DESIGN OF EXPERIMENTS FACILITATE PROCESS OPTIMIZATION … · Constant incubation (10 min) and DE concentration (60 g L-1) Filter capacity not tested I-optimal 16 -run design Product

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May 19th 2016 | Faculty Club Leuven | Session: Quality by Design and MoreRobin Kastilan, Jörg Schuphan, Rainer Fischer, Johannes Felix Buyel

DESIGN OF EXPERIMENTS FACILITATE PROCESS OPTIMIZATION IN BIOPHARMACEUTICAL PROTEIN PRODUCTION – CLARIFICATION AS A CASE STUDY


Acknowledgments

Team: Department of Integrated Production Platforms at the Fraunhofer-Institute for Molecular Biology and Applied

Ecology IMEInstitute for Molecular Biotechnology at the RWTH Aachen University

Funding: Fraunhofer Future FoundationFraunhofer Internal Programs: Attract

European Research Council: Future-Pharma grant


AGENDA

Applied research in Germany Importance of biopharmaceutical proteins

DoE approach to increase filter capacity during clarification Pichia pastoris fermentation broth

Nicotiana tabacum extract


Organizations covering the product development process

APPLIED RESEARCH IN GERMANY


Research landscape

Helmholtz-Association

Industry

Max-Planck-Society Fraunhofer-SocietyUniversities

Lead/Innovation Product/Application


Fraunhofer society and Fraunhofer IME in numbers

Largest society for applied research in Germany >22,000 employees

Budget of >2 billion €/a Structured in >60 institutes

Clustered in seven alliances E.g. life sciences

Fraunhofer IME ~650 employees

~70 mio. € budget Focus on biopharmaceuticals

IME

man

agem

ent (

Prof

. D

r. R

aine

r Fis

cher

)

IPGMP

Molecular Biology Division (260)

AachenPBT, IBT, IMT, IPP (160)

MünsterFAG (25)

GiessenBRE (30)

FrankfurtTMP (25)

HamburgSCP (20)

Applied Ecology Division (113)

SchmallenbergECC, ECT, ESA, EFA,

BAM (113)

Global Divisions (200)

Newark (DE)CMB (105)

Santiago (CL)CSB (95)IMB

(RWTH Aachen University) (65)


Covers each step in the value chain up to clinical phase II

Drug discovery- SCP- HTS assay development- Small molecule libraries

Lead development- IMT, BRE- Human, insect and MO protein repertoires- mAbs, antibiotics

Pre-clinical studies- IMT, PBT- Mice, rabbit, goat- In house animal facilities

Process development- PBT, IPP- Multi-platform expression screening- DoE screening

GMP-manufacture of APIs- IPP- Process scale-up- Bulk API release

Clinical phase trials- TMP- Linked to university hospital- Access to volunteers and patients

Fraunhofer IME along the pharma value chain

ProductLead


mAbs, enzymes, artificial proteins

IMPORTANCE OF BIOPHARMACEUTICAL PROTEINS


Use of biopharmaceutical proteins – Example: HIV/AIDS

PhRMA, in: PhRMA (Ed.), PhRMA annual membership survey, Pharmaceutical Research and Manufacturers of America, Washington, D.C., USA 2014, pp. 1-84.


R&D cost development in the pharma sector

DiMasi, J. A., Grabowski, H. G., Hansen, R. W., Innovation in the pharmaceutical industry: New estimates of R&D costs. J. Health Econ. 2016, 47, 20-33.PhRMA, in: PhRMA (Ed.), PhRMA annual membership survey, Pharmaceutical Research and Manufacturers of America, Washington, D.C., USA 2014, pp. 1-84.www.fda.gov


Strain development

Upstream production

Harvest Clarification Purification

Typical process flow

Batch

Versatile active pharmaceutical ingredients Cancer therapy

Vaccines Metabolic diseases

Yield

Purity

QualityProduct


Pichia pastoris broth and tobacco extract

DOE APPROACH TO INCREASE FILTER CAPACITY DURING CLARIFICATION


Process overview

Clarification should be scalable and cost effective avoid centrifuges Costs for filters are high due to particle burden

