FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See Section 10 and Rule 13)
A NOVEL CELL LINE DEVELOPMENT PROCESS TO PRODUCE RECOMBINANT PROTEINS USING TWIN VECTOR EXPRESSION
SYSTEM
Intas Biopharmaceuticals Limited
An Indian company having its registered office at:
Plot No: 423/P/A/GIDC
Sarkhej-Bavla Highway
Moraiya, Tal.: Sanand
Ahmedabad-382 213
Gujarat, India
The following specification describes the invention.

RELATED APPLICATIONS
This application is related to Indian Provisional Application 3323/MUM/2010 filed 7 Dec, 2010 and is incorporated herein in its entirety.
FIELD OF THE INVENTION
The present invention relates to a process of designing gene constructs and developing stable cell line(s) for high level expression of recombinant proteins using twin vector expression system.
BACKGROUND OF THE INVENTION
The importance of recombinant proteins for therapeutic applications has been increasingly highlighted over the past two decades. Mammalian expression systems are generally used to produce around 60% of all the recombinant therapeutic proteins because mammalian cells are able to provide human-like post translational modifications of the recombinant proteins making them similar in their biochemical properties to the naturally occurring human proteins. The emerging demand for high quality recombinant proteins drives the research and development of stable mammalian cell based expression systems for enhancing productivity while maintaining human-like quality.
The development of stable and high producer cell lines for the production of the protein of interest (POI) involves the need of selecting the right vector and host systems, finding the appropriate gene cloning strategy, transfection method and selection of clones that combines stable integration, gene amplification, efficient transcription with efficient folding, processing and secretion capabilities without affecting the host cell multiplication or its physiological state.

Dihydrofolate reductase (DHFR) and Glutamine Synthetase (GS) are the most commonly used expression systems in Chinese hamster ovary (CHO) and mouse myeloma cell lines (SP2/0 and NSO) - which are the common mammalian host cells used for expression of recombinant proteins. The production of recombinant cell lines involves multiple steps that begin with the cloning of the gene of POI in a mammalian expression vector system. Selection is achieved by introducing the vector into mammalian host cells. The vector carries a copy of the gene that complements the auxotrophy (used as selection markers) along with the POI gene. For the selection of transformed cells, cells after transfection are cultured in the absence of specific suitable metabolites (hypoxanthine and thymidine for DHFR system and glutamine for GS system) to prevent the growth of non-transformed cells. The presence of two selectable marker genes allows stringent and stable selection of recombinant cell lines.
An alternative to the auxotrophic selection method is the utilization of genes having resistance to antibiotics like geneticin (G418), hygromycin, zeocin, blasticidin or puromycin on the vector containing the POI gene. With this method, transfected cells are selected with medium containing the appropriate antibiotic. However, a major advantage of both the DHFR and GS selection systems is that they allow for amplification of the integrated recombinant genes thereby enhancing POI production.
The vector containing POI is co-transfected with DHFR plasmid often co-integrates into the host genome, and hence subsequent amplification of DHFR gene in presence of methotrexate (MTX) results in co-amplification and enhanced gene copy number of desired gene. The POI gene co-amplifies in this manner resulting in co-expression of both the genes using host cell machinery.
Kaufman et al (Mol. Cell. Biol. 1985, vol.5 (7), pp. 1750 - 1759) reported the expression of human tissue plasminogen activator (t-PA) at high levels that can be achieved in CHO cells by co-transfection and subsequent co-amplification of the

transfected sequences. Expression vectors containing the t-PA cDNA gene and a dihydrofolate reductase (DHFR) cDNA gene were co-transfected into CHO DHFR-deficient cells. Transformants expressing DHFR were selected by growth in DHFR+ transformants in increasing concentrations of methotrexate resistant cells which had amplified both DHFR genes and t-PA genes over 100-fold. These cell lines expressed elevated levels of enzymatically active t-PA.
EP0236059 discloses the production of erythropoietin by cells co-trans fected with an erythropoietin-expression vector and another vector having a selective marker.
US6777205 provides a process wherein a eukaryotic cell line produces recombinant human EPO obtained by transfection with an expression vector that comprises a gene coding human EPO. The invention further provides a unique promoter and terminator as expression control elements. The vector in the present invention is pDHFR.
WO2009/010534 describes a method for obtaining cells that stably express a protein of interest, even when cultivated in the absence of selective pressure. DHFR is used as a surrogate marker. The transfected cells are not selected based on resistance to a toxic compound, but based on fluorescence as measured by FACS using fluorescent MTX.
WO2009/080720 describes a vector nucleic acid for expressing polypeptide of interest in a mammalian cell, comprising (a) at least one expression cassette for expressing a polypeptide of interest (POI); (b) an expression cassette comprising a mammalian selectable marker (MSM) gene; (c) an expression cassette comprising a mammalian amplifiable, selectable marker (MASM) gene; wherein the expression cassette (POI) is flanked 5' by the expression cassette (MASM), the expression cassette (MSM) is located 3' from the expression cassette (POI) and wherein the expression cassettes (MASM), (POI) and (MSM) are arranged in the same 5' to 3' orientation. Also

