Risk Mitigation in Cell Culture: How to Prevent Viral Contamination

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1 Platzhalter Bild Risk Mitigation in Cell Culture: How to Prevent Viral Contamination Upstream Day New Technology Platforms for Cell & Microbial Cultures May 2013 Claire Roulin, Application Specialist Purification Technologies

2 Integrated & Orthogonal Virus Clearance Technology Platform UV inactivation Low ph inactivation Adsorption Size exclusion UV-C system Very effective for small non-enveloped viruses FlexAct VI Targets large enveloped viruses Membrane chromatography Targets all viruses Virus filters Targets all viruses FlexAct VR

3 A few questions to start... Have you ever had a virus/bacteriophage contamination in your culture process? Do you consider your raw material as potential source of contamination? What is your strategy for risk mitigation?

4 Impact of Contamination Adventitious viruses/phages do infect mammalian/bacterial cell expression systems several bioreactor viral contamination reports (Genentech 96, Genzyme 09) 0.1µm filter not sufficient to retain small viruses Detection limit of test methods > 10 virus particles per liter => raw materials (media components) were most likely the source of the infection high cost of contamination : plant shutdown loss of goods security of supply at risk communication to customers and regulatory agencies estimated cost of contamination: approx $/liter * * Lutz et al., Biotechnology Progress, 2000.

5 Three Pillars of Pathogen Safety Selection Non-animal/plant origin Supply chain control Testing High LOD New viruses? Reduction Robustness Upstream prevention Pathogen safety Adapted from T.R. Kreil, Global Pathogen Safety Conference, Münich Nov 2012

6 Influence of Virus Geometry on Inactivation Viral envelope protein Viral lipid membrane Viral capsid Viral genome (DNA or RNA) -> Small non-enveloped viruses are more resistant to inactivation

7 The Virus Clearance Toolbox in Upstream

8 Gamma Irradiation Principle

9 Gamma Irradiation Facility Example Picture Courtesy of MDS Nordion

10 Gamma Irradiation Efficacy Studies done in frozen fetal bovine serum. Nims et al, Biologicals 39 (2011)

11 Gamma Irradiation SWOT Strengths Traditionally used for serum Known technology Weaknesses Not point-of-use Volume is limited Short term radicals can affect proteins Limited efficacy for small viruses Opportunities Small media batches in bags Threats Effect on cell culture performance at higher doses Scale-down

12 Heat Treatment Principle Heat causes destruction of tertiary and secondary protein structure. It is most effective with moisture (liquid systems) Heat treatment Contact time Temperature Applications Dry Heat 72 hours 80 C Used for lyophilised proteins e.g. coagulation factors Pasteurisation 10 hours 60 C-80 C Protein solutions with stabilizers e.g. albumin Autoclaving 30 min 1h 121 C-134 C Sterilization of microbial culture media (except some supplements) High Temperature Short Time msec 75 C-102 C Popular method for cell culture media application

13 Heat Treatment HTST System

14 Heat Treatment SWOT Strengths Fast High flow rates > 1000 L/h Known technology Weaknesses Not applicable to heat labile components High capital expenses Cleaning is a challenge Small viruses need very high temperature Opportunities Large media batches for commercialized products Threats Impact of product properties on inactivation (moisture, proteins, concentration...) Temperature control

15 UV Irradiation Principle Nucleic acid UV-C damage: Cyclobutan Pyrimidine Dimer Capsid Glycoprotein

16 UV Irradiation Flow distribution in the module

17 UV Irradiation Scale-up of UVivatec Scale Up UVivatec Lab performance data: Irradiation intensity: 60 W/m² Flow rates from 6 to 20 L/h UVivatec Process performance data: Irradiation intensity: 194 W/m² Flow rates from 30 to 120 L/h per module Parallelization of modules possible -> up to 480 L/h per Process system Custom-made GMP device

18 UV Irradiation Parvovirus reduction

19 UV irradiation SWOT Strengths Adapted to difficult to filter solutions Scalable Especially effective for small viruses Single-use module Weaknesses Flow rates still not high enough? Reusability of disposables Immature technology Opportunities Sensitive and difficult to filter components (containing serum or hydrolysates) Threats Footprint and costs could be an issue for high volumes Impact on culture and product

20 Nanofiltration Principle Size exclusion - 20 nm nominal pore size Targeted against all viruses Robust & efficient virus clearance step antibody parvo virus In downstream: high product transmission AND high virus retention required -> need for very selective membranes Flow rates LMH at 2 bar

21 Nanofiltration Virosart CPV Filter Family Virus filter platform based on PES - Homogeneous double layer Parvo & Retrovirus retentive membrane - Log 10 4 for PPV (20 nm range) - Log 10 6 for Retroviruses (50 nm range) - Protein transmission IgG > 95% Water based integrity test (with Sartocheck) Every membrane lot tested for phage retention Easy scale-up Typical buffer flow rate: 120 LMH at 2 bar

22 Nanofiltration Classes of cell culture media - serum-containing media - serum-free media - protein-free media - chemically-defined media - synthetic low molecular weight media => amino acids, sugars, buffer salts only sustained trend towards increasingly defined media - improved stability - improved consistency - decreased risk of infection

23 Nanofiltration Types of cell culture media Growth media (or seed media) - complete formulation for cell growth - often contain surface active agents to prevent cell damage (Pluronics, PVA) Feed or perfusion media - supplement media (nutrient addition) - added during cultivation to prolong cultivation cycle - typically do not contain surface active agents

24 Nanofiltration Filterability of Seed vs. Feed LonzaCC Media with Virosart CPV 45 Powerfeed A PowerCHO 2 LMH [L/m² bar h] L/m²

25 Nanofiltration SWOT Strengths Most robust clearance method Single-use Simultaneous removal of mycoplasma Easy to use and scale No impact on cell culture Opportunities Perfusion processes Small volume supplements (Slow) filtration of sterile media New generation of virus filters Weaknesses Flow rate limited for batch filtration Limited filterability for e.g. serum High consumable costs Threats Economical feasibility in large scale

26 Future trends Any alternatives to HTST? Requirement within media treatment: Low foot print Cost? 1-2 $/L Low costs (compared to DSP) Easy scalable solution Fast process step Volume? URS? Time? L 2-24h Possible solutions: Alternative technologies? UV Inactivation up to >1000 L/h? New generation of media virus filters

27 Many thanks to Volkmar Thom Anika Meyer Jürgen Drewelies Membrane R&D Product Manager Virus Clearance CCMedia team And YOU for your attention! If you have more questions: