Title of Invention	METHOD FOR DETERMINING THE POTENCY OF G-CSF
Abstract	The present invention relates to a standardised method of bioassay for detecting the effective concentration of granulocyte colony stimulating factor and estimation of ED50 and specific activity of G-CSF using human acute myeloid cell line. The invention also mentions further use of this technique for estimation of potency of G-CSF samples during various stages of purification process.

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FORM 2 THE PATENT ACT 1970 (39 of 1970) & The Patents Rules, 2003 PROVISIONAL SPECIFICATION (See section 10 and rule 13) 1. TITLE OF THE INVENTION: "Novel use of Indicator Cell Line for Bioassay" 2. APPLICANT (S): (a) NAME: FDC Limited (b)NATIONALITY: Indian company incorporated under the Companies Act 1956 (c) ADDRESS: 142-48, S.V. Road, Jogeshwari (W), Mumbai - 400 102, Maharashtra, India. 3. PREAMBLE TO THE DESCRIPTION The following specification describes the invention. FIELD OF THE INVENTION: The present invention relates to a standardised method of bioassay for detecting the effective concentration of granulocyte colony stimulating factor and estimation of ED50 and specific activity of G-CSF using human acute myeloid cell line. The invention also mentions further use of this technique for estimation of potency of G-CSF samples during various stages of purification process. BACKGROUND OF THE INVENTION: Various methods available for determination of protein concentration, purity, and structural configuration can be applied to G-CSF preparations as well. General protein estimation methods such as Lowry's, Biuret or absorbance assay in UV range estimate the total protein concentration of purified sample. Whereas methods such as, SDS PAGE and RP-HPLC determine the purity and concentration of the sample accurately. Specific methods such as G-CSF ELISA are available which estimate the concentration as well as binding properties (and hence the potency) of the molecule to its receptor. Methods such as MALDI-TOF mass spectroscopy and CD- spectroscopy can reveal, in detail, the structural configuration and integrity of the molecule required for its proper biological activity. But none of these methods can estimate the biological activity of the sample as none of them take into account the actual functional properties of the G-CSF molecule. All the methods mentioned above are based on properties such as structure, molecular weight, ionic charge and purity of the sample molecule. The biological activity of the sample can only be estimated by in vivo (animal based) or in vitro (cell based) bioassays. In vivo, the G-CSF molecule stimulates the survival and proliferation of myeloid progenitor cells as well as their differentiation and maturation towards neutrophilic granulocytes. In addition, G-CSF stimulates release of mature neutrophils from bone marrow and brings about their activation. In vivo, bioassay of G-CSF makes use of this fact and depends upon the increase in number of circulating Neutrophils in the blood of injected neutropenic mouse. As per all other animal based bioassays, this assay also has limitations such as use of animals, variations in the results due to variations in the animal models etc. In vitro, bioassays are considered as a "Gold Standards" for biological activity of the cytokine.(Cytokine assay by Daniel G. Remickl,* and Theresa L. Whiteside2 IThe University of Michigan Medical School, Catherine Road, Ann Arbor, MI 48109-0602, USA 2Pathology Department, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, USA). These assays are not only more sensitive than immunoassays but are the only techniques, which is biologically relevant without making use of any animal models. Bioassays not only detect the presence of cytokine but it measures the biological activity, which gives in vitro simulation of in vivo activity of the molecule. For this reason in vitro bioassays are indispensable for characterization and studies of any cytokine. In case of G-CSF, many cell lines are available that respond to G-CSF. Few such cell lines are NFS-60 (US 5,919,757, Glycopegylation of recombinant therapeutic proteins produced E.coli, Novel G- CSF Conjugates by Shawn DeFrees Zhi-Guang Wang3, Ruye Xing3, Arthur E. Scott3, Jin Wang3, David Zopfl,3, Dominique L. Gouty4, Eric