1

2 3

High particle burden


Broth/extract Addition of diatomaceous earth Incubation Filtration

Evaluate the potential of a new filtration unit

Potential benefits Single-use improved containment

Increased capacity Simplified process

Potential drawbacks Product loss due to binding to

diatomaceous earth

DE-filtration


Application to P. pastoris fermentation broth

Diatomaceous earth (DE) body-feed filtration pH 6.0 (manufacturer´s recommendation)

DE concentration of 30−75 g L-1

Incubation time 10 min.

Feed WCM: 118 g L-1

Turbidity: 45,000 NTU Excellent capacity, but…

…no product recovery

WCM: wet cell mass; NTU nephelometric turbidity units


Troubleshooting via DoE – Step 1

Binding to DE can be due to electrostatic interaction pH 6.0−8.0

NaCl 0−250 mM Constant incubation (10 min) and

DE concentration (60 g L-1)

Filter capacity not tested I-optimal 16-run design

Product recovery partially restored pH effect dominant

Salt effect significant, but irrelevant

Recovery p-valueModel 4.31-13

pH 7.25-14

Salt 0.011176pH2 1.27-10

Salt2 0.018383Lack of Fit 0.769588

R2 0.995981Adj. R2 0.99452

Pred. R2 0.991626



Factors pH 7.5−9.5

DE conc. 30−60 g L-1

Incubation 10 min.

I-optimal, 16-run Unexpectedly high recovery

Different broth conditions Two extreme value for capacity

Flawed data entry Unknown reason

Recovery p-valueflawed

p-value corrected

Model 1.08-5 9.84-8

pH n.a. 0.003084DE concentration 1.08-5 1.73-8

pH × DE conc. n.a. 0.006655Lack of Fit 0.5896 0.8498

R2 0.7599 0.9429Adj. R2 0.7428 0.9287

Pred. R2 0.6513 0.8601



Recovery Agent is

beneficial

Model will be refined

Capacity

Model has good quality (all R2 >0.95)

Factor unit had to be changed

Work in progress

Recovery p-valueModel 0.0025

pH 0.1389Agent 0.0038

pH × agent 0.0014Agent2 0.0274

Lack of Fit 0.5020R2 0.7217

Adj. R2 0.6290Pred. R2 0.3725


Different equipment setups possible Select the simplest and most

effective (1)

pH 8.0 Salt 500 mM

No product losses Limited access to equipment

Select only one setup instead of categoric factor

Skip pH as factor for initial screening

Application to tobacco extract

C1 & C2: control setups with conventional filters


DoE screening

Factors I-optimal, 14-run

DE concentration 25−60 g L-1

Incubation time 10−90 min

Product recovery not affected Capacity increased with DE conc.

Incubation time significant but irrelevant

Next steps

Scale-up Transfer to other products


Summary

DoE approach useful to identify significant and relevant process parameters

Parameter selection and design depend on

User experience Prior knowledge

Process constraints Personal preferences

Availability of material …

Knowing physical/technicalconstraints can help to interpret

extreme values


…questions, comments and suggestions are highly welcome…

THANK YOU VERY MUCH FOR YOUR ATTENTION.

Dr. rer. nat. Johannes Buyel, M. Sc.Head of Department Integrated Production PlatformsGroup leader Fraunhofer-Attract FAST-PEPFraunhofer Institute for Molecular Biology and Applied Ecology IME/Institute for Molecular Biotechnology of the RWTH Aachen UniversityForckenbeckstraße 6, A 30752074 Aachen

Tel.: ++49 241 6085 13162Fax: ++49 241 6085 10000E-mail: [email protected]

[email protected]

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DESIGN OF EXPERIMENTS FACILITATE PROCESS OPTIMIZATION … · Constant incubation (10 min) and DE concentration (60 g L-1) Filter capacity not tested I-optimal 16 -run design Product