provided are host cells, comprising said vector and methods for producing a polypeptide using respective host cells.
US2010021911 shows that DHFR-deficient CHO cells grow slower compared to CHO wild type cells even in the presence of hypoxanthine and thymidine supplemented medium. To correct the growth defect, the invention introduces DHFR expression cassette.
Production of monoclonal antibody (mAb) requires the simultaneous expression of two genes, encoding both light chain (LC) and heavy chain (HC) and a selectable marker. In general two types of transfection strategies have been employed (1) where both the LC and HC genes are expressed under the control of individual promoters on the same vector and (2) where cells are co-transfected with two separate vectors encoding either HC or LC gene. In the latter scenario, different selectable markers may be associated with either HC or LC plasmid, for example HC expression selected by DHFR co-expression and LC via neomycin selection or use of two different antibiotic resistance genes on separate vectors.
Page et al (See Nat. Biotechnol. 1991, vol. 9, pp. 64 - 68) cloned the light and heavy chain cDNAs for the humanized monoclonal antibody Campath - 1H and expressed them in Chinese hamster ovary (CHO) cells using a dihydrofolate reductase (DHFR) amplification procedure. Each cDNA was positioned under control of the strong human beta actin promoter/ polyadenylation signals and used to evaluate alternative vector design and amplification procedures. By employing a dual selection co-transfection strategy, initial transformants accumulated antibody levels of 0.5μg/ml was obtained after 4 days continuous culture. When subjected to successive rounds of selection in medium containing stepwise increments to methotrexate, stable cell lines were obtained that secreted up to 200ug/ml of Campath - 1H during the same period. This reflects a productivity of 100μg/106 cells/day and demonstrates the potential of engineering CHO cells for the production of recombinant antibodies.

The present invention is of a novel and efficient process for developing a stable cell line for high level expression of recombinant proteins using co-transfection of two expression vectors, wherein the protein of interest (a single gene) has been cloned into two mammalian expression vectors having Neomycin resistance gene in one vector and DHFR gene in another vector utilized for mini-pool selection and gene amplification, respectively. The said cloning strategy is useful for cloning and expression of a gene selected from the group consisting of erythropoietin and its analogues, colony stimulating factors, interferons, growth factors, hormones, blood factors, receptors, single chain fragment variable (scFv), antibody fragments (Fab), antigens, enzymes, and the like. The utility of the current invention is in the field of developing stable and high yielding cell line(s) in a shorter time period suitable for industrial production at large scale.
SUMMARY OF THE INVENTION
The present invention provides a process for developing a stable cell line for high level expression of protein of interest (POI) comprising co-transfection of two vectors constructs, wherein (a) the first vector construct comprises gene of protein of interest (POI) and a DHFR amplification gene and (b) the second vector construct comprises the same gene of protein of interest as in the first vector and a selection marker other than DHFR wherein the said cell line is selected from the group consisting of a human cell line, CHO cell line, murine cell line and hybridoma selected from murine cell line.
In one aspect, the selection marker on the second vector construct is selected from the group consisting of neomycin, hygromycin, zeocin, blasticidin or puromycin.
In another aspect, the invention provides a process for generating a stable cell line for high level expression of protein of interest which comprising the steps of: a) selection of clones based on selection marker application b) amplification using

various concentration of methoxtrate c) screening and selection of clones that produce high level expression of protein of interest and d) establishing/selecting the clone following protein characterization
In yet another aspect, wherein the process for the selection of clone(s) producing the desired yield of POI as described herein is achieved in at least two rounds of gene amplification.
The present invention further provides a process of producing POI, which comprises
of: a) culturing a cell line obtained according to the process described herein under
conditions which permit expression of the said POI; b) harvesting the said POI and c)
purifying the said POI.
The present invention also encompasses a process for developing a stable cell line for
high level expression of POI, harvesting and purifying the said POI.
The present invention also provides a co-transfection process comprising the
permutation of two vector constructs whereby (a) the first vector construct comprises
a protein of interest and a DHFR expression cassette and (b) the second vector
construct comprises a protein of interest and a selection marker other than DHFR.
In a further aspect of the present invention, wherein the protein of interest is selected from the group consisting of erythropoietin and its analogues, colony stimulating factors, interferons, growth factors, hormones, blood factors, receptors, scFv, antibody fragments (Fab), antigens, enzymes, and the like.
The present invention further provides a pair of cloning or expression vector constructs for developing a stable cell line for high level expression of recombinant protein of interest (POI) comprising:
(a) a first vector construct comprising the gene of POI and a DHFR amplification gene; and

(b) a second vector construct comprising the same gene of POI and a selection marker
other than DHFR.
It is provided that the stable cell line for high level expression of recombinant protein
of interest (POI) is selected from the group consisting of human, CHO and murine
cell lines.
The first vector construct comprises the gene of POI and the amplification gene which
is selected from the group consisting of DHFR, Glutamine Synthetase (GS) and
Thymidine Kinase (TK) genes.
The second vector construct comprises the gene of POI and the selection marker other
than DHFR selected from the group consisting of neomycin, hygromycin, zeocin,
blasticidin and puromycin.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure la shows the pOptiVEC-TOPO® vector (with DHFR gene) used for cloning of
POI
Figure lb shows the pcDNA3.3-TOPO® vector (with Neomycin resistance gene)
used for cloning of POI
Figure 2 1 kb ladder (Lane 1) and purified PCR product (Lane 2) of POI
Figure 3 shows restriction digestion analysis of pOptiVEC TOPO®- POI construct
(with Ndel & Xmal enzymes)
Figure 4 shows restriction digestion analysis of pcDNA3.3 TOPO - POI construct
(with Sad & Hindlll enzymes)
Figure 5 shows POI Expression profile by ELISA (post transfection & G418
selection)
Figure 6 shows POI Expression profile after MTX amplification
Figure 7 shows SDS PAGE-Western blot of partially purified POI
Figure 8 shows RP-HPLC Profile of partially purified POI from the supernatant
DESCRIPTION OF THE INVENTION

The present invention relates to a process for developing a stable cell line for high level expression of recombinant proteins using co-transfection of two mammalian expression vectors wherein the desired protein of interest (POI) along with a signal sequence and a reading frame are cloned/ inserted in both the vectors independently.
The term "expression vector" as used herein refers to a DNA molecule, linear or circular, comprising a segment encoding a protein of interest (POI) operably linked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences. A vector may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, a tag etc. Vectors are generally derived from plasmid or viral DNA, or may contain elements of both. The term "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in the promoter and proceeds through the coding segment to the terminator. Replication of expression vectors in a host organism can be autonomous or through integration into the host genome.
The term "signal sequence" as used herein refers to a sequence of amino acids bound to the N-terminal portion of a protein which facilitates the secretion of the mature form of the protein outside the cells. The definition of a signal sequence is a functional one. The mature form of the extracellular protein lacks the signal sequence which is cleaved off during the secretion process.
The term "reading frame" as used herein refers to nucleic acid sequence encoding a non-regulatory protein required to encode a particular protein of interest without intervening stop codon.

Promoter as referred herein is a DNA sequence recognized by RNA polymerase in order to initiate the gene transcription and generate the corresponding messenger RNA.
In another embodiment of the present invention, the first vector (Figure la) comprises the desired protein of interest POI, signal sequence and DHFR gene as an amplification marker. In place of DHFR, other selectable markers such as Glutamine Synthetase (GS) and Thymidine Kinase (TK) genes are also used. Preferably, DHFR gene is used as the amplification marker.
In one embodiment of the present invention the second vector (Figure lb) comprises the POI (same gene as in the first vector), signal sequence and Neomycin selection marker. Other antibiotic resistance genes used in place of neomycin are hygromycin, zeocin, blasticidin or puromycin. The preferred antibiotic resistance gene is Neomycin used as the selection marker in the present invention.
The term "DHFR" refers to a polypeptide that is a member of dihydrofolate reductase family (EC 1.5.1.3) that reduces folic acid to dihydrofolic acid to tetrahydrofolic acid, using NADPH as electron donor which is precursor for synthesis of Hypoxanthine, Thymidine, Glycine and Purine.
The term "GS" refers to glutamine synthetase enzyme responsible for the biosynthesis of glutamine from glutamate and ammonia. This enzymatic reaction provides the sole pathway for glutamine formation in a mammalian cell. In the absence of glutamine in the growth medium, the GS enzyme is essential for the survival of mammalian cells in culture.
Geneticin,G418 is an aminoglycoside antibiotic, used as a dominant selective agent in cell transfection which interferes with the function of 80S ribosomes and blocks

protein synthesis in eukaryotic cells. G418 in the present invention is used as an antibiotic for neomycin resistance gene (selection marker).
In one embodiment of the present invention, the cell line used is selected from the group consisting of a human cell line, a Chinese Hamster Ovary (CHO) cell line, a murine cell line and hybridoma derived from murine cell line.
In a preferred embodiment, the nucleic acid encoding the POI has been inserted into vectors comprising Neomycin and DHFR independently which then is co-transfected . into mammalian cells grown as a suspension culture. Both the vectors can also be co-transfected in the same manner to other adherent and suspension cell lines. In an embodiment of the present invention, the process of cell line development for expression of a POI comprising the steps of:
• Derivation of the POI gene (with or without a signal sequence and additional segments using PCR and/or chemical synthesis);
• Cloning of desired gene into both the vectors at TOPO®/multiple cloning site;
• Co-transfection of mammalian cells using different combination of twin vectors (with single vector as control);
• Selection of clones upon application of various concentration of an antibiotic [Geneticin(G418)];
• Gene amplification using various concentration of drug MTX;
• Limit dilution cloning and clone selection by ClonePixFL™ (Genetix, Molecular Devices)
• Screening and selection of high producer and stable clones;
• Protein characterization;
• Establishment of research cell bank and master cell bank candidate for commercialization of the POI.

Yet another embodiment of the present invention is to increase the yield of recombinant protein in one or two rounds of gene amplification.
It is envisaged that the process described herein enhances the gene copy number. The concentration of methotrexate (MTX) used was 250 to 1000nM to achieve desire gene copy numbers with the above said gene construction and transfection process whereas in general the concentration varies from 25 to 10000nm to achieve the desired protein yield.
In yet another embodiment, the present invention provides a process for producing POI, said process comprising the step of: a) culturing a cell line obtained according to the process described herein under conditions which permit expression of said POI; b) harvesting said POI and c) purifying said POI.
In preferred embodiments, the POI includes but not limited to a group consisting of erythropoietin and its analogues, colony stimulating factors, interferons, growth factors, hormones, blood factors, receptors, scFv, antibody fragments (Fab), antigens, enzymes, and the likes thereof.
The present system has the advantage of developing stable cell lines much more rapidly [approximate time 1 -2 months] than the usual step-wise amplification [4-8 months]. In addition, higher production levels can be obtained with the said strategies compared to the existing systems. Accordingly, the present invention provides a process for developing high-expressing stable cell lines quickly and efficiently which offers significant reduction of developmental cost and time.
The following examples illustrate in detail about the present invention and the means of carrying out the invention to obtain a stable cell line for high level expression of recombinant proteins using co-transfection of two mammalian expression vectors

wherein the desired protein of interest (POI) along with signal sequence and reading frame is cloned/ inserted in both the vectors independently.
Examples Method
The examples described in detail below used the gene encoding POI which was chemically synthesized with signal and other essential sequences at 5' (start codon, promoter and enhance elements) and 3' (stop codon and terminator) ends. The gene was codon optimized for CHO cells to achieve optimal protein expression. The synthetic construct was amplified by PCR using forward and reverse primers and cloned into both pcDNA3.3-TOPO® and pOptiVEC-TOPO® vectors at T/A cloning site. Twin vector strategy was employed to have two selection markers, i.e. Geneticin and DHFR in individual vector in order to increase stringency and select the stable and high expressive clones. The well-established TOPO TA Cloning® strategy was employed to ligate the desired PCR product at T/A cloning site. Prior to CHO transfection, both the constructs were characterized by restriction digestion analysis as well as DNA sequencing and were used for cell line development.
The steps given below were sequentially followed in cell line development of POI:
• Derivation of the POI gene (with or without a signal sequence and additional segments using PCR and/or chemical synthesis)
• Cloning of the POI gene into both the vectors at multiple/ TOPO® cloning site
• Co-transfection of mammalian cells using different combination (ratio) of twin vectors
• Selection of mini-pools upon Geneticin (G418) antibiotic application
• Gene amplification using various concentration of MTX
• Limit dilution cloning by high throughput Clonepix
• Screening and selection of high producer stable clones
• Protein purification and characterization

