© 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
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• 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
HARVEST
BufSoln
Flu
sh
ENDONUCLEASE
Cell Expansion
WVS
FILTRATION
CHROMATOGRAPHY
UF/DF STERILE FILTRATION
DRUG SUBSTANCE
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• 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)
WCB
10x
Ret
HARVEST
BufSoln
Flu
sh
ENDONUCLEASE
Cell Expansion
WVS
FILTRATION
CHROMATOGRAPHY
UF/DF STERILE FILTRATION
DRUG SUBSTANCE
hcp
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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
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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.
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• 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
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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?
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Modern vaccines purified through complex DSPCompared to older vaccines
Typical hcp amounts in a Live Virus Vaccine?
700g/dose!1-50g/dose!
WCB
10x
Ret
HARVEST
BufSoln
Flu
sh
ENDONUCLEASE
Cell Expansion
WVS
FILTRATION
CHROMATOGRAPHY
UF/DF STERILE FILTRATION
DRUG SUBSTANCE
Wouldn’t recommend this!
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• 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!
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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?
70% Coverage of hcp?
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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?
100% Coverage of hcp?
50% Coverage of hcp?
What coverage is acceptable?
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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
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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
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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
UF/DF STERILE FILTRATION
HCP
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Cell Lysis
Preparation of HCP Antigen for Immununisations Cont.
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
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Can characterize on 2D gel to compare lysate with product, including blotting with Ab
Preparation of HCP Antigen for Immununisations Cont.
Low & High wt. lysate
Product containing hcp
Lyse Free HCP
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• 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
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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
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The Serum can then be assessed to understand the coverage of product hcp
Characterising the Serum
✓50%70%+
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• 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?
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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
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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
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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
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• 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