The Challenges of Developing HCPs Methods for Vaccines

© 2018 GreyRigge Assoc. Ltd. All rights reserved

15 May 2018

The Challenges of Developing HCPs Methods for Vaccines A Risk Based Approach to HCP Monitoring

Host Cell Protein ConferenceMay 14-16, 2018Dubrovnik, Croatia

Dr. Lee Smith

© 2018 GreyRigge Assoc. Ltd. All rights reserved

Types of Vaccines & Their Cell Substrate

| 15-MAY-20182

• Mammalian, Insect

• Vero, HEK293, sf9

• Bacterial culture

• E. coli

• Subunit or VLP

• E. coli, yeast

• Inactivated whole virus

• Live attenuated virus

• Live attenuated viral vector

• DNA vaccine

• Subunit or VLP

© 2018 GreyRigge Assoc. Ltd. All rights reserved| 15-MAY-20183

• 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

Live Virus Vaccine Processes

WCB

10x

Ret

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sh

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Cell Expansion

WVS

FILTRATION

CHROMATOGRAPHY

UF/DF STERILE FILTRATION

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.

Live Virus Vaccine Processes & Host Cell Protein (hcp)

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10x

Ret

HARVEST

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Cell Expansion

WVS

FILTRATION

CHROMATOGRAPHY


DRUG SUBSTANCE

hcp


Live virus expression can be:1) lytic2) secreted3) intracellular

Enveloped Live Virus Expression

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.

Enveloped Live Virus Expression cont.


• 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

for the smallpox vaccine-weighs only 9 femtograms, or

quadrillionths of a gram.

Host Cell Protein Levels

How much protein?10 Femtograms per particle1 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.

Typical hcp amounts in a Live Virus Vaccine?


Modern vaccines purified through complex DSPCompared to older vaccines

Typical hcp amounts in a Live Virus Vaccine?

700g/dose!1-50g/dose!

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Cell Expansion

WVS

FILTRATION

CHROMATOGRAPHY


DRUG SUBSTANCE

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

How was 700ug/dose of hcp acceptable?

• 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

Should I use an OTS Host Cell Protein Immunoassay?

100% 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

Could I use OTS Host Cell Protein Immunoassay?



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

Determining your Coverage


Null Cell Line Approach – to obtain hcp profile similar to the product

Expand the cells, do not infect and process supernatant through the DSP.

Preparation of HCP Antigen for Immununisations

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

Preparation of HCP Antigen for Immununisations Cont.

We can expand cells and simply lyse them through Freeze/Thaw or sonication

releasing the hcp. These are then processed them through the DSP

10

x

Re

t

HARVEST

BufSoln

Flu

sh

ENDONUCLEASE

Cell Expansion

Lysis

FILTRATION

CHROMATOGRAPHY


HCP


Cell Lysis


We can expand cells and simply lyse them and immunize with the

crude hcp.

Crude HCP

Cell Expansion

Lysis

Depth filtration can be used to remove large particulates from the

crude lysate

10

x

Re

t

BufSoln

Flu

sh

UF/DF

UF/DF with a suitable membrane cutoff can be used to split the

lysate into high and low molecular weight hcp


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


Low & High wt. lysate

Product containing hcp

Lyse Free HCP


• The Low & High M. Wt

lysate can be immunized

separately or in

combination

• This serum will be

immunized in line with

an agreed protocol

Immunisations


The animals are bled to obtain and

test the serum for binding to the

hcp

Immunisations cont.

Cascade immunization can be considered if

Ag components immunodominant

& the FT re-immunised


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

Characterising the Serum

✓50%70%+


• 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?

Development of the ELISA & Long-Term Thinking

• 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.

HCP ELISA Method Qualification & Validation


HCP Protein Conc (ug/mL)

HCP ELISA Method Qualification & Validation cont.

HCP Protein Conc. (ug/mL)

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

- Specificity


Once the method is developed and qualified, the method must be transferred

The transfer would be to a GMP QC laboratory

The QC laboratory would perform the validation

This would use the data derived from the qualification as well as routine performance data to set acceptance criteira for the validation

The validation would be based upon the same ICH Q2(R1) requirements for a quantitative impurity assay

Method Transfer

Qualifying Lab

QC Lab

Contract Lab

Another Production

Site


• 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

Summary

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The Challenges of Developing HCPs Methods for Vaccines