R. Sjoberg4, Krishnasamy Panneerselvam4, Els CM. Brinkman-Van der Linden2,4, Robert J. Bayer4, Mads A. Tarp5, and Henrik Clausen5 ), M-NFS-60 (Production of bioactive human granulocyte stimulating factor in transgenic rice cell suspension cultures by Shin-Young Hong a, Tae-Ho Kwon a, Yong-Suk Jang b, Soo-Ho Kim c, Moon-Sik Yang a, a Division of Biological Sciences and Research Institute for Bioactive Materials, Chonbuk National University, Jeonju 561-756, Republic of Korea b Bank for Cytokine Research, Chonbuk National University, Jeonju 561-756, Republic of Korea, Institute for Molecular Biology and Genetics, Chonbuk National University, Jeonju 561-756, Republic of Korea.); AML-193 (Establishment of Human Acute Myeloid Leukemia Cell Line Kasumi-1 with 8:21 Chromosome Translocation by Hiroya Asou, Satoshi Tashiro, Kazuko Hamamoto, Akira Otsuji, Kenkichi Kita, and Nanao Kamada); WEHI, HL-10 Kasumi-1 (Differentiation of IL-3 Dependent NFS-60 Cell Line and Adaption to growth in Macrophage Colony Stimulating Factor by Ilona Nakoinz, Mei-Ting Lee, John F Weaver and Peter Ralph from Depertment of Cell Biology and Analytical Development, Cetus Corporation, Emeryville CA 94608)etc. Out of these cell lines murine myeloblasts cell line NFS-60 is a commonly used cell line for the bioassay and potency estimation of G-CSF in various samples. The cell line requires IL-3 for its growth and continual maintenance and also responds to G-CSF and GM-CSF ATCC (Product Description Sheet of Kasumi-1). But this cell line is not available with the most important cell repository ATCC. Another cell line is derived from IL-3 dependent murine cell line NFS-60 by growing the cell line in presence of 10% L cell conditioned media as a source of M-CSF or pure M-CSF for few continual passages. The subline such derived called M-NFS-60 is available with ATCC. The cell line requires M-CSF (62ng/ml) for growth and maintenance and does not show any growth in absence of M-CSF. It gives proper response to M-CSF and IL-3 but a very weak response to G-CSF. When this cell line was used for bioassay of G-CSF, the response of cell line for G-CSF was very weak. Also, the response is not consistent over the concentration range of G-CSF, but shows a variation after few passages. Because of this the results of G-CSF bioassay were not consistent and the method could not be used confidently for potency estimation of G-CSF sample. Also, the presence of M-CSF in regular maintenance and assay media of M-NFS-60 cell line caused the hindrance in the bioassay of G-CSF and complicated the result analysis. This problem led to the development of G-CSF bioassay using new human acute myeloid cell line i.e. Kasumi-1. Kasumi-1 cell line is derived from the peripheral blood of a 7-year-old Japanese boy with Acute Myeloid Lymphoma (AML) in relapse after bone marrow transplantation. The cell line was procured from ATCC. The particular cell line was selected for G-CSF assay development because, as per ATCC's product description, the cell line shows response to IL-3, IL-6 GM-CSF and G-CSF. The cell line shows neither granulocytic nor eosinophilic maturation in vitro liquid culture by addition of DMSO, G-CSF or IL-5. The most important properties considered for selection of this cell line were its responsiveness to G-CSF, no compulsory requirement of any cytokine in the growth and maintenance media for routine maintenance, suspension cell line with minimal media requirement and its human origin. The cell line after obtaining from ATCC was expanded in the laboratory to get the sufficient number of token and working stocks. These stock cultures were then used for the various experimental procedures, which led to the standardisation of a bioassay procedure for G-CSF. In work leading up to the present invention, the Kasumi-1 cell line was used to establish the G-CSF bioassay in order to estimate the potency of various samples of G-CSF in terms of ED50 and specific activity, which are obtained during purification process of the protein. SUMMARY OF THE INVENTION: Accordingly, an aspect of present invention contemplates a method of bioassay of G-CSF by making use of a more stable, easy to maintain cell line Kasumi-1 as an indicator cell line for G-CSF bioassay. The invention covers various parameters used for standardization of G-CSF