Host Cell Protein Conference May 14-16, 2018 Dubrovnik, Croatia The Challenges of Developing HCPs Methods for Vaccines A Risk Based Approach to HCP Monitoring 15 May 2018 Dr. Lee Smith © 2018 GreyRigge Assoc. Ltd. All rights reserved Types of Vaccines & Their Cell Substrate • Inactivated whole virus • Mammalian, Insect • Live attenuated virus • Vero, HEK293, sf9 • Live attenuated viral vector • DNA vaccine • Bacterial culture • E. coli • Subunit or VLP • Subunit or VLP • E. coli, yeast 2 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved Live Virus Vaccine Processes • Similar in principle to most biologics, an upstream and a downstream • However, while many processes will have a viral removal step, this is different for a live virus vaccine process WCB Cell Expansion HARVEST ENDONUCLEASE WVS Buf Soln Flush x Ret 10 FILTRATION UF/DF STERILE FILTRATION CHROMATOGRAPHY DRUG SUBSTANCE 3 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved Live Virus Vaccine Processes & Host Cell Protein (hcp) • There are a number of stages throughout the process where host cell proteins are removed. • It is likely that hcp co-purify with the virus product. hcp WCB Cell Expansion HARVEST ENDONUCLEASE WVS Buf Soln Flush x Ret 10 FILTRATION UF/DF STERILE FILTRATION CHROMATOGRAPHY DRUG SUBSTANCE 4 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved Enveloped Live Virus Expression Live virus expression can be: 1) lytic 2) secreted 3) intracellular The virus, if it buds off the cell membrane, it will incorporate host cell proteins into the envelope of the virus product The product will contain free hcp as well as hcp incorporated into the product Therefore we can’t remove all hcp without removing virus 5 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved Enveloped Live Virus Expression cont. The product in the harvest may be surrounded by hcp with also some of the hcp incorporated into the virus envelope. Even when the process removes much of the hcp, some of it will remain in the product. 6 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved Host Cell Protein Levels • How much hcp is too much for a vaccine? • There is no specified amount and it is case by case • A risk assessment needs to be made and justified to regulators for the product taking into account: ➢ Vaccine dosing regime such as: • Dose level • number of doses required ➢ For a live infectious virus even a billion particles may represent a small amount of virus protein relative to hcp protein in the product. ➢ A single particle of vaccinia-a virus that forms the basis How much protein? for the smallpox vaccine-weighs only 9 femtograms, or 10 Femtograms per quadrillionths of a gram. particle 1 fg = 0.000000001ug 7 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved Typical hcp amounts in a Live Virus Vaccine? It depends on how long the vaccines have been around Measles vaccines first used in vaccination programs in 1960’s Vaccine still being used now Frequently these vaccines might almost be described as formulated harvest What would the hcp content be? Very High? None-the-less, these vaccines are very safe. 8 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved Typical hcp amounts in a LiveWCB Virus Vaccine?Cell Expansion HARVEST ENDONUCLEASE WVS Buf Modern vaccines purified Soln Flush through complex DSP x Ret 10 Compared to older vaccines FILTRATION UF/DF STERILE FILTRATION CHROMATOGRAPHY DRUG SUBSTANCE 1-50g/dose! 700g/dose! Wouldn’t recommend this! 9 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved How was 700ug/dose of hcp acceptable? • Primarily there is no benchmark to say it isn’t safe • The hcp vaccine from prior dose ranging studies (the high dose) demonstrated hcp levels caused no adverse events in toxicology and phase I clinical studies • Argued hcp content was linked to vaccine dose due to the hcp being integral within the membrane • All particles may not be infectious; non- infectious particles still contain hcp • Commitment to improve process to remove hcp in lysate as process evolves Plus method didn’t detect all hcp! 10 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved Should I use an OTS Host Cell Protein Immunoassay? It depends on the ability of the ELISA kit to detect all of the hcp in the product If the coverage is good then it becomes an option to stay with the OTS ELISA kit 100% Coverage of hcp? 70% Coverage of hcp? 11 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved Could I use OTS Host Cell Protein Immunoassay? It depends on the ability of the ELISA kit to detect all of the hcp in the product If the coverage is good then it becomes an option to stay with the OTS ELISA kit 100% Coverage of hcp? 50% Coverage of hcp? What coverage is acceptable? 