Host Cell Proteins

Passion. Innovation. Life
Host Cell Protein (HCP)
Bharti Warghude
Date: 8th Feb, 2019

OBJECTIVE
1. What are HCPs
2. Why are they so important
3. How to detect and remove these from our Drug substance/ solution
4. Regulatory requirements to check the limits of HCP

INTRODUCTION
Process related protein impurities during the manufacturing of biologics.
Endogenous proteins.
(Already exist as components of living cells).
Unique to respective host.
(Specific to the cell line or the host used for example E.coli and CHO cell line).
Closely associated with the host expression system itself.
[For example, E. coli has 4,300 genes (Blattner et al., 1997), whereas mammalian cells such as Chinese Hamster Ovary (CHO) and a commonly used
mouse myeloma cell line (NS0) have about 30,000 genes (Gibbs et al., 2004; Waterston et al., 2002)].
Depend upon the manner in which the biologic of interest is expressed.
[For example, recombinant proteins produced in E. coli may be expressed directly in the cytoplasm or secreted into the periplasm (these can be
engineered to be deposited as inclusion bodies), whereas in mammalian cells it may be secreted into the culture medium].

WHY DO WE NEED TO DETECT THE HCPS?
HCPs carry potential clinical safety risks in addition to those that might be related to the intended recombinant
protein itself.
[These risks are difficult to evaluate since pre-clinical models (both animal & human) are rarely informative].
These are unwanted.
[The foreign or ‘‘non-self’’ nature of HCP derived from non-human expression systems suggests that almost any individual HCP has the potential to
elicit an immune response in humans, (Janeway et al., 2005)].
Note: Those proteins that are common b/w the species wont be an issue.
Immunogenic effect.
(HCPs contamination can result in adverse toxic or immunological reactions).
To much of it can effect our health.
(The degree of immunogenicity on a long-term basis is practically impossible to determine and thus might be a relatively severe threat to the patient’s
health).
Proteolytic enzymes from the host may effect our product.

HOW ANTIBODIES ARE GENRATED?
1. Rabbits or goats are most commonly used for generation of these antibodies (anti-HCP Ab).
2. It usually takes several immunizations to reach a maximum immunological response; the process could take 60–90 days
to complete depending on the frequency of immunization.
3. When the titer reaches the maximum, Western blot will be used to test the coverage of HCPs from these antibodies.
4. These will be pooled to develop the ELISAAssay.

SUPPLIMENTARY TEST TO CHECK HOW GOOD IS OUR ANTIBODY OR THE REAGENT
Western Blotting
1. Western blot is an antibody-dependent detection method (Speicher, 2008).
2. In this method, HCPs are first separated in a polyacrylamide 1 or 2D gel and transferred to a PVDF or
nitrocellulose membrane.
(The primary antibodies raised against HCPs, will be incubated on this membrane).
3. These primary antibodies form complexes with the HCPs on the membrane.
4. These HCP-antibody complex could be detected by labeling the primary antibody with an enzyme like
Horseradish Peroxidase (HRP) (Speicher, 2008) and successive reaction with a substrate that emits certain color
(Blue- TMB Substrate).

CONVENTIONALLY USED METHODS TO DETECT HCPs
i) ELISA
ii) MS/MS

I ) ELISA

II) MS/MS

COMPARISON HCP PATTERNS FOR DIFFERENT TECHNIQUES USED

LIMITATIONS
1. During the production process several factors, including:
The genes of the host cell,
The way of product expression, and
The purification steps
Influence the HCP composition and abundance. Therefore, it is difficult to capture each and every type of
HCPs.
2. Several studies report that HCPs often are co-purified along with the product itself by interacting with the
recombinant protein.
3. It is difficult or impossible to design non-clinical or in vitro experiments to demonstrate which HCPs and at
what level may confer risk to humans.

KIT USED AT ENZENE
• Immunoenzymatic Assay from Cygnus Technologies (Part of Maravai Lifesciences)
For both the measurement of CHO (Catalogue no. F550) and E.coli (Catalogue no. F410) Host Cell
Proteins.

REGULATORY REQUIREMENT
Range of HCPs in biologic products reviewed by FDA is 1–100 ppm (also mentioned in the ICH Guidelines).
The limits of HCP varies with the route of administration:
1. Subcutaneous (Subcut) (under the skin)
2. Intramuscular (IM) (directly into muscle)
3. Intravenous (IV) (administered into a vein)
4. Intravitreal (medicine into the eye)

14. REFERENCES
• Host Cell Proteins in Biologics Development: Identification, Quantitation and Risk Assessment (Wang
Wang et al., 2009)

THANK YOU 

Host Cell Proteins

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