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 !!