HCP Presentation Croatia 15 May 2018

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

• 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

• 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

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CHROMATOGRAPHY DRUG SUBSTANCE

• 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

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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

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.

• 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

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.

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1-50g/dose! 700g/dose!

Wouldn’t recommend this!

• 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!

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?

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?

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

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

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

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We can expand cells and simply lyse them and immunize with the crude hcp.

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Lysis Crude HCP

Depth filtration can be used to remove large particulates from the crude lysate

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x x Ret 10 lysate into high and low molecular weight hcp UF/DF

Low & High wt. lysate

Lyse Free HCP

Product containing hcp

Can characterize on 2D gel to compare lysate with product, including blotting with Ab

• The Low & High M. Wt lysate can be immunized separately or in combination

• This serum will be immunized in line with an agreed protocol

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

The Serum can then be assessed to understand the coverage of product hcp

• 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?

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.

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

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

• 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. If you get this wrong and proceed, you may waste years

• Who develops the assay and provides long term supply once the product is licensed is a vital consideration and need to be considered early