bioassay using Kasumi-1 cell line. Further, the invention discloses in vitro assay method for potency estimation of G-CSF in various samples. The growth tendency of M-NFS-60 and Kasumi-1 in presence and absence of M-CSF and G-CSF is also mentioned in the present invention. The invention also shows the preference and response of Kasumi-1 and M-NFS- 60 cell lines towards the cytokines M-CSF and G-CSF and correlates it to the standardisation of G-CSF bioassay results. Abbreviations: M-CSF:- Macrophage Colony Stimulating Factor G-CSF:- Granulocyte Colony Stimulating Factor GM-CSF:- Granulocyte Macrophage Colony Stimulating Factor IL- 3:-Interleukin3 IL-5:-Interleukin5 IL-6:- Interleukin 6 ED50:- Effective Dose 50 PBS:- Phosphate Buffered Saline RPMI-1640:- Rosewell Park Memorial Institute 1640 CO2:- Carbon dioxide DMSO:- Dimethyl sulphoxide ATCC:- American Type Collection Centre MTT:- (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide SDS PAGE:- Sodium dodesyl sulphate polyacrylamide gel electrophoresis RP-HPLC:- Reverse Phased High Performance Liquid Chromatography UV:- Ultra violet ELISA:- Enzyme Linked Immunosorbant Assay MALDI-TOF:- Matrix Assisted Laser Desorption/ionisation time-of-flight CD:-Circular Dichromism BRIEF DESCRIPTION OF FIGURES: Figure 1 is a graphical representation of growth of M-NFS-60 in presence and in absence of M-CSF and G-CSF. M-NFS-60 cells were washed three times in PBS and seeded to media containing M-CSF, G-CSF, M-CSF + G-CSF and no cytokine at a density of 1X105 cells/ml. Flasks incubated for 6 days and response of M-NFS-60 to these conditions was estimated by reduction of Alamar Blue incorporated in growth media. Figure 2 is a graphical representation of growth of Kasumi-1 in presence and in absence of M-CSF and G-CSF. Kasumi-1 cells were washed three times in PBS and seeded to media containing M-CSF, G-CSF, M-CSF+G-CSF and no cytokine at a density of 1X105 cells/ml. Flasks were incubated for 6 days and response of Kasumil to these conditions was estimated by reduction of Alamar Blue incorporated in growth media. Figure 3 is a graphical representation of response of Kasumi-l and M-NFS-60 to M-CSF and M-CSF+G-CSF. Kasumi-l and M-NFS-60 cells were washed three times in PBS and seeded to media containing M-CSF and M-CSF+G-CSF at a density of 1X105 cells/ml. Flasks were incubated for 6 days and response of cells to these conditions was estimated by reduction of Alamar Blue incorporated in growth media. Figure 4 is a graphical representation of response of Kasumi-l and M-NFS-60 to G-CSF and M-CSF+G-CSF. Kasumi-l and M-NFS-60 cells were washed three times in PBS and seeded to media containing M-CSF and M-CSF+G-CSF at a density of 1X105 cells/ml. Flasks were incubated for 6 days and response of cells to these conditions was estimated by reduction of Alamar Blue incorporated in growth media. Figure 5 is a graphical representation of effect of seeding density of Kasumi-l for G-CSF bioassay. Kasumi-l cells were harvested and seeded at three different cell densities of 1 X105, 5 X 105 and 1 X 106 cells/ml. Figure 6 is a graphical representation of effect of diluent of Kasumi-l for G-CSF bioassay. Kasumi-l cells were harvested and seeded at a density of 5 X 105 cells/ml. Protein sample diluted in complete media and Elix water is added to the culture wells covering a concentration range of 10" to 10 pg/ml. Figure 7 is a graphical representation of effect of incubation time of Kasumi-l for G-CSF bioassay. Kasumi-1 cells were harvested and seeded at a density of 5 X 105 cells/ml in three different microtiter plates. Protein sample diluted in complete media is added to the culture wells covering a concentration range of 10" to 10 pg/ml. One plate is harvested at time intervals of 24, 48 and 96 hrs. Figure 8 is a graphical representation of effect of volume of G-CSF dilution on Kasumi-1 for G-CSF bioassay. Kasumi-1 cells were harvested and seeded at a density of 5 X 105 cells/ml. Protein sample prepared from three different stock solutions of 6mg/ml, 3mg/ml and 2.33mg/ml and diluted in complete media are added to the seeded plates in three volumes of 20, 50ml and 75ml respectively covering a range of 10" to 10 pg/ml. DETAILED DESCRIPTION OF THE INVENTION: The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects there of may be more fully