April 15, 2014 (Vol. 34, No. 8)

Anton Posch, Ph.D.
Thomas Berkelman, Ph.D. Bio-Rad Laboratories

Large molecule, protein-based biopharmaceuticals can have significant advantages over conventional small molecule pharmaceuticals including greater specificity, lower toxicity, and higher efficacy. Despite their potential advantages, biopharmaceutical and biosimilar drug developers face at least one unique challenge: eliminating residual impurities—i.e., host cell proteins (HCPs)—in the final drug substance. Inability to detect potential HCPs that may co-purify with the final drug substance may diminish drug efficacy and pose a risk to patient safety.

Regulatory bodies worldwide require careful monitoring of residual HCP impurities in manufactured biopharmaceuticals. The HCP ELISA/immunoassay is most widely used for HCP monitoring given its high sensitivity and its potential to detect a wide range of impurities. The ELISA/immunoassay, known as an anti-HCP ELISA, relies on a polyclonal antibody reagent mixture capable of detecting a wide range of potential HCP impurities.

This range of HCP detection—immunocoverage—is often evaluated using methods of limited resolution such as SDS-PAGE (1D electrophoresis/1DE) followed by Western blotting of the HCP mixture. Owing to the limited resolution of this method, the FDA has recently stated that 1DE and Western blotting is not suitable for evaluating the anti-HCP ELISA.

The FDA now recommends that for all drug application submissions biopharmaceutical developers use 2DE (two-dimensional electrophoresis wherein proteins are separated by isoelectric point then molecular weight) and Western blotting to evaluate their anti-HCP antibody/ELISA coverage. To assess anti-HCP antibodies by 2DE and Western blotting, the biggest technical issue is the lack of reproducibility between replicate 2DE gels

This problem is further compounded if the protein transfer from the 2DE to the Western blot membrane is not efficient, leading to erroneous match rate estimates. Additionally, imaging and software options that simplify overlay analyses (between total protein staining post-2DE and Western blotting) and standardize match rate determinations would be helpful.


Reliable and Accelerated Anti-HCP Antibody Evaluations

Bio-Rad Laboratories recently developed an efficient workflow (Figure 1) that enables highly reliable and accelerated evaluations of anti-HCP antibodies in less than two days. Bio-Rad’s workflow can also be applied during anti-HCP antibody development to evaluate the effect of various immunization schemes on the range of detectable impurities or generate optimal antibody mixes from various immunization schemes.

The workflow’s first step features the PROTEAN i12 IEF system for 2DE. This is the only instrument that enables optimization of up to 12 variables affecting isoelectric focusing performance in a single experiment for highly reproducible first-dimension protein separations. Another key workflow improvement that improves reproducibility is the use of the same 2DE separation for both total protein staining and Western blotting.

Using SYPRO Ruby for total protein staining does not interfere with Western blotting, thus eliminating the need to run and compare two different 2DE gels for protein detection and Western blotting. SYPRO Ruby is also better than dye labeling as it does not covalently modify proteins, which can affect match rate reliability. This approach not only effectively halves the number of gels that need to be run, but more importantly, it enables a like-for-like comparison between the total protein results and those of the Western blot.

Since only a single membrane is required for analysis, the efficient transfer of HCPs from the 2DE gel onto the membrane is critical. The Bio-Rad workflow incorporates specialized SDS-PAGE chemistry known as TGX that is optimized for high transfer efficiency and fast run times on Bio-Rad’s Trans-Blot Turbo Protein Transfer System that leaves no detectable protein on post-transfer gels.

Consequently, low amounts of immunoreaction on the Western blot can be assigned with confidence to the lack of coverage provided by the antibody reagent being tested, rather than being due to the inefficient transfer of HCPs. Dedicated image analysis software in the final step of the process overlays the total protein signal data with that of the antibody binding results. As both procedures use the same membrane, the overlays match and antibody coverage can be calculated with high accuracy.


Figure 1. An optimized workflow using 2D gel electrophoresis and Western blotting for validating anti-HCP antibodies.

Same Membrane Analyses for Anti-CHