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

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 (YJKIM@greencross.com) Biologics World Korea 2015

Agenda Company Introduction QbD and Cell Culture Process Development Case Studies

Green Cross History 1967 Established as Sudo Microorganism Medical Supplies Co. 1971 Production of the Nation s First Plasma Fractionation Product 1983 1988 Hepatitis B Vaccine for the 3rd Time in the World (Hepavax) World s 1st Epidemic Hemorrhagic Fever Vaccine (Hantavax) 1993 World s 2nd Varicella Vaccine (Suduvax) 2009 Construction of Ochang Plant for Bioproducts 2009 Construction of Hwasun Plant for Vaccine Products 2009 Approval of Seasonal Influenza Vaccine (GC Flu) H1N1 Influenza Vaccine (GreenFlu) 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

Sales Trends Profile Summary Sales $950M Established 1967 $807M $887M IPO(KOSPI) 1978 Employees Market Capital 1,500 $1.37B $71.6M Operating Profit 2013 $84.6M 2014 $85.4M 2015(E)

Investment in R&D Sales Composition R&D Spending $91M Rx 19.4% Others 16.5% Vx 31.6% PD 32.5% R&D Spending as a % of Sales $71M 8.8% $80M 9.0% 9.6% 2013 2014 2015(E)

Facility Overview Head Office and R&D Center Eumseong Plant Seoul Yongin - OTC Hwasun Plant Ochang Plant - Vaccine (FLU and Others) - Plasma Fractionation - Recombinant

R&D Capability Plasma Fractionation Vaccine Antibody Engineering Recombinant Protein

What is Quality by Design? Risk Assessment DOE QbD Process Understanding QRM Design Space Statistics MVDA Systematic Approach

Overall QbD Approach FDA s GMPs for the 21 st Century and the PAT initiative QbD related guidelines ICH Q8, 9, 10, 11 European Journal of Pharmaceutics and Biopharmaceutics 81 (2012) 426-437

Systematic approach to Applying QbD Pharmaceutical Quality by Design(QbD) Principles SeerPharma Pty Ltd

Upstream Process Development Advances in The Development of Biopharmaceuticals, Invitrogen

Upstream Manufacturing Process Trends in Biotechnology Vol.28 No.5

Cell Culture Process Operating Parameters Affect Process Performance & Product Quality mabs 2:5, 466-477; September/October 2010; 2010 Landes Bioscience

Identification of Operational Parameters Potential Critical Process Parameters in Cell Culture Production Temperature ph Agitation Dissolved oxygen Medium constituents Feed type and rate Howard L. Levine, Ph.D. BioProcess Technology Consultants, Inc. FIP Quality International 2007 Conference

Combination of Risk Assessment & Statistical Design of Experiments (DoE)

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 Incubator: CO 2 Incubator and Orbital Shaker

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

Design of Experiments Run Initial VCD Temp. CO 2 Duration (E5 cells/ml) ( ) (%) (hr) Remarks 1-1 -1-1 -1 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 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 Resolution IV 4 center points Augmentation Axial points

Prediction Profiler

Prediction Formulas Response Polynomial equation of the response in terms of coded factors Statistical significance R 2 R 2 Adj p-value VCD CP*+3.23A-6.09B+2.39C+4.41D-0.58AB-4.32B 2 +2.06C 2-3.08BD-4.61D 2 0.983 0.970 <0.0001 Viability CP*-10.57B+5.56C-2.2D-7.96B 2 +5.86BC-2.19BD-4.11D 2 0.961 0.942 <0.0001 A: Initial VCD, B: Temp., C: CO 2, D: Duration CP*: prediction of the response at the Center Point

Design Space (Contour Plots) *NOR: Normal Operational Range

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

Operational Parameters Analysis of the Production Bioreactor 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

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

Prediction Profiler

Regression models for the production bioreactor responses Response Polynomial equation of the response in terms of coded factors Statistical significance R 2 R 2 Adj p-value Yield CP*-416A+156B+662C-439AB-327AC-435BC 901A 2 +101ABC 0.998 0.991 0.0074 Impurities#1 CP*+1.07A+1.59B+6.04C+3.25AC+1.61BC-8.79C 2 0.937 0.843 0.0216 Impurities#2 CP*+7207A+5040B+2401C+3425AB+967AC+700BC+5116C 2 0.994 0.979 0.0026 QA#1 CP*+3.72A+5.90B+1.97C-2.00AB+1.54AC 0.77BC 8.62B 2 2.07ABC 0.999 0.999 0.0009 QA#2 CP*+11.91A+7.92B+1.61C 0.88AB+1.55AC-0.02BC-13.39A 2 2.51ABC 0.999 0.999 0.0009 QA#3 CP*+8.25A+5.99B 2.11AB 12.54A 2 0.915 0.858 0.0023 QA#4 CP*+1.1A+1.13B+0.15C 0.19AB+0.33AC+0.24BC 2.25B 2 0.64ABC 0.996 0.982 0.0141 QA#5 CP*+8.68A+4.83B+0.41C+0.44AB 1.52AC+3.36BC 9.07A 2 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

Design Space (Contour Plots) NOR NOR NOR X : ph, Y : Temp. X : Prod. shift timing, Y : ph X : Prod. shift timing, Y : Temp. *NOR: Normal Operational Range

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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