understood and appreciated. According to the first aspect of the invention, there is provided a method or a bioassay of G-CSF for the estimation of potency of G-CSF in terms of ED50 and specific activity using a human acute myeloid cell line i.e. Kasumi-1. In a preferred embodiment, cytokine used is of human origin expressed as a recombinant protein in prokaryotic system. Preferably, a protein of 95% or more purity is used for a bioassay and other experimental purposes in the present invention. In another aspect of the invention the bioassay method invented is then validated for its performance under various different experimental conditions. Such validated method of bioassay is then used for monitoring of the purification process of G-CSF molecule obtained as a recombinant protein expressed in prokaryotic systems. The bioassay after standardisation of various parameters under considerations (total cell count, time of incubation, volume of protein solution, diluents etc) is conducted as follows: In a preferred aspect of the invention, the effective amount of Kasumi-1 cells (subcultured 24-48 hrs. prior to assay) is added to RPMI 1640 medium with 20 % v/v FBS and plated at density of l00ml/well in each well of 96-well microtiter plate excluding the wells of all four edges (to avoid the edge effect). The effective amount of cells was standardised and kept close to 5X105 cells/ml of complete media. To these wells was then added, in duplicates, 50ml of G-CSF dilution performed in complete growth media covering a range of G-CSF concentration of 10"2 to 106 pg/ml of diluent (i.e. complete growth media). The controls were set up in duplicates containing only cells and complete growth media but no cytokine. All the wells of edges were filled with 100ml of media only to protect form edge effect. The plate was then incubated in 5% v/v C02 in air at 37°C, 85% humidity for 48 hrs. The extent of Kasumi-1 cell proliferation due to amount of G-CSF in the given well was determined by using a redox indicator MTT. The graph of absorbance vs. G-CSF concentration was plotted and ED50 and specific activity was determined using GraphPad software. The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention. EXAMPLE 1 Cell Bank Preparation for Kasumi-1 and M-NFS-60 A vial of Kasumi-1 cell line procured from ATCC is stored in vapour of liquid nitrogen till revival. The vial is rapidly thawed at 37°C in a water bath and cells transferred to 5ml of RPMI 1640 + 20 % FBS v/v (Complete media). The cells are centrifuged at 25°C, 125g for 10 minutes. The supernatant is discarded and the cells are seeded to 5ml of fresh complete media in T-25 flasks. The total cell count and % viability is determined by haemocytometer and Nucleocounter. Flasks are incubated in 5% v/v CO2 in air at 37°C, 85% humidity till high-density culture is obtained. Token, master and working stocks are prepared using 95% of above mentioned complete media and 5% cell culture grade DMSO as a cryoprotectant. Working stock vials are used for all further experimentation and bioassay purpose. Similarly a vial of M-NFS-60 cell line procured from ATCC is stored in vapour of liquid nitrogen till revival. The vial is rapidly thawed at 37°C in a water bath and cells transferred to 5ml of RPMI 1640+10 % FBS v/v supplemented with M-CSF 62ng/ml of media (Complete media). The cells are centrifuged at 25°C, 125g for 10 minutes. The supernatant is discarded and the cells are seeded to 5ml of fresh complete media in T-25 flasks. The total cell count and % viability is determined by haemocytometer and Nucleocounter. Flasks are incubated in 5% v/v C02 in air at 37°C, 85% humidity till high-density culture is obtained. Token, master and working stocks are prepared using 95% of above mentioned complete media and 5% cell culture grade DMSO as a cryoprotectant. Working stock vials are used for all further experimentation and bioassay purpose. EXAMPLE 2 Growth Curve of M-NFS-60 and Kasumi-1 Cryovials of M-NFS-60 and Kasumi-1 cell line are expanded and sufficient numbers of cells are obtained to start the growth curve. Growth curves are set at 1X105 cells/ml for both the cell lines and resazurin based redox indicator Alamar Blue (AB) is used as a growth indicator. RPMI 1640 with 10% FBS and RPMI 1640 with 20% FBS are used as basal media for M-NFS-60 and Kasumi-1 respectively. Both the cells are harvested from previous growth flasks by centrifugation at 125g, 25°C for 10 minutes in Sigma cooling centrifuge and washed thrice with PBS to remove the traces of growth media. Cells are then resuspended in 1 ml of basal media and counted using Chemometec Nucleocounter on l00ml aliquots. Using this cell suspension, 20 ml of cell suspension is prepared at a density of 1 X 105 cells/ml in respective basal media. Alamar Blue is added to this cell suspension at a concentration of 5% of the final volume. 