IBPL February 2008 Dhananjay B. Patankar, Ph.D. Dhananjay B. Patankar, Ph.D. Intas Biopharmaceuticals Intas Biopharmaceuticals Ahmedabad, India Ahmedabad, India Bioassay For Granulocyte Colony Bioassay For Granulocyte Colony Stimulating Factor (GCSF) Stimulating Factor (GCSF)
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IBPL February 2008
Dhananjay B. Patankar, Ph.D.Dhananjay B. Patankar, Ph.D.Intas BiopharmaceuticalsIntas Biopharmaceuticals
Ahmedabad, IndiaAhmedabad, India
Bioassay For Granulocyte Colony Bioassay For Granulocyte Colony Stimulating Factor (GCSF)Stimulating Factor (GCSF)
Cell ProliferationCell ProliferationInhibition of Cytopathic EffectInhibition of Cytopathic EffectcAMPcAMPCalciumCalciumReporterReporter--genegene
IBPL February 2008
GCSFGCSF
Naturally produced by endothelium, macrophages Naturally produced by endothelium, macrophages and other immune cells.and other immune cells.
174 amino acids, 18.8 174 amino acids, 18.8 KDaKDa rDNArDNA molecule. 4 molecule. 4 antiparallelantiparallel ∝∝ helices with two disulphide bonds; helices with two disulphide bonds; natural molecule is natural molecule is glycosylatedglycosylated..
Stimulates bone marrow to produce granulocytes Stimulates bone marrow to produce granulocytes and stem cells and to pulse them out into the blood. and stem cells and to pulse them out into the blood. Also stimulates proliferation, differentiation, Also stimulates proliferation, differentiation, maturation and survival of maturation and survival of neutrophilsneutrophils..
IBPL February 2008
GCSF in IndiaGCSF in India
2 Indian manufacturers since number of years (Dr. Reddy, Intas)
Under development at several companies
Neupogen, other imported products
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Types of Types of rDNArDNA GCSF moleculesGCSF molecules
Established 88/502 (yeast derived) as International Standard
Assigned potency of 10000 IU per ampoule
Lyophilized product
Supplied by National Institute for Biological Standards and Control (NIBSC)
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WHO standard establishment WHO standard establishment collaborative studycollaborative study
Tested three preparations (yeast, CHO and E.coli derived) of rh GCSF.
By 29 different laboratories in 11 countries
Types of assays usedBone marrow cells proliferation -colony count or 3H incorporationCell line based (NFS-60, AML-193,32D, FDC-PG)- 3H incorporation or MTTImmunoassay-ELISA or IRMA
Assay designAssay design44--dose in quadruplicatedose in quadruplicateMedia & cell controlMedia & cell control2 samples & 1 ref. per plate2 samples & 1 ref. per plate
Potency calculationPotency calculationParallel line modelParallel line modelCalculation as per Ph. Eur.Calculation as per Ph. Eur.Potency calculated from common slopePotency calculated from common slopeFiducial limits using Fieller’s theoremFiducial limits using Fieller’s theorem
IBPL February 2008
Typical of graph inTypical of graph in--house assayhouse assay
ANOVA as per Ph. Eur.ANOVA as per Ph. Eur.InIn--house developed Excel worksheethouse developed Excel worksheet
Worksheet “validated” through test Worksheet “validated” through test modules compared against commercial modules compared against commercial softwaresoftware
Significance tests for Regression, Significance tests for Regression, Linearity, ParallelismLinearity, Parallelism
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InIn--house method compared to house method compared to EP methodEP method
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Graph Comparing EP monograph and Graph Comparing EP monograph and Intas methodIntas method
0
0.5
1
1.5
2
2.5
-0.5 0 0.5 1 1.5 2 2.5 3 3.5log IU/ml
Opt
ical
Den
sity
NIBSC EPGCSF EPNIBSC IntasGCSF Intas
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Specific Activity by EP method and Specific Activity by EP method and In house methodIn house method
10.0418.7%RSD
1.221.17Average Specific
Activity (E+08)
29n
In house methodEP method
IBPL February 2008
Preliminary Comments on EP methodPreliminary Comments on EP method
Specific activity by both methods matching
EP range is too wide where first three points will be under saturation.
Incubation time of 48 hrs can be reduced to 24 hrs if dose range is high (less time, less evaporation).
1.1. Cell countingCell counting%RSD for 9 repeats of same sample < 5%%RSD for 9 repeats of same sample < 5%
2.2. Multi channel and Repeat pipettingMulti channel and Repeat pipetting%RSD for 96%RSD for 96--well pipetting < 5%well pipetting < 5%
3.3. Serial Multichannel DilutionSerial Multichannel Dilution%RSD across each dilution <10% %RSD across each dilution <10% Linear with R^2 >0.99, Slope = log 2 (within 10%)Linear with R^2 >0.99, Slope = log 2 (within 10%)
SpecificitySpecificityCompare to excipient mix / inCompare to excipient mix / in--process matrixprocess matrixDenatured productDenatured productUnrelated proteins handled in premisesUnrelated proteins handled in premises
Linearity and RangeLinearity and RangeVerify linear response across the dose range usedVerify linear response across the dose range usedR^2 > 0.95 at least 1 dose level beyond on each R^2 > 0.95 at least 1 dose level beyond on each side (obtained >0.98)side (obtained >0.98)
Accuracy Accuracy Samples of different strengths. Corrected sp. act. Samples of different strengths. Corrected sp. act. should be within 30% of standard strengthshould be within 30% of standard strength
Acceptance criteria: Calculated specific activity Acceptance criteria: Calculated specific activity not affected (within intermediate precision)not affected (within intermediate precision)
Cell seeding density varying up to 20% Cell seeding density varying up to 20% MTT incubation time: 100 MTT incubation time: 100 –– 150% of standard150% of standardIncubation temperature with SDS: 5Incubation temperature with SDS: 5ººC C variationvariationCell passage number from bank <5 to >20Cell passage number from bank <5 to >20
P1V2
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Assay TrendingAssay Trending
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Slope values of standardSlope values of standardLimits set by 2 sigma of average ( 52Limits set by 2 sigma of average ( 52-- 148 % )148 % )
Absorbance values of standardAbsorbance values of standard at highest doseat highest doseLimits set by 2 sigma of average ( 56Limits set by 2 sigma of average ( 56--144 % )144 % )
Absorbance values of standardAbsorbance values of standard at lowest doseat lowest doseLimits set by 2 sigma of average (51Limits set by 2 sigma of average (51--149 %)149 %)
IBPL February 2008
Cell control valuesCell control valuesLimits set by 2 sigma of average (44Limits set by 2 sigma of average (44--156%)156%)
Pegylated Vs Native ProteinPegylated Vs Native Protein
IBPL February 2008
AcknowledgementsAcknowledgements
Biocharacterization group of R&D and QC Biocharacterization group of R&D and QC for all of the data presentedfor all of the data presented
Shubhangi Argade for help in compiling Shubhangi Argade for help in compiling data & preparing slidesdata & preparing slides
Hatim Motiwala for the trend analysisHatim Motiwala for the trend analysis
Sanjeev Kumar (Sanjeev Kumar (ZydusZydus) & Benjamin ) & Benjamin Stephen (Reliance) for information about Stephen (Reliance) for information about their assay approachtheir assay approach