Applying QbD to Biotech Process Validation

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TECHLOGIC

BioTechLogic, Inc.BIO TECH

LOGICBIO®

TECHIO LOGICLogic, Inc.

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g QbD to Biotech Process n :udy in applying QbD to

– Process Design

IVT Validation Week 28-30 March 201128 30 March 2011

Kurtis Epp, John KandlBioTechLogic, Inc.

Agenda

• Process Backgrou• Risk Assessment• DOE• Parameter Evaluat• Conclusions

TECHLOGICBIO TECHLOGICBIO

®BioTechLogic, Inc.

und

tion

Slide 2Company Confidential

Process Back

The scope of this case stupdata obtained from small-Chromatography runs perlimits in experiments planlimits in experiments planDesign of Experiments (D



kground

udy is to provide and evaluate y p-scale Ion Exchange rformed within defined parameter ned and executed according toned and executed according to

DOE).


Process Back



kground


Risk AssessmOperating Parameter Normal Operating

UV at Start of AEX Fraction Collection > 0.50 AUUV at End of AEX Fraction Collection < 20% of maximum p

Elution Gradient 8% per CVElution Gradient (20 – 100% B oveEquilibration volume ≥ 5 CVLoad flow rate 100 – 120 L/h (TargeWash flow rate 100 – 120 L/h (TargeElution flow rate 100 – 120 L/h (TargePre-Equilibration flow rate 100 – 120 L/h (Targe

F1 F3: 0 2Fraction Volume

F1 – F3: 0.2 F4+: 0.1 CV

Wash volume ≥2 CVFraction Mixing Speed 65 rpmFraction Mixing Time 20 - 30 min

Pool Mixing Time 10 – 15 minPool Mixing Time

Pool Mixing Speed 100 rpm

Pre-Equilibration volume ≥ 4 CVWFI Rinse volume ≥ 3 CVWFI Rinse flow rate 100 – 120 L/h (TargeEquilibration flow rate 100 – 120 L/h (Targeq ( g

In-Process Control LimitsColumn Load 15 – 25 g / L rColumn Bed Height 30 ± 3 cmColumn Backpressure during Equilibration, Load, Wash, and Elution < 3 bar

Effl t H t d f E ilib ti ± 0.3 pH Units of EqEffluent pH at end of Equilibration p qBuffer pH

Effluent Cond. at end of Equilibration ± 1 mS/cm Units of EBuffer Conduc

Effluent UV at the end of Equilibration ZeroFraction Pooling Criteria (RP-HPLC) ≥ 95% Main P



mentg Range Potential Effect(s) of Failure SEV OCC DET RPN

U Product loss, quality 9 4 2 72peak height Product loss, quality 9 4 2 72V Inconsistent quality 8 2 4 64r 10 CV) Inconsistent quality 8 2 4 64

Product loss, quality 9 2 1 18et: 110 L/h) Inconsistent quality 4 1 3 12et: 110 L/h) Inconsistent quality 4 1 3 12et: 110 L/h) Inconsistent quality 4 1 3 12et: 110 L/h) Longer equilibration 4 1 3 12CVCVV Product loss, quality 9 1 1 9

Inconsistent quality 5 1 1 5Inconsistent quality 4 1 1 4

n Inconsistent quality 4 1 1 4

n Non-homogeneity, inconsistent sampling/yield 4 1 1 4sampling/yieldNon-homogeneity, inconsistent sampling/yield 4 1 1 4

Longer equilibration 3 1 1 3Longer equilibration 3 1 1 3

et: 110 L/h) Longer equilibration 1 1 1 3et: 110 L/h) Product loss, quality 3 1 1 3) , q y

resinm

quilibration q

Equilibration ctivity

Peak


Risk Assessm

The parameters selectedpthe UV at start of fractioncollection, gradient slopeD d t i blDependent variables anaand purity by RP-HPLC.

TestinInput parameter

Lower

UV at the beginning of fraction collection (AU) 0.40

UV at the end of pooling (% from peak max) 10

Gradient slope (% per CV) 4.0

Resin Load, (g/L resin) 15



ment

d for evaluation in this study were yn collection, UV at end of fraction e, and resin load.

l d t i i ldalyzed were protein recovery yield

ng limitsgOutput parameters

Upper

0.60

Purity by RP-HPLC (≥ 95%)Step yield (≥ 75%)

30

12.0

25


DOE

Pattern Exp. No.:

UV at the staof fractioncollectionp collection

(AU)−−−− 1 0.40000 2 0.50000 3 0 50000 3 0.5−++− 4 0.40000 5 0.5++−− 6 0.6

7 0 6+−−+ 7 0.6++++ 8 0.6+−+− 9 0.6−−++ 10 0.40000 11 0.5−+−+ 12 0.4

Note: It is important to randomizeindependence of your observationmistake by the operator.



art n UV at the end

of pooling Gradient

slope Resin Load (g/L)(% peak max) (% per CV) (g/L)

10 4 1520 8 2020 8 2020 8 2030 12 1520 8 2030 4 1510 4 2510 4 2530 12 2510 12 1510 12 2520 8 2030 4 25

e the run order to assure the ns and reduce the chances of a


Experimental

• A summary of the varIon Exchange ChromOutputs step yield anOutputs, step yield anlisted in the following