5ml of above cell suspension is added to 4 different T-25 flasks for each cell line. For each cell line rhM-CSF and rhG-CSF are added to 2 of these flasks separately at a final concentration of 62 ng/ml. Both the above cytokines are added at final concentration of 62ng/ml to the 3rd flask and 4th flask is maintained as without any cytokine. All the flasks are incubated in CO2 incubator at 37°C, 5% CO2, 86% RH for next 6 days. During this incubation period, l00ml samples are withdrawn, in duplicates from all the flasks and plated in Tarson 96 well plates at intervals of 0, 24,48,72,96,120 hours. Absorbance of these plates is taken using Multiskan EX ELISA reader at 570 and 630nm. % Reduction of alamar blue is calculated for all the samples at all intervals (Table 1) as per the calculations given by AlamarBlue product insert from Biosource. The results obtained are analysed by GraphPad software and graphs are plotted in various manner to check the effect of both cytokines on the cell lines in combination and in single use. Above analysis gave following results and conclusions- 1. % Reduction of AB by M-NFS-60 cell line in absence of either of cytokine is negligible. M-NFS-60 shows maximum growth in presence of M-CSF and very low growth in presence of G-CSF but absence of M-CSF. Thus the cell line compulsorily requires M-CSF (minimum concentration 62ng/ml) for maintenance as well as growth even in presence of G-CSF. (Fig. 1) 2. No increase in the AB reduction is observed in presence of both cytokines as compared to M-CSF only which means that response of M-NFS-60 to G-CSF is very weak. (Fig. 3) 3. Considerable reduction of AB is observed for Kasumi-1 cell line even in absence of either of the cytokine, which means that none of the cytokine is required for regular maintenance and growth of the cell line. (Fig. 2) 4. Considerable increase in the AB reduction is observed when M-CSF is present in the growth media of M-NFS-60 cells as compared to G-CSF only or no cytokine, which means that response of M-NFS-60 to M-CSF is very strong. 5. Considerable increase in the AB reduction is observed when G-CSF is present in the growth media of Kasumi-1 cells as compared to M-CSF only or no cytokine, which means that response of Kasumi-1 to G-CSF, is very strong. (Fig. 2) 6. No increase in the AB reduction is observed in presence of both cytokines as compared to G-CSF only which means that response of Kasumil to M-CSF is very weak. (Fig. 4) 7. From all the above observations it is clear that M-NFS-60 cell line is strongly responsive to M-CSF and very weakly responsive to G-CSF whereas Kasumi-1 cell line is strongly responsive to G-CSF and not at all responsive to M-CSF. EXAMPLE 3 Standardisation of cell density for G-CSF bioassay using Kasumi-1 To obtain proper results of any bioassay it is necessary to determine the optimal cell density of the culture under given experimental conditions. Stock solution of rhG-CSF is prepared in preincubated sterile Elix water at the concentration of 3 mg/ml. Serial 10 fold dilutions are performed from this stock in sterile Elix water covering a range of 3X10"2 to 3X106 pg/ml. Kasumi-1 cells are harvested from the culture flask by centrifugation at 25°C, 125g for 10 minutes. The Supernatant is discarded and the cell pellet is resuspended in 1ml of preincubated complete media. The cell density of this suspension is determined by haemocytometer and three cell suspensions are prepared in preincubated complete media to get a final cell density of 1 X 105 cells/ml, 5 X 105 cells /ml and 1 X 106 cells/ml. Cell density of these suspensions is determined by Nucleocounter to assure that cell density adjusted is as desired. lOOul each of above cell suspensions is plated in 96 well plate. 