Safety Testing of Chinese Hamster Ovary (CHO) Cell Banks

Safety Testing of Chinese Hamster Ovary (CHO) Cell Banks

Biosafety testing within the manufacturing process should be established in the early stages of drug development. We offer biosafety testing services throughout development and manufacture: ` ` Master Cell Bank (MCB) ` ` Working Cell Bank (WCB) ` ` End of Production Cell Bank (EPC) `Bulk ` Harvest ` ` Genetic Stability of Cell Banks ` ` Final Product and GMP Lot Release All of our biosafety testing is compliant with appropriate GMP, GLP and GCP quality standards. Further information on our range of services is available on our website: www.biooutsource.com Introduction To Safety Testing For CHO Cell Banks CHO cells are by far the most commonly used production cell lines in the biopharmaceutical industry. These cells are used for a number of reasons including the good safety record of no known incidence of patient infection from a contaminant in a product manufactured on these cells. The guidelines for the safety testing of these products were globally harmonized in 2003 to allow a world-wide single panel of tests to assist drug developers to move through regulatory submissions. Since 2007, BioOutsource has performed biosafety testing of over 200 recombinant protein products from Chinese Hamster Ovary (CHO) cell banks. The general testing strategy outlined in this document complies with international regulatory guidelines and remains current owing to our on-going dialogue with worldwide regulatory authorities. 1 The guidelines outline a safety testing programme which is dependent on three aspects; testing of the starting materials which includes the cell banks, testing of the in-process materials which are typically the bulk harvest and the ultimate stage which is validation of the virus removal ability of the downstream purification process. The full profile of safety testing is developed over a number of years as the clinical trials are engaged and the product moves to commercialisation. BioOutsource scientists have a wealth of experience and knowledge and will advise on the most appropriate and cost-effective testing strategy to meet scientific and regulatory requirements. The package of testing recommended by our scientists take into consideration the history of the cell line, raw materials used during production, product dosage, therapeutic application, clinical stage, patient population, in addition to other factors which may impact the strategy. A general overview of testing requirements is described in this document. From the International Congress on Harmonisation in 2003 each regulatory jurisdiction prepared its own safety testing guideline which all have slightly different wording. Therefore the guidance for testing cell banks is described in European Medicines Agency and the United States Food and Drug Administration Guidance documents. Our scientists have devised a package of recommended safety testing and characterisation for CHO projects to ensure the ultimate safety of the final product which will satisfy both agencies. A general overview of tests required during production of a recombinant product is detailed below. Test MCB WCB EPC Bulk Harvest Drug Substance Contents Introduction to Safety Testing for CHO Cell Banks 3 Table 1 General Testing in Compliance with ICH Q5A 3 Master and End Of Production Cell Bank Testing 3 Table 2 MCB and EPC Testing 4 Working Cell Bank (WCB) Testing 5 Table 3 WCB Testing 5 Bulk Harvest (Unprocessed Bulk) Testing 5 Table 4 Unprocessed Bulk Harvest Testing 5 Purified Bulk Harvest and Final Product Testing 5 Table 5 Purified Bulk Harvest and Final Product Testing 6 Genetic Stability Testing 6 Identity + + + - - Mycoplasma + + + + - Sterility + + + - + In vitro assays + - + + - In vivo assays + - + + - Antibody tests + - - - - Retrovirus infectivity + - + + - Reverse transcriptase + - + + - Electron microscopy + - + + - Species specific tests + - - - - Residual DNA - - - - + Table 1 General testing requirement in compliance with ICH Q5A Table 6 Genetic Stability Testing 6 Additional Testing 6 2 3

Master and End of Production Cell Bank Testing Cells used in the production of biologic products are initially banked as a Master Cell Bank (MCB), which consists of a bank of fully characterized cells processed together to ensure uniformity and stability. Characterisation and testing of the MCB for bacterial and viral contaminants should be performed as described in International Conference on Harmonisation (ICH) Q5A prior to initiation of a Phase I clinical trial 1. In addition to testing for the presence of microbiological contaminants, extensive screening for the presence of both exogenous and endogenous viral contamination should be performed. Description Sterility test and qualification of sterility test by direct inoculation (Eu Pharm current edition 2.6.1. sterility, US Pharmacopoeia <71>) (Technical Note T1) Test for mycoplasma including mycoplasmastasis assay (EP 2.6.7. and USP <63> ) (Agar, broth and indicator cells) (Technical Note T1) Appearance SP-GSM-6981 4 Test 1% of cell bank (minimum 2 vials) plus 1.5x number tested for qualification SP-GSM-7002 6 1 vial, sufficient for test and stasis Morphology by light microscopy TSOP179 1 Live cells required Identity Random amplified polymorphic DNA barcoding assay for cell line identity (Technical Note T2) Adventitious Agents 28 day in vitro assay using 3 detector cell lines (Technical Note T3) TSOP182 6 In vivo adventitious agent assay: adult and suckling mice and embryonated eggs as outlined in ICH Q5A (Technical Note T4) Retroviruses (Technical Note T5) Detection of murine xenotropic retroviruses by Mus dunni co-cultivation followed by MiCl S+L- assay TSOP382 4 1 vial containing minimum 1e6 cells Minimum 1e7 cells (plus 20 ml supernatant per detector cell line = 60 ml supernatant) SP-GSM-7006 8 20 ml at 1e7 cells /ml TSOP131 6 Live cells required Fluorescent Product Enhanced Reverse Transcriptase TSOP023 6 2 ml cell free supernatant Examination of 200 cell profiles by Transmission Electron Microscopy (TEM) TSOP190 8 Live cells required Species Specific Viruses MCB testing is performed once and the testing package is repeated on the End of Production Cell Bank (EPC) in order to determine that the cells remain unchanged from the original cells, and to detect any low level contamination which may have been present but undetected in the MCB. Testing the EPC also allows detection of latent infections as well as any contaminants which may have introduced during the production process. Testing of EPC used in production should be performed prior to submitting a Product Licence Application (PLA). A summary of the testing required for for MCB and EoPCB is described in Table 2. Please note the sample volumes listed in the tables below are a guide and may vary for specific projects. Please speak to our scientists, who can advise on the required sample volumes for each project. Hamster Antibody Production Test (HAP), 5 viruses (Technical Note T6) On request 12 1e7 cells/ml in 2 x 5 ml spent supernatant Mouse Antibody Production Test (MAP), 16 viruses (Technical Note T7) On request 12 1e7 cells/ml in 2 x 5 ml spent supernatant Bovine and Porcine virus screen by real-time PCR (Technical Note T8) TSOP042 4 1 vial containing minimum 5e6 cells Detection of MVM by real-time PCR (Technical Note T9) TSOP042 2 1 vial containing minimum 1e6 cells Detection of Vesivirus by real-time PCR (Technical Note T9) TSOP042 2 1 vial containing minimum 1e6 cells Working Cell Bank (WCB) Testing The WCB is derived from a vial of the MCB, and may be used as a starting cell substrate for drug production. Less extensive testing is required for the WCB, as the MCB has already been fully characterized, hence a package of limited testing should be performed to confirm identity, ensure that the bank is sterile and demonstrate absence of mycoplasma. In some cases full testing may be performed on the WCB as an alternative to the MCB. An outline of testing required on the WCB is shown in Table 3. Description Sterility test by direct inoculation (Eu Pharm current edition 2.6.1 sterility, US Pharmacopeia <71> (Technical Note T1) SP-GSM-6958 4 Test 1% of cell bank (minimum 2 vials) Test for mycoplasma (Eu Pharm, current edition 2.6.7. mycoplasmas and USP - NF Mycoplasma test, USP <63> current edition) (Technical Note T1) SP-GSM-7002 6 Test 1 vial + 23 ml supernatant Identity Random amplified polymorphic DNA barcoding assay for cell line identity (Technical Note T2) Table 3 WCB testing Bulk Harvest (Unprocessed Bulk) Testing TSOP382 2 1 vial containing minimum 1e6 cells A representative of the unprocessed bulk, which consists of pooled harvests of cells and culture media from the fermenter or production reactor, should be analysed for the presence of virus. Data from at least 3 lots of bulk harvest is required for submission to the regulatory authorities. Certain specifics relating to the process, materials and intended product may need to be taken into consideration when determining the bulk harvest testing schedule, however, in general, the following testing regime would be recommended. Description Microbiological examination of non-sterile products: microbial enumeration test (Eu Pharm current edition 2.6.12) (Bioburden) Test for Mycoplasma including mycoplasmastasis (Eu Pharm, Current edition 2.6.7. mycoplasmas and USP - NF