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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)
IBPL February 2008
Intas BioPharmaceuticals Ltd.Intas BioPharmaceuticals Ltd.
Part of US$ 250 Mio INTAS group
Fully integrated recombinant drug development, manufacturing and marketing company
Product range: cytokines, growth factors, hormones, pegylated compounds
Also active in contract research and manufacturing services
IBPL February 2008
Intas Intas BioPharmaceuticalsBioPharmaceuticals
Company initiation Company initiation 20002000Launched rHu GLaunched rHu G--CSFCSF 20042004Launched rHu EPO 2005Launched rHu EPO 2005Launched Launched rIFNrIFN alfa 2balfa 2b 20062006Launched PEGLaunched PEG--GCSFGCSF 20072007Received EUReceived EU--GMP cert.GMP cert. 20072007
~6 Biosimilar products in pipeline~6 Biosimilar products in pipeline
IBPL February 2008
Bioassays at IntasBioassays at Intas
ApplicationsApplicationsProduct ReleaseProduct ReleaseProduct Labeling (some Product Labeling (some cases)cases)Stability StudyStability StudyProduct ComparabilityProduct ComparabilityBiosimilarityBiosimilarity
Types of AssaysTypes of Assays
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
IBPL February 2008
Types of Types of rDNArDNA GCSF moleculesGCSF molecules
Lenograstim – CHO cell derivedSargrastim – Yeast derived Filgrastim – Bacteria derived
(methionylated)
IBPL February 2008
WHO Reference StandardWHO Reference Standard
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)
IBPL February 2008
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
IBPL February 2008
Cell Proliferation AssayCell Proliferation AssayPrinciplePrinciple
Dose dependent proliferation of cells (e.g. myeloblastic cells NFS 60 , leukemic cells HL60, WEHI-3B).
Stimulatory effect is measured by the reduction of tetrazolium reagent by dehydrogenase enzymes found in metabolically active cells.
The resulting colored formazon product is quantified spectrophotometrically.
Activity is calculated using suitable reference standard
IBPL February 2008
EP Draft Monograph ProtocolEP Draft Monograph Protocol
Addition of 20μl of 0.5% MTS
Read at 490 nm
35,000 cells (in 50 μl) + G-CSF at 11 different doses (in 50 μl) of two fold dilutions
(800 to 0.78 IU / ml) + 50 μl media
Day 3
Day 1
Incubation at 37°C+1°Cwith 6±1%CO2 for 44-48 hrs
Incubation for 4 hrs
Statistical Analysis by 4PL or parallel line models
IBPL February 2008
Methods currently in useMethods currently in use
Cell line: M-NFS-60, HL-60
Variations in: Dose range, cell no., time of incubation, detection dye
Differences in replicates & statistical design
Analysis: Parallel line, 4-PL with EC50 ratio
IBPL February 2008
SpecificationsSpecifications
Neupogen PI: 1.0E+8 (± 0.6)
EP monograph: NLT 1.0E+8
In-house release: NLT 1.0E+8
IBPL February 2008
Development of Assay at IntasDevelopment of Assay at Intas
IBPL February 2008
Optimization MatrixOptimization Matrix
1.1. GCSF concentration rangeGCSF concentration range2.2. Cell seeding densityCell seeding density3.3. Time for IncubationTime for Incubation
Other parameters studiedOther parameters studied4.4. Plate effects Plate effects 5.5. Dye staining parametersDye staining parameters
IBPL February 2008
Concentration Range (lower)…Concentration Range (lower)…
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
-0.5 0.0 0.5 1.0 1.5 2.0Lo g ( IU / m l )
IBPL February 2008
Concentration Range (higher)…Concentration Range (higher)…
0.2
0.3
0.4
0.5
0.6
-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0Log(IU / ml)
OD
at 5
70 n
m
IBPL February 2008
Seeding Cell Density…Seeding Cell Density…
13.0608.5395.40312.2088.418%RSD
0.2350.1030.0770.1920.107STDEV
1.7981.2111.4201.5751.272Average
1.7681.1031.5011.8601.320
2.0951.1441.4691.4391.219
1.5221.2811.3471.5071.154
1.8051.3161.3621.4941.396Specific
activity
40,00030,00025,00020,00015,000Cells / well
IBPL February 2008
PlatePlate--ToTo--Plate Variation…Plate Variation…
1.561.721.331.351.271.181.201.39Max / Min
1.6120.8435
1.441.2410.8121.650.7474
1.490.7930.7541.381.0311.0981.3770.733
0.9560.8410.9441.020.8491.0851.2480.8052
1.0570.7210.9731.170.8790.9291.4991.0181
Exp 8Exp 7Exp 6Exp 5Exp 4Exp 3Exp 2Exp 1Plate
Standard on each plate vs standard on 1Standard on each plate vs standard on 1stst plateplate
IBPL February 2008
Plate Pattern (sample placement)…Plate Pattern (sample placement)…
Plate bias experiments
0
0.2
0.4
0.6
0.8
1
1 2 3 4 5 6 7 8 9
Absorbance at 1.25 IU Slope Specific Activity
P6V2
Note: data for different product
IBPL February 2008
Assay in practiceAssay in practice
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
0.700
0.800
0.900
1.000
1.100
1.200
1.300
0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 0.800 0.900 1.000
log10 Dilution
Mea
sure
d O
ptic
al D
ensi
ty
NIBSC Bulk 1 Bulk 2
IBPL February 2008
Statistical AnalysisStatistical Analysis
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
IBPL February 2008
InIn--house method compared to house method compared to EP methodEP method
IBPL February 2008
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
IBPL February 2008
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).
IBPL February 2008
Qualification & ValidationQualification & Validation
IBPL February 2008
Analyst PreAnalyst Pre--QualificationQualification
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%)
IBPL February 2008
Assay ValidationAssay Validation
LinearityPrecisionSpecificityAccuracyPrecision –Repeatability & Intermediate precisionRobustness
IBPL February 2008
ValidationValidation
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
IBPL February 2008
Validation Validation -- Specificity ExperimentsSpecificity Experiments
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0.2 0.4 0.6 0.8 1 1.2 1.4
Log (IU/ml)
Abs
orba
nce
Native proteinNative protein exposed at 60 deg C for 15 hours1:1 Native protein (of the treated and untreated)
P1V3
IBPL February 2008
Validation Validation -- Accuracy ExperimentsAccuracy Experiments
P1V4
121.740.8850191.851.256711.541.248001.561.5610041.632.1112531.602.4715091.703.43200
201.255.06400
% Difference from 100%
Corrected SA
Measured SA
Strength (%)
IBPL February 2008
Validation Validation -- PrecisionPrecision
Repeatability: multiple plates same day. Repeatability: multiple plates same day. RSD 11% (Acc criteria 15%)RSD 11% (Acc criteria 15%)
Intermediate precision: 3 days, 2 analysts. Intermediate precision: 3 days, 2 analysts. RSD 22% (Acc Criteria 30%)RSD 22% (Acc Criteria 30%)
P1V5
IBPL February 2008
Validation Validation -- RobustnessRobustness
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
IBPL February 2008
Assay TrendingAssay Trending
IBPL February 2008
Slope values of standardSlope values of standardLimits set by 2 sigma of average ( 52Limits set by 2 sigma of average ( 52-- 148 % )148 % )
0.008
0.0101
0.0122
0.0143
0.0164
0.0185
0.0206
0.0227
0.0248
0.0269
0.029
0.0311
0.0332
0.0353
0.0374
0.0395
0.0416
0.0437
0.0458
0.0479
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101
105
109
113
Batch Number
Slop
e Va
lue
Upper Limit
Average
Lower Limit
IBPL February 2008
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 % )
0.25
0.270.29
0.310.33
0.350.37
0.390.41
0.430.45
0.470.49
0.51
0.530.55
0.570.59
0.610.63
0.650.67
0.690.71
0.730.75
0.77
0.790.81
0.830.85
0.870.89
0.910.93
0.950.97
0.991.01
1.03
Batch Number
Abs
orba
nce
(570
nm)
Upper Limit
Average
Lower Limit
IBPL February 2008
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0.55
0.6
0.65
0.7
0.75
0.8
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101
105
109
113
Batch Number
Abs
orba
nce
(570
nm)
Upper Limit
Average
Lower Limit
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%)
Upper Limit
Average
Lower Limit
00.020.040.060.080.1
0.120.140.160.180.2
0.220.240.260.280.3
0.320.340.360.380.4
0.42
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101
105
109
113
Batch Number
Abs
orba
nce
(570
nm)
IBPL February 2008
For 95% CI and Specific Activity valuesFor 95% CI and Specific Activity valuesFor C.I.For C.I. Limits set by EP (74Limits set by EP (74--136%)136%)
For S.A.For S.A. Upper limit set by 3 sigma of average (179%)Upper limit set by 3 sigma of average (179%)
0
0.5
1
1.5
2
2.5
3
3.5
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113 117
Batch Number
Sp.A
ct (x
10E
8 IU
/mg)
0
20
40
60
80
100
120
140
160
180
200
95%
CI
SampleSpecific activity(*10E+8IU/mg)% Lower
% Upper
Upper Limit S.A.
Lower Limit C.I.Lower Limit S.A.
Upper Limit C.I.
IBPL February 2008
Evolution of inEvolution of in--house assay at Intashouse assay at Intas
By 2 σ of trends for Cell control, absolute OD values, slope
----Assay acceptance criteria
By 3 σ of trendNot specifiedNot specifiedSp. activity upper limit
1 x 108 1 x 108 1 x 108 Sp. activity lower limitGeometric mean of 6Geometric mean of 2SingleReported result
ParallelismParallelismParallelismLinearityLinearityLinearity
RegressionRegressionRegressionStatistical Criteria
4,44,44,4Replicates & dose levels
Up gradationCommercial ProductionR&D
IBPL February 2008
Product analysisProduct analysis
IBPL February 2008
BiosimilarityBiosimilarity
0
0.5
1
1.5
2
2.5
3
3.5
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47Assay
Pote
ncy
Intas Product Commercial Product
IBPL February 2008
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
log10 Dilution
Mea
sure
d O
ptic
al D
ensi
ty
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
IBPL February 2008
Thank you !!Thank you !!
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