50^1 of the each G-CSF dilution is then added to these wells in duplicate. Negative control is maintained for each cell density by adding only water without G-CSF to 2 wells. The plate is incubated in CO2 incubator at 37°C, 5% C02, 86 % RH for 72 hrs. At the end of incubation period, the assay is harvested by adding 15ml of MTT (Cell Proliferation Kit ATCC Cat. No.30-101 OK) to all the wells and the plate is again incubated in CO2 incubator for 3-4 hrs. At the end of incubation, purple coloured precipitate of MTT is dissolved by adding l00ml of detergent (Cell Proliferation Kit ATCC Cat. No.30-1010K) to all the wells. The plate is incubated at RT in dark for overnight for colour development. Next day absorbance of the plate is taken using Multiskan EX ELISA reader (Labsystems) at 570 nm. Results obtained are analysed by GraphPad software. From the results and graphs of above analysis (Fig. 5), it is observed that cell density of 5X105 cells/ml gives best results. Readings of 1X105 cells/ml are below and 1X10 cells/ml are above the standard range of MTT assay kit. Hence cell density of 5X105 cells/ml is maintained as a desired cell density for all further experiments. EXAMPLE 4 Standardization of diluent for G-CSF bioassay using Kasumi-1 Diluent used for the dilution of given protein sample can affect the results of bioassay to great extent. Along with protein when diluent is added to culture wells, it dilutes the nutrients of growth media and can affect the growth of indicator cell line. Growth rate and sensitivity of cell line to various diluents are also important parameters to consider while standardizing the diluent. Hence it is necessary to determine the appropriate diluent for bioassay. Stock solution of rhG-CSF is prepared at the concentration of 3mg/ml in preincubated sterile complete media and sterile Elix water. Serial 10 fold dilutions are performed from this stock in sterile complete media and Elix water respectively covering a range of 3X10" 2 to 3X106 pg/ml. Kasumi-1 cells are harvested from the culture flask by centrifugation at 25°C, 125g for 10 minutes. The supernatant is discarded and the cell pellet is resuspended in 1ml of preincubated complete media. The cell density of this suspension is determined by haemocytometer and cell suspension is prepared in preincubated complete media at a density of 5 X 105 cells/ml. Cell density of this suspension is determined by Nucleocounter to assure that cell density adjusted is as desired. 100)4.1 each of above cell suspension is plated in 96 well plate. 50ml of the each G-CSF dilution prepared in media and Elix is added to these wells separately in duplicates. Negative control is maintained in duplicates for each diluent by adding respective diluent without G-CSF. The plate is incubated in CO2 incubator at 37°C, 5% C02, 86 % RH for 72 hrs. at the end of incubation period, the assay is harvested by adding 15ml of MTT to all the wells and the plate is again incubated in CO2 incubator for 3-4 hrs. At the end of incubation, purple coloured precipitate of MTT is dissolved by adding l00ml of detergent to all the wells. The plate is incubated at RT in dark for overnight for colour development. Next day absorbance of the plate is taken using Multiskan EX ELISA reader (Labsystems) at 570 nm. Results obtained are analysed by GraphPad software. From the results and graphs of above analysis (Fig. 6) it is observed that optical density readings obtained with Elix dilutions are in very low range. This is because the nutrients of the media are getting diluted with Elix, which is reducing the growth of the Kasumi-1 cells. Results of assay with media dilutions are observed to be in proper range and hence complete media is maintained as a diluent for all further experiments. EXAMPLE 5 Standardisation of incubation time for G-CSF bioassay using Kasumi-1 Incubation time is the critical parameter in the standardisation of bioassay. The assay should be harvested at proper time when cells have grown to their maximum limit and cell death has not begun because of lack of nutrients and increased acidity. Stock solution of rhG-CSF is prepared in preincubated sterile complete media at the concentration of 3 |ag/ml. Serial 10 fold dilutions are performed from this stock in sterile complete media covering a range of 3X10" to 3X10 pg/ml. Kasumi-1 cells are harvested from the culture flask by centrifugation at 25°C, 125g for 10 minutes. The Supernatant is discarded and the cell pellet is resuspended in 1ml of preincubated complete media. The cell density of this suspension is determined by haemocytometer and cell suspension is prepared in preincubated complete media at a density of 5 X 105 cells/ml. Cell density of this suspension is determined by Nucleocounter to assure that cell density adjusted is as desired. lOOul each of above cell suspension is plated in three separate 96 well plates. 