogenetic protein-2 (rBMP-2),the method (300) comprising: a. incubating a single-cell colony of a plurality of mammalian cells in a serum- free media for a pre-defined time period to produce a spent media, each mammalian cell includes a construct (150) having a vector (130) and a nucleic acid molecule (110) comprising SEQ ID No. 1; and b. purifying the spent media to obtain rBMP-2.

17. The method (300) as claimed in claim 16, wherein the step of purifying the spent media includes subjecting the spent media to at least one of ultrafiltration, dialysis, affinity chromatography, and column chromatography.

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