Mycoplasma test, USP <63> current edition ) (Technical Note T1) Adventitious Agents SP-GSM-6984 4 10 ml SP-GSM-7002 6 40 ml 14 day in vitro assay using 3 detector cell lines (EP and FDA compliant) TSOP138 3 In vivo adventitious agent assay: adult and suckling mice and embryonated eggs as outlined in ICH Q5A Retroviruses Up to maximum of 20 ml per detector cell line SP-GSM-7006 8 20 ml at 1e7 cells /ml Virus detection and quantitation by thin section TEM TSOP200 8 20 ml Species Specific Viruses Detection of MVM by real-time PCR TSOP042 2 1 ml Table 4 Unprocessed Bulk Harvest Testing Genetic Analysis Confirmation of sequence of transgene (coding region) by mrna sequencing Design and qualification of a real-time PCR for copy number determination of the transgene TSOP134 6 1 vial containing minimum 1e6 cells TSOP044 4 - Purified Bulk Drug Substance and Final Product Testing The purified bulk drug substance is a sample taken at the end of the production process after downstream purification activities have been performed. These samples are not usually analysed for the presence of virus if the host cell line has been tested and found to be negative, and, the unprocessed bulk testing has been shown to be negative. Determination of insert gene copy number by quantitative real time PCR TSOP044 2 1 vial containing minimum 1e6 cells Restriction enzyme analysis by Southern Blot TSOP174 6 1 vial containing minimum 5e6 cells Table 2 MCB and EPC testing Depending on the manufacturing process these may be individual samples or pooled samples from different unprocessed bulk harvests. While certain specifics in relation to the process, materials, and intended product may need to be taken into further consideration with the testing schedule, in general, the following testing regime is recommended. 4 5

Description Technical Notes T1. Qualification of the cell bank sample in the mycoplasma and sterility tests is required once on the MCB and first batch of Bulk Harvest and does not need to be repeated on future samples unless there is a change in the materials or manufacturing method. For the sterility test, if antibiotics have been used in cell culture, an alternative membrane filtration sterility test can be performed. Genetic Stability and Identity Testing ICH Guidelines indicate that the stability and integrity of the insertion sequence needs to be characterized to ensure stable production of the correct protein. There are a number of steps which are required to be undertaken to ensure the cell line and production system is acceptable. The minimal characterisation required is to assess the MCB and the EPC. Testing can also be carried out at other times but is particularly useful where there are changes to the fermentation process. BioOutsource offers the following genetic stability assays and can advise on the most appropriate approach for your product. Description Confirmation of sequence of transgene (coding region) by mrna sequencing TSOP134 4 1 vial containing minimum 1e6 cells Random amplified polymorphic DNA (RAPD) analysis TSOP047 2 1 vial containing minimum 1e6 cells DNA barcoding assay for cell identity (Technical Note T2) TSOP382 4 1 vial containing minimum 1e6 cells Determination of insert gene copy number by quantitative real time PCR TSOP044 2 1 vial containing minimum 1e6 cells Restriction enzyme analysis by Southern Blot TSOP174 6 1 vial containing minimum 5e6 cells Table 6 Genetic Stability Testing Additional Testing Qualification and Validation of s `` Microbiological examination of non-sterile products: microbial enumeration test EU Pharm current edition 2.6.12 Bioburden Sterility test and qualification of sterility test by direct inoculation EU Pharm current edition 2.6.1 Sterility and US Pharmacopeia <71> Endotoxin BioOutsource has extensive experience in the qualification and validation on all assays used to support safety testing of a licensed product. BioOutsource Certification SP-GSM-6984 3 SP-GSM-6981 4 Detection of Endotoxin using chromogenic LAL Kit TSOP201 1 General Safety/Abnormal Toxicity Abnormal Toxicity test EU Pharm current edition SP-GSM-6964 1 Residual Impurities (Bulk Drug Substance only) Detection of residual CHO DNA by real time PCR TSOP048 1 1 ml Determination of CHO Host Cell Proteins (HCP) by ELISA assay (Technical Note T10) Table 5 Purified Bulk Harvest and Final Product Testing TSOP104 2 1 ml 10mL Bulk drug substance Final product, as per parenteral guidelines, dependant on batch size 1 ml of Drug Substance 1 ml of Final Product No more than 7 human does of final product BioOutsource has both GMP and GLP certification; last inspection dates being 27th and 28th July and 16th September 2015 respectively. Following several