50ul of the each G-CSF dilution prepared in media is then added to all the plates in duplicate. Negative control is included in each plate containing only cells and media and no G-CSF. All three plates are incubated in C02 incubator at 37°C, 5% CO2, 86 % RH. One plate is removed after 24hrs., 48hrs. and 72hrs. and harvested by adding 15ml of MTT to all the wells and the plate is again incubated in C02 incubator for 3-4 hrs. At the end of incubation, purple coloured precipitate of MTT is dissolved by adding l00ml of detergent to all the wells. The plate is incubated at RT in dark for overnight for colour development. Next day absorbance of the plate is taken using Multiskan EX ELISA reader (Labsystems) at 570 nm. Results obtained are analysed by GraphPad software. From the results and graphs of above analysis (Fig 7), it is observed that for 24 hrs. incubation very less cell growth has occurred whereas for 72 hrs. incubation, excess cell growth has occurred. Also, media is not completely used for 24 hrs. incubation and longer time will be required to get the results with 72hrs. incubation. Results of 48 hrs. incubation are observed to be in proper range giving best results. Considering all these points, 48 hrs. incubation is maintained for all further experiments. EXAMPLE 6 Standardisation of volume of protein dilution for G-CSF bioassay using Kasumi-1 Volume of protein dilution going in each well of microtiter well plate is also important as it is diluting the cell concentration in each well. If the cell density is reduced too much because of volume of protein sample, it can affect the growth rate of cells and hence the results of assay. Stock solution of rhG-CSF is prepared in preincubated sterile complete media at three different concentrations of 6mg/ml, 3 g/ml and 2.33mg/ml. Serial 10 fold dilutions of above three stocks are performed in sterile complete media covering a range of 6X10"2 to 6X106 pg/ml, 3X10-2 to 3X106 pg/ml and 2.33X10-2 to 2.33X106 pg/ml. Kasumi-1 cells are harvested from the culture flask by centrifugation at 25°C, 125g for 10 minutes. The Supernatant is discarded and the cell pellet is resuspended in 1ml of preincubated complete media. The cell density of this suspension is determined by haemocytometer and cell suspension is prepared in preincubated complete media at a density of 5 X 10s cells/ml. Cell density of this suspension is determined by Nucleocounter to assure that cell density adjusted is as desired. 100ml each of above cell suspension is plated in 96 well plate. 20ml, 50ml and 75ml of above dilutions are then added for 6mg/ml, 3mg/ml and 2.33mg/ml stock solutions respectively so that actual highest concentration that is obtained in the well is lug/ml. Negative control is included for each protein volume where only media (of respective volume) without G-CSF is added to the wells. The plate is incubated in CO2 incubator at 37°C, 5% CO2, 86 % RH for 48 hrs. At the end of incubation period, the assay is harvested by adding 15ml of MTT to all the wells and t le plate is again incubated in CO2 incubator for 3-4 hrs. the end of incubation, purple coloured precipitate of MTT is dissolved by adding l00ml of detergent to all the wells. The plate is incubated at RT in dark for overnight for colour development. Next day absorbance of the plate is taken using Multiskan EX ELISA reader (Labsystems) at 570 nm. Results obtained are analysed by GraphPad software. From the results and graphs of above analysis (Fig. 8), it is observed that protein volume of 50ml gives best results hence it is maintained for all further experiments. Thus various experimental parameters of G-CSF bioassay using Kasumi-1 cell line are standardised to the optimal conditions (Table 2). 19

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