QbD based Development and Characterization of a Cell Culture Process for Therapeutic Proteins

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QbD based Development and

Characterization of a Cell Culture

Process for Therapeutic Proteins

2015. 06. 09

Green Cross Corp.

Green Cross Research Center

Yong Jae Kim ([email protected])

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Agenda

• _{Company Introduction}

• QbD and Cell Culture Process Development

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1967

1971

1983

1988

1993

2009

Production of the Nation’s First Plasma Fractionation Product

World’s 1st Epidemic Hemorrhagic Fever Vaccine (

Hantavax

)

World’s 2nd Varicella Vaccine (

Suduvax

)

Established as Sudo Microorganism Medical Supplies Co.

Hepatitis B Vaccine for the 3rd Time in the World

(Hepavax

)

Approval of Seasonal Influenza Vaccine (

GC Flu

)

H1N1 Influenza Vaccine (

GreenFlu

)

2009

_{Construction of Ochang Plant for Bioproducts}

2009

Construction of Hwasun Plant for Vaccine Products

2010

Approval of Recombinant Human Factor VIII (

GreenGene F

)

2011

_{Approval of}

_Shinbaro

₍

_{Herbal Drug}

₎

2012

_{Approval of}

_Hunterase

₍

_{Hunter Syndrome}

₎

2013

_{Construction of R&D Center}

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Sales Trends

Established

IPO(KOSPI)

Employees

Market Capital

1967

1978

1,500

$1.37B

2013

$807M

2014

$887M

$950M

Operating

Profit

$71.6M

$84.6M

$85.4M

2015(E)

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Investment in R&D

2013

2014

2015(E)

$71M

$80M

$91M

8.8%

9.0%

9.6%

R&D Spending

as a % of Sales

Sales Composition

R&D Spending

PD

32.5%

Vx

31.6%

Rx

19.4%

Others

16.5%

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Seoul

Ochang Plant

Eumseong Plant

Hwasun Plant

Head Office and R&D Center

Yongin

Facility Overview

- Plasma Fractionation

- Recombinant

- Vaccine (FLU and

Others)

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R&D Capability

Recombinant Protein

Vaccine

Antibody Engineering

Plasma Fractionation

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What is Quality by Design?

QRM

DOE

MVDA

Statistics

Process

Understanding

Risk Assessment

Design Space

QbD

Systematic

Approach

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Overall QbD Approach

European Journal of Pharmaceutics and Biopharmaceutics 81 (2012) 426-437

FDA’s “GMPs for the 21

st

Century” and the PAT initiative

QbD related guidelines

ICH Q8, 9, 10, 11

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Systematic approach to Applying QbD

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Upstream Process Development

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Upstream Manufacturing Process

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Cell Culture Process Operating Parameters

Affect Process Performance & Product Quality

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Identification of Operational Parameters

• Potential Critical Process Parameters in Cell Culture Production

–

Temperature

–

pH

–

Agitation

–

Dissolved oxygen

–

Medium constituents

–

Feed type and rate

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Combination of Risk Assessment &

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Process Development and Characterization

Case I : Flask Inoculum Expansion

• Project: Therapeutic Proteins I

• Cell line: CHO-DG44

• Medium: Commercial Medium

• Culture vessel: Shake Flask

• Culture volume: 100mL

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Operational Parameters Analysis of the

Flask Inoculum Expansion

Failure Mode Effect Analysis (FMEA)

- Severity of the excursion: S

- Occurrence of the excursion: O

- Detection of the excursion: D

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Design of Experiments

Run Initial VCD Temp. CO2 Duration Remarks (E5 cells/mL) (℃) (%) (hr) 1 -1 -1 -1 -1 Resolution IV 4 center points 2 -1 -1 +1 +1 3 -1 +1 -1 +1 4 -1 +1 +1 -1 5 +1 -1 -1 +1 6 +1 -1 +1 -1 7 +1 +1 -1 -1 8 +1 +1 +1 +1 9 0 0 0 0 10 0 0 0 0 11 0 0 0 0 12 0 0 0 0 13 +1 0 0 0 Augmentation Axial points 14 -1 0 0 0 15 0 +1 0 0 16 0 -1 0 0 17 0 0 +1 0 18 0 0 -1 0 19 0 0 0 +1 20 0 0 0 -1