• Runs that did not meeare shaded to indicate



Results

riable parameters for each of the atography runs as well as the

nd eluate purity by RP HPLC arend eluate purity by RP-HPLC are table.

et the defined acceptance criteria pe run failure


Experimental

Run Resin Load GradientRun Number

Resin Load (g/L)

Gradient Slope (%)

1 15 4

2 20 8

3 20 8

4 15 12

5 20 85 20 8

6 15 4

7 25 4

8 25 128 25 12

9 15 12

10 25 12

11 20 811 20 8

12 25 4



Results

UV @ Start % Peak Step Yield (%)

RP-HPLC Purity (%)UV @ Start

Collection Height End Collection

(%) Purity (%) Acceptance Criteria≥ 45% ≥ 95%

0.4 10 53 95

0.5 20 56 97

0.5 20 50 97

0.4 30 59 96

0 5 20 51 970.5 20 51 97

0.6 30 53 97

0.6 10 52 96

0 6 30 40 970.6 30 40 97

0.6 10 50 98

0.4 10 45 97

0.5 20 51 970.5 20 51 97

0.4 30 60 97


Statistical dat

• For evaluation of statistion protein quality and qsoftware package was uSquares model for EffecSquares model for Effec

• The model was run sepof yield and purity by RP

• The fractional factorial minclusion of all single facinteractions as model efinteractions as model efresource and time constwas not possible.



ta analysis

ically significant effects of factors uantity, the JMP statistical used applying the Standard Least ct Leveragect Leverage. arately for two output parameters P-HPLC.model was initially run with the ctor and some two-factor ffects (Resolution IV) Due toffects (Resolution IV). Due to traints, a higher resolution study


Statistical Model PStep Yieldp

• The final model used for effects and two factor int

• There were two significai ifi t i t ti Tsignificant interaction. T

graphically represented iplots and interaction conp



Parameter Estimates for

assessment all four primary p yteractions listed above. nt main effects and one h l ihese conclusions were in the main effects leverage tour plots.p


Main Effect Lever

a. UV at Start Collection

c. Gradient Slope



rage Plots for Step Yield

b. UV at End Collection

d. Resin Load


Statistical Model PPurity by RP-HPLy y

• The final model used forprimary effects (excludintwo-factor interactionstwo factor interactions.



Parameter Estimates for C

r assessment included all four ng resin load) as well as three


Two-Factor InteraPurity by RP-HPLy y

Step Yield (%)

Gradient Slope (%/CV)

0

UV @ End (%)

a. UV at End Collection x Gradient Slope



action Contour Plots for C

Step Yield (%)

Resin Load (g/L resin)

0

UV @ Start (AU)

b. UV at Start Collection x Gradient Slope


Confirmation • There is one area of th

represents process failp pyield, run 8. In order toacceptance criterion isoperated within its defioperated within its defimodified and two additaugment the initial des

• We chose to tighten thas it was determined tosignificant primary effesignificant primary effeanalysis.

• None of the experimenpfailed the acceptance cconfirms the predicted study



study.

of Resultshe tested design space that lure space with regard to step p g po ensure that the step yield always met when the process is ned PARs the design space wasned PARs, the design space was tional runs were performed to sign.e gradient slope upper limit to 11% o be easy to control and the most ect for step yield in our initialect for step yield in our initial

ntal runs for the tightened limits gcriterion for step yield. This design space from the initial


Design SpaceSpaceSpace



e vs Defined Control

Based on the models generated for h f th d fi deach of the defined process

outputs, a three-dimensional plot was generated to graphically show the process control space with

d h i llregard to the experimentally defined design space


Conclusions

• The results of this DOdesign space from the

• Based on our findings h f th thwe chose for the three

appropriate with the ex• As a result we tighteneAs a result we tightene• The IPC limits for resin

set appropriately.



E study confirm the predicted y pe initial study.

we determined that the IPC limits i t te input parameters were

xception of Gradient Slope. ed our control limitsed our control limits.n load were also determined to be


Final Paramet

Normal OpeOperating Parameter

Normal Ope(NO

UV at the beginning of pooling, % from peak max Target: >p g p

UV at the end of pooling, % from peak max Targe

Gradient slope, % per CV TargeGradient slope, % per CV Targe

Performance Parameter In

Resin Load, g/L

* Note: The claimed experimentally confirmed range is slightly tighter than thsupport the higher end of the range.



ter Limits

rating Range Experimentally Confirmedrating Range OR)

Experimentally Confirmed Ranges

> 0.5 AU 0.4 – 0.6 AU

t: 20% 10 – 30%

et: 8% 4 – 11%*et: 8% 4 11%n-Process Acceptance Criterion

15 – 25 g/L

he design space in that it takes the worst-case data point to


Thank You

BioTechLogic Inc serBioTechLogic Inc. serbiopharmaceutical indwith companies to mwith companies to mresource and comme



rves therves the dustry by collaborating eet their developmenteet their development,

ercialization needs.

www.biotechlogic.comwww.processvalidation.com


Applying QbD to Biotech Process Validation

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