successful MHRA inspections, we were rated with a low risk rating and have continued GMP and GLP compliance and certification; valid until 2018. We were also successfully audited by the U.S. Food and Drug Administration on 25th and 26th January 2016. The inspection confirmed that our Glasgow site, is compliant with the principles and guidelines of GMP. Furthermore, not a single 483 observation for non-compliance was issued. T2. Isoenzyme analysis of cell lines is no longer available, due to supply issues. DNA barcoding assay is available as an alternative to isoenzyme analysis of cell lines. T3. For the MCB, the more sensitive 28 day in vitro assay is recommended. T4. For in vivo tests, various options for inoculation include both the yolk sac and allantoic membrane in eggs, suckling mice, intracranial and intramuscular route in adult mice, as well as the option to include guinea pigs. Animal Volume Required Concentration Yolk sac 6 ml 1 x 10 7 cells/ml Allantoic 6 ml 1 x 10 7 cells/ml Suckling Mice 2 ml 1 x 10 7 cells/ml Adult Mice Intracranial route 1 ml 1 x 10 7 cells/ml Intramuscular route 2 ml 1 x 10 7 cells/ml Guinea Pigs 26 ml 4 x 10 5 cells/ml T5. CHO cell lines contain an endogenous, defective, noninfectious retrovirus. When testing for retrovirus, a number of approaches can be taken and it is not necessary to perform all of the procedures. a. The electron microscope is a powerful tool used to visualize retroviral particles (for example budding C-type, R-type and intracisternal A-type particles) b. The PERT assay is used to detect enzymatic activity (which could provide a positive result as hamster cells contain an endogenous, defective, non-infectious retrovirus). c. Cell based infectivity assays for detection will determine if virus detected is infectious. In the event that infectious virus is detected, cells can be co-cultivated with a human cell line to determine if infection of human cells is possible. The PERT test may produce a positive result, and should only be performed following the infectivity and TEM tests. T6. The HAP test will detect the following viruses; Lymphocytic Choriomeningitis virus (LCMV), Pneumonia Virus of Mice (PVM), Reovirus Type 3, Sendai Virus and Simian virus type 5 (SV5). T7. The MAP test will detect the following viruses; Ectromelia Virus; Lymphocytic Choriomeningitis virus, Hantaan Virus, Pneumonia Virus of Mice (PVM), Reovirus Type 3, Mouse Theilers Encephalomyelitis Virus, Lactic Dehydrogenase Virus, Sendai Virus, Minute Virus of Mice (MVM), Mouse Adenovirus, Mouse Cytomegalovirus, Mouse Hepatitis Virus, Mouse Rotavirus, Polyoma Virus, Thymic Virus and K virus. T8. If at any point during production, cells have come into contact with bovine serum or porcine trypsin full testing for a range of bovine and porcine viruses should be performed. Bovine Polyomavirus, in particular, has been requested by the European regulators, as it has been found as a frequent contaminant of foetal bovine serum. T9. It is recommended that testing for the presence of Minute Virus of Mice (MVM) should be performed on production harvests and the EPC. This virus has previously been detected as a contaminant in CHO fermentations, and testing for its presence can be performed by inclusion of an additional cell line susceptible to MVM infection in the in vitro assay, for example 324K cells, or alternatively by performing a real time PCR assay. Similarly vesivirus has also been detected in biological materials used in product manufacture. T10. A validated HCP assay is required for the final product testing of licenced biologics. The assay listed here is a screening assay for early phase clinical material. Further discussion will be required if BioOutsource is to develop a specific HCP assay for late phase material. Please contact us for further information regarding this. s 1. ICH Q5A (R1) Viral Safety Evaluation of Biotechnology products derived from cell lines of human or animal origin. 6 7

Sartorius Stedim BioOutsource Global Facilities For further contacts, visit www.biooutsource.com. Glasgow, UK Cambridge, USA Pangyo, South Korea Glasgow 1 Technology Terrace, Todd Campus, West of Scotland Science Park, Glasgow, G20 0XA, United Kingdom Phone +44.141.946.4222 Fax +44.141.946.4552 Cambridge 245 First St, Cambridge, Boston, MA 02142, United States Phone +1.857.209.3414 Follow Us www.biooutsource.com/biosafety/ Pangyo 8th Floor, Solid Space B/D, PanGyoYeok-Ro 220, BunDang-Gu 463-400 SeongNam-Si, GyeongGi-Do, South Korea Phone +82.31.622.5700 Fax +82.31.622.5799 Specifications subject to change without notice. Printed in the EU on paper bleached without chlorine. Publication No.: S--1540-e161002 Order No.: 85037-554-21 Ver. 11 2017