Host Cell Protein (HCP) Antibody Generation


Host Cell Protein Antibody Generation Overview

Biopharmaceuticals require products to be free of process-related impurities including Host Cell Protein (HCP) contaminants. These HCPs could be left behind during the purification process from the expression hosts, such as E. coli, insect, or mammalian cells and responsible for serious adverse effects in humans.

To examine the presence of residual contamination during the bioprocessing and the final biopharmaceutical product, development of custom polyclonal antibody reagents with maximum coverage and sensitivity against native HCP extracts is required. Please see US FDA Guidelines for Bioanalytical Validation.

Rockland’s product portfolio provides well characterized and sensitive generic reagents for immunodetection of host cell proteins as well as multiple customizable options for process-specific HCP antibody development as a service. These options allow flexibility and control to the sponsor for developing early stage through late stage HCP detection reagents.

 

 Bioprocessing and Host Cell Protein Reagents

 

 
Figure 1: Schematic drawing showing examples of customizable options for HCP antibody development.  
Separation of the HCP population to increase detection coverage and multiple host species are available. Elective variations in the immunization strategies can aid in detection of low abundant and non-immunogenic antigens. Customizing the antibody generation to the sponsor requirements, enables the selection of most suitable strategy for effective detection of process-specific HCP. 
 

 

 

 

Standard Host Cell Protein Antibody Reagent Generation

The Standard method is a process is where the two sets of animals are immunized using high and low molecular weight Host Cell Protein (HCP) extract. After immunization and primary boosting, antisera is collected. The developed anti-HCP antibodies are evaluated to determine coverage of potential HMW and LMW host cell contaminants . The final HCP antibody product is a pool of anti-HMW and anti-LMW antibodies having broad coverage of the HCP antigens in the final antibody reagent. Coverage is demonstrated by 1D western blot analysis or 2D western blot analysis against the HCP extract. HCP-detection assay development is available. To obtain the optimal level of coverage multiple host animals may be utilized. The following species are available; Rabbit, Chicken, Sheep, Goat and Donkey. 


 Standard Host Cell Protein


 


 

Cascade Host Cell Protein Antibody Reagent Generation

Cascade immunization is a method where the animals are boosted using complete Host Cell Protein (HCP) extract. After immunization and primary boosting, antisera is collected. The developed anti-HCP antibodies are purified and used to immunoprecipitate the immunodominant antigens from the HCP extract. The remaining HCP antigens are used to boost the animals in what is called a cascade immunization. This method of immunization has been shown to improve the coverage of the final anti-HCP antibody reagent by driving an immune response and development of antibodies against the weaker immunogens or lower concentration proteins in the HCP. Coverage is demonstrated by western blot or 2D Analysis against the HCP extract. HCP-detection assay development is available. To obtain the optimal level of coverage multiple host animals may be utilized. The following species are available; Rabbit, Chicken, Sheep, Goat and Donkey

Cascade Antibody Generation

 


 

1D SDS-PAGE with Western Blotting

One Dimensional SDS-PAGE/Western Blot analysis is performed on test and production bleed s to evaluate the host antibody-mediated immune response to HCP lysate. Both a standard stained gel (Coommassie, Silver or Oriele Stain) and western blot are performed as part of the evaluation. Rockland can customize the testing and analysis methodology to meet your HCP Reagent needs.

1D Analysis

 

 


 

2D SDS-PAGE with Western Blotting


Protein contaminant detection can be visualized on 1D electrophoresis with western blotting. However, the immunogenicity of individual HCPs can vary widely and highly immunogenic proteins can disguise or “mask” the effects of other, similar size proteins. The implementation of 2D SDS-PAGE assessment, by which complex protein mixtures are separated according to isoelectric point and molecular weight followed by western blotting, can deliver a more complete insight into the capability of an anti-HCP polyclonal antibody with respect to immuno-coverage of protein components of the host cell lysate. Rockland can customize to meet your 2D Analysis with both a Midi (8 X 13 cm) and Large Gel Format (17 X17 cm)

2D Gel

 

 


 


Bioprocessing Related Products   Catalog #   Applications   Size
Anti-Bovine Serum Albumin (Rabbit) Antibody   201-4133-0025   ELISA, WB, IHC   25 uL
Anti-Bovine Serum Albumin (Mouse) Monoclonal Antibody (BSA)   201-301-033   ELISA, WB, IHC, IP   500 uL
Anti-Streptavidin (Rabbit) Antibody   200-401-095   ELISA, WB, IHC   100 uL
Anti-Biotin (Mouse) Monoclonal Antibody   200-301-098   ELISA, WB, IHC, IF   100 uL
Anti-Protein A (Goat) Antibody   100-1177   ELISA, WB, IHC   2 mL
Anti-Protein G (Rabbit) Antibody   100-401-138   ELISA, WB   2 mL
Anti-Bovine Igg (H&L) (Rabbit) Antibody   201-4102   ELISA, WB, IHC   50 mg

 

 

Host Cell Protein Related Products   Catalog #   Applications   Size
E.Coli Host Cell Protein Control Protein   000-001-J08   ELISA, WB   50 ug
Anti-E.Coli  Host Cell Protein [Total] (Rabbit) Antibody   100-401-J07   ELISA, WB, IHC   100 uL
Anti-E.Coli  High Molecular Weight Fraction Host Cell Protein Antibody   200-401-J05   ELISA, WB   100 ug
Anti-E.Coli Low Molecular Weight Fraction Host Cell Protein Antibody   200-401-J06   ELISA, WB   100 ug
Anti-E.Coli  Host Cell Protein [High And Low Molecular Weight Combined] Antibody   200-401-M61   ELISE, WB   100 ug
Anti-E.Coli  Host Cell Protein Combined Biotin Conjugated Antibody   200-406-M61   ELISA, WB   100 ug
Chemiluminescent Western Blot Kit For Detection Of E.Coli Host Cell Proteins   KCAJ07   WB   1 Kit
             
             
 

HCP Analysis of a Small Drug- Protein in Process Sample Poster PDF

Download the HCP Analysis of a Small-Drug Protein in Process Sample Poster 


 Small HCPs in a 12 kDa Protein Drug Analyzed by GeLC-MS/MS Poster PDF

 

Download the Small HCPs in a 12kDa Protein Drug Analyzed by GeLC-MS/MS Poster 

 

2D-SDS PAGE Analysis Methods For Determining HCP Coverage Using Process Derived anti-HCP Polyclonal Antibodies

2D-SDS PAGE Analysis Methods For Determining HCP Coverage Using Process Derived anti-HCP Polyclonal Antibodies

 

Production and Characterization of High Coverage, Process Specific Pichia P. HCP Antibodies
Production and Characterization of High Coverage, Process Specific Pichia P. HCP Antibodies

15-02_HCP_Poster_Side_Bar

Download the Multi-assay Functional Antibodies in HCP Detection poster

 

HCP Development Poster

Download the Enhanced Standardized HCP poster  

 

Host Cell Protein Workflow 

Producing an HCP Antibody: Workflow