• Establishment of research and master cell bank for commercialization of product.
Example 1
Cloning of desired gene into both the vectors at multiple cloning site
The desired gene expression cassette was designed to contain all essential elements
required for protein expression and extracellular secretion.
The desired gene obtained in a synthetic vector was amplified using forward and
reverse primers.
The Pfu polymerase having proofreading property was used for amplification of
desired gene.
PCR product was analyzed on 1 % agarose gel and seen in the gel at expected
position. The desired PCR product was purified from the gel and was further used for
cloning into expression vectors.
The purified PCR fragment was then subjected for addition of A-overhang since it had to be cloned into TOPO® T/A cloning vectors. A-overhang was obtained with 1U of Taq DNA polymerase at 72 C for 20 minutes in a PCR machine. Subsequently, A-overhanged PCR product was ligated into TOPO®, T/A cloning vectors (both). The ligated products were used to transform E. coli DH5-alfa electro competent cells. Cells were transformed by electroporation and plated on Soya Yeast (SY) agar plates containing 100μg/ml Ampicillin antibiotic for selection purpose. About 100-125 colonies were obtained on each plate/ combination from which 10-20 were picked up for plasmid DNA isolation and restriction analysis to identify the recombinant clones.
10-20 colonies from each combination were inoculated to 10ml SY broth containing 100μg/ml Ampicillin, incubated at 37 C for overnight with 200 revolutions per

minute (rpm) shaking speed. Next day, plasmid DNA was isolated (from 2.0ml culture) using Promega wizard mini-kit and protocols (Catalogue No. A1460). Finally, DNA was eluted in 75ul of elution buffer.
All samples were subjected to restriction digestion for characterization and verification of the clones. Restriction enzymes Sad and HindIII were used for pcDNA3.3 TOPO®-POI, whereas Ndel & Xmal for pOptiVEC/TOPO-POI clones. Samples were incubated at 37 C for ~4 hours and electrophoresed on 1 % agarose gel at 100 volts for 1.5 hour.
Two clones from each combination were found positive and showed presence of desired DNA fragments (Figures 3 and 4)- Subsequently, these clones were verified by DNA sequencing. Both the vectors were linearized with Pvul enzyme and ethanol purified prior to transfection.
Example 2
Co-transfection of mammalian cells using different combination of twin vectors
Among CHO cells, the dihydrofolate reductase-deficient (DHFR~) CHO DG44 cells are routinely used to establish cell lines for production of recombinant proteins. These were developed by Dr. Chasin at Columbia University using gamma rays to eliminate the entire DHFR locus. In non-mutated cells, DHFR is an essential enzyme for de novo synthesis of glycine, purines and thymidylate. This allows DHFR to be used as a dominant selectable marker and a gene amplifier for the expression of proteins in DHFR" cell lines. The DHFR" mutation in DG44 cells is stable and irreversible, which makes it a suitable mammalian cell line for production of recombinant proteins. The DG44 cell line is derived from suspension Chinese hamster ovary (CHO-S) cells (Urlaub et al., 1983). DG44 cells were adapted to suspension culture in CD-DG44 Medium.