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Prediction Formulas

Response Polynomial equation of the response in terms of coded factors

Statistical significance R2_R2 _Adj_p-Value

VCD CP*+3.23A-6.09B+2.39C+4.41D-0.58AB-4.32B2_+2.06C2_{-3.08BD-4.61D}2_{0.983 0.970 <0.0001}

Viability CP*-10.57B+5.56C-2.2D-7.96B2_{+5.86BC-2.19BD-4.11D}2_{0.961 0.942 <0.0001}

A: Initial VCD, B: Temp., C: CO2, D: Duration

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Design Space (Contour Plots)

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Process Development and Characterization

Case II : Production Bioreactor

• Project: Therapeutic Proteins II

• Cell line: CHO-DG44

• Medium: In-house medium

• Culture system: Glass type STR

• Culture volume: 2.5 L

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Failure Mode Effect Analysis (FMEA)

- Severity of the excursion: S

- Occurrence of the excursion: O

- Detection of the excursion: D

- Risk Priority Number(RPN) = S X O X D

Operational Parameters Analysis of the

Production Bioreactor

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Design of Experiments

Run Temp. pH Timing of Prod. shift (℃) (E5 cells/mL) 1 -1 -1 -1 2 -1 -1 +1 3 -1 +1 -1 4 -1 +1 +1 5 +1 -1 -1 6 +1 -1 +1 7 +1 +1 -1 8 +1 +1 +1 9 0 0 0 10 0 0 0 11 0 0 0

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Response Polynomial equation of the response in terms of coded factors Statistical significance R2_R2 _Adj_p-Value

Yield CP*-416A+156B+662C-439AB-327AC-435BC–901A2_{+101ABC 0.998 0.991 0.0074}

Impurities#1 CP*+1.07A+1.59B+6.04C+3.25AC+1.61BC-8.79C2_{0.937 0.843 0.0216}

Impurities#2 CP*+7207A+5040B+2401C+3425AB+967AC+700BC+5116C2 _{0.994 0.979 0.0026}

QA#1 CP*+3.72A+5.90B+1.97C-2.00AB+1.54AC–0.77BC–8.62B2_{–2.07ABC 0.999 0.999 0.0009}

QA#2 CP*+11.91A+7.92B+1.61C–0.88AB+1.55AC-0.02BC-13.39A2_{–2.51ABC 0.999 0.999 0.0009}

QA#3 CP*+8.25A+5.99B –2.11AB–12.54A2_{0.915 0.858 0.0023}

QA#4 CP*+1.1A+1.13B+0.15C–0.19AB+0.33AC+0.24BC–2.25B2_{–0.64ABC 0.996 0.982 0.0141}

QA#5 CP*+8.68A+4.83B+0.41C+0.44AB–1.52AC+3.36BC–9.07A2_{–1.23ABC 0.999 0.995 0.0040}

A: Production Temp., B: Production pH, C: Timing of production shift CP*: prediction of the response at the Center Point

Regression models for the production

bioreactor responses

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X : pH , Y : Temp. X : Prod. shift timing , Y : pH X : Prod. shift timing , Y : Temp.

*NOR: Normal Operational Range

Design Space (Contour Plots)

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Summary

• The concept of QbD aims at gaining a deeper knowledge of the product and

the manufacturing process starting with product development.

• Case studies describe the application of QbD principles to the flask inoculum

expansion step and the production bioreactor step of therapeutic protein,

which resulted in the definition of a design space for the cell culture process.

• Characterization studies were performed using a multi-fractional DoE

approach to determine the impact of the identified potential CPPs, both

individually and as interactions, on CQAs

• The design space was set in such a way to ensure consistent delivery of a cell

culture process for the requirements set for the therapeutic proteins.

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