12 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved Determining your Coverage If using immunoassays, a Western/fluorescent 2D blot using the OTS ELISA kit can be used to determine the amount of coverage If the coverage is considered inadequate (we’ve used < 70%) then new serum/antibodies should be prepared Preparation of serum/antibodies for a live viral vaccine product is a significant technical challenge 13 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved Preparation of HCP Antigen for Immununisations Null Cell Line Approach – to obtain hcp profile similar to the product Expand the cells, do not infect and process supernatant through the DSP. However, some virus will secrete into Sn so little HCP released. For lytic or non-lytic virus this is a lesser concern. HCP from harvest can then be purified However, the hcp may purify differently to when virus is present 14 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved Preparation of HCP Antigen for Immununisations Cont. Cell Lysis & Processing We can expand cells and simply lyse them through Freeze/Thaw or sonication releasing the hcp. These are then processed them through the DSP Cell Expansion Lysis HARVEST ENDONUCLEASE Buf Soln Flush x Ret 10 FILTRATION UF/DF STERILE FILTRATION CHROMATOGRAPHY HCP 15 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved Preparation of HCP Antigen for Immununisations Cont. Cell Lysis We can expand cells and simply lyse them and immunize with the crude hcp. Cell Expansion Lysis Crude HCP Depth filtration can be used to remove large particulates from the crude lysate Buf Soln Flush UF/DF with a suitable membrane cutoff can be used to split the x Ret 10 lysate into high and low molecular weight hcp UF/DF 16 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved Preparation of HCP Antigen for Immununisations Cont. Low & High wt. lysate Lyse Free HCP Product containing hcp Can characterize on 2D gel to compare lysate with product, including blotting with Ab 17 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved Immunisations • The Low & High M. Wt lysate can be immunized separately or in combination • This serum will be immunized in line with an agreed protocol 18 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved Immunisations cont. Cascade immunization can be considered if The animals are bled to obtain and Ag components immunodominant test the serum for binding to the hcp & the FT re-immunised 19 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved Characterising the Serum The Serum can then be assessed to understand the coverage of product hcp 50%70%+ ✓ 20 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved Development of the ELISA & Long-Term Thinking • Once the serum is suitable then the ELISA must be developed • Careful thought must be given to who and how this is developed. Have they suitable quality systems for long term supply? • The method must be developed with a view to long term supply (20 yrs +) • Key reagents such as labels, secondary antibodies • Reference preparation (hcp Ag) • Materials (plates) • Also, will a large amount of polyclonal serum be made and stored long term? • Will new serum be raised on a regular basis and characterised? 21 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved HCP ELISA Method Qualification & Validation If the method is developed externally, the ELISA would then need to be qualified in line with ICH Q2(R1) guidance. Hcp is a impurity that is measured quantitatively. 22 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved HCP ELISA Method Qualification & Validation cont. There will be a hcp standard curve Control samples (H, M, L) The Qualification should focus on putting samples through the standard curve. The Std. curve will be sigmoidal but your samples should be reading off linearly. During Qualification you will determine - Linearity - Precision - Accuracy (or relative bias) - Range - Limit of Quantitation HCPHCP Protein Protein Conc. Conc (ug/mL (ug/mL)) - Specificity 23 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved Method Transfer Once the method is developed and qualified, the method must be transferred The transfer would be to a GMP QC laboratory Qualifying QC Lab The QC laboratory would perform the Lab validation This would use the data derived from the qualification as well as routine performance Another data to set acceptance criteira for the Contract Production validation Lab Site The validation would be based upon the same ICH Q2(R1) requirements for a quantitative impurity assay 24 | 15-MAY-2018 © 2018 GreyRigge Assoc. Ltd. All rights reserved Summary • Developing a hcp method for a live viral vaccine brings particular challenges • The levels of host cell protein in a product are case by case and must be justified based upon good scientific rationale, data & a risk based approach • Off the shelf methods are not product specific and may not give an adequate level of hcp coverage for your product and must be assessed • Preparation and characterization of the antigen is pivotal.