Prior to transfection, the CHO/DG44 cells were revived in CD-DG44 complete medium (with 4.0mM Glutamine). To revive the cells, frozen vial stored in Liquid Nitrogen (LN2) was withdrawn and revived in 30ml complete medium in a 125ml shake flask and incubated at 37 °C with 8% Carbon dioxide (CO2) and at a shaking speed of 140 rpm (Biomatrix shaker). Cells were sub-cultured 4-5 times maintaining seed density at 0.33xl06 cells/ml at each passage,.
CHO/DG44 suspension cells were co-transfected with both the vector constructs comprising POI gene using liposome (Invitrogen) Max reagent. One day prior to transfection, cells were seeded with 0.33xl06 cells/ml and allowed to grow for approximately 24 hours so that cells will reach log phase. Approximately 8.0-9.0μg of total DNA was mixed with 15μl of Max reagent in four different combinations of the construct and transfection reagents (A to D) as shown in Table 1 and allowed for complex formation. Transfection samples were mixed to the suspension cells after 20 minutes incubation at room temperature, subsequently all flasks were incubated at 37°C with 8 % CO2 and shaking at a speed of 140 rpm in CO2 incubator. Supernatant collected (after 48 hours of post-transfection) from each flask was checked for protein expression by ELISA. Indirect sandwich ELISA was carried out to check the transfection results. ELISA strips (NUNC) were coated with 300ng/well of coating monoclonal antibody (Anti-POI mAb). Blocking was done with 3 % bovine serum albumin for 1 hour at 37 °C after which samples were applied to the wells. Different concentrations of POI were used as standard to correlate the expression yield of desired POI. After incubating with the test sample and the standard, 200ng/well of primary antibody, rabbit polyclonal antibody (Anti-POI) was used. Assay was detected using anti-rabbit-HRP conjugated antibody (the secondary antibody). Finally, the substrate tetramethylbenzidine (TMB) was added and color development was measured at 450 nm in an ELISA reader (BioTek Instruments). The standard curve was plotted to calculate the expression yield of desired POI.

Table 1 - Protein expression - post transfection

Sample 0> Expression Yield in (u.g/I/48 hours)
A 200
B 370
C 500
D 260
Control (No DNA) 0.0
Example 3
Selection of Mini-pools upon Geneticin (G418) antibiotic application
After 48 hours of transfection, cells were passaged at 0.33xl06 cells/ml to their respective flasks A to D for 3 passages without Hypoxanthine and Thymidine (HT) supplement subsequently. Cells were subjected to G418 selection at 250, 500, 750 & 1000u.g/ml (Except Pool A) and maintained for 4 to 5 passages. Protein expression was checked by ELISA. In comparison to all mini-pools (Except Pool A), cells selected at 500u.g/ml G418 gave highest expression which were further used for gene amplification. The G418 selection summary and expression profile of all mini-pools are given below (Table 2 & Figure 5).
Table 2: Productivity of minipools during selection in G418 medium

Stages Pool A PooIB Pool C PoolD
G418@500ug/ml Yield (mg/1/72 hours) 1.66 6.23 1.51
Example 4
Gene amplification using various concentration of MTX

The high producer mini-pools selected at 500μg/ml of G418 were subsequently subjected for gene amplification to increase the gene copy number and thus the protein expression. Various concentration of the drug MTX ranging from 25nM to 2000nM was applied and mini-pools were selected on the basis of POI yield and growth behavior. The MTX concentration was increased from low to high and their expression yield was analyzed by ELISA (Table 3 & Figure 6). The high producer mini-pool was selected from each combination and subjected further for protein identity test and limit dilution cloning.
Table 3: Expression yield after MTX amplification as analyzed by ELISA

Stages Pool A PoolB PoolC PoolD
Post Transfection (mg/l/48h) 3.20 0.37 0.50 0.26
G418 Selection (mg/l/72h) 1.66 6.23 1.51
Expression (mg/l/72h) 13.50 15.00 24.20 24.70
MTX Cone. 2000nM 500nM 250nM lOOOnM
Mini-pool C5 & D8 (derived from pool C & D) were studied in the shake flask in batch mode upto 5 days to check the growth and expression yield. The peak cell density reached approximately 1.5 x 106 to 2.0 xlO6 cells/ml in both the mini-pools. Expression yield of C5 and D8 was found to be 30 to 40 mg/L and 40 to 50 mg/L, respectively. Considering growth, quality and quantity attributes mini-pool D8 was selected for limit dilution cloning and clone selection. The automated clone selection work was done using ClonePixFL (Genetix).
Example 5
Limit dilution cloning & selection of high producer clone(s)

As mentioned above, mini-pool D8 (derived from pool D) was selected for dilution cloning and for high throughput selection. All materials used in the clone selection process were sterile and animal components free. The Clonepix high throughput device was used for dilution cioning of the mini-pool.
The cell viability and cell number of D8 was determined with a GUAVA® ViaCountc analysis. The viability was >80 % and cells were seeded into semi-solid medium. Approximately 1700 clones were picked up with ClonePixFL device. A production ELISA analysis (for relative production) was performed with all the clones and the following top twelve producer positive clones, were scaled up (from 96-well) and cryopreserved.
Table 4 - Expression profile of high produce clone

S.No Clone ng/ml
1. 1G3 2194
2. 1H1 2731
3. 1H3 2189
4. 1H5 1383
5. 2H1 826
6. 2H3 1126
7. 1G3B6 1815
8. 1G3B10 1642
9. 1G3B11 1696
10. 1G3C6 5118
11. 1G3G1 2897
12. 1G3G3 6374
Example 6
Clone screening and selection process
Various studies including growth profile, stability, growth in shake flask, bioreactor suitability, and various analytical tools such as SDS-PAGE western for product identity, IEF for isoform profile analysis, in-vitro assay for activity and RP-HPLC/ELISA for product quality and quantity were carried out to identify the best clone for further development. The sub-clones obtained after limit dilution cloning

from DHFR platform were analyzed for their quality and productivity. All clones were scaled up to 30ml in a 125ml shake flask and grown for 4 to 10 days in a batch mode. The yields were estimated by RP-HPLC/ ELISA performed on the shake flask batches of batch mode culture. Example 7
Expression Yield of top six clones
The top six 1G3, 1H3, 2H3, 1G3C6, 1G3G1 and 1G3G3 clones obtained after limit dilution cloning were analyzed for their quality and productivity. All clones were scaled up to 30ml in a 125ml shake flask and grown for 8 days in a batch mode. Samples were withdrawn at various time points for evaluation of product quantity, quality and growth profiles.
The yields were estimated by RP-HPLC/ELISA performed on the shake flask after 8 days of batch mode culture. The productivity ranged between 44 to 75 mg/1 (Table 5). Table 5 - Protein Yield of Selected Clones (Shake flask)

Days Clones Concentration (mg/1)
End of Batch-Day-8 1G3 44.17

1G3C6 71.22

1G3G1 57.65

1G3G3 48.48

2H1 56.88

2H3 74.07
Example 8 Purification
The harvest was subjected to purification by using various chromatographic techniques followed by RP-HPLC analysis. Purified protein sample was compared along with the standard POI, to see the comparability of POI produced by the said invention. (Figure 8).

Claims We Claim
1. A process of designing gene constructs for developing a stable cell line for
high level expression of recombinant protein of interest (POI), the process
comprising co-transfection of two different vector constructs, wherein
(a) the first vector construct comprises the gene of POI and a DHFR amplification gene; and
(b) the second vector construct comprises the same gene of POI and a selection marker other than DHFR.

2. The process of claim 1, wherein the said cell line is selected from the group consisting of human, CHO and murine cell lines.
3. The process of claim 1, wherein the said selection marker is selected from the group consisting of neomycin, hygromycin, zeocin, blasticidin and puromycin.
4. A process of developing a stable cell line for high level expression of recombinant protein of interest comprising the steps of:

a) selection of clones upon selection marker application;
b) amplification with drug methoxtrate;
c) screening and selection of clones that produce high level expression of protein of interest; and
d) establishing/selecting the clone following protein characterization.
5. The process of claim 4 wherein the selection of clone(s) is achieved in at least
two rounds of gene amplification.

6. A process of producing a POI, said process comprising the steps of: a) developing a stable cell line for high level expression of the said POI as in claim 1; b) harvesting the POI; and c) purifying the POI.
7. The process as claimed in claim 1, wherein the co-transfection allows the permutation of the two vector constructs (a) the first vector construct comprises of gene of POI and a DHFR amplification gene and (b) the second vector construct comprises of the gene of protein of interest and a selection marker other than DHFR.
8. The process of claim 1, wherein the protein of interest is selected from the group consisting of erythropoietin and its analogues, colony stimulating factors, interferons, growth factors, hormones, blood factors, receptors, scFv, their analogues and derivatives.
9. A pair of cloning or expression vector constructs for developing a stable cell line for high level expression of recombinant protein of interest (POI) comprising:

(a) a first vector construct comprising the gene of POI and a DHFR amplification gene; and
(b) a second vector construct comprising the same gene of POI and a selection marker other than DHFR.

10. The stable cell line of claim 9, wherein the cell line is selected from the group consisting of human, CHO and murine cell lines.
11. The second vector construct of claim 9, wherein the said construct is selected from the group consisting of neomycin, hygromycin, zeocin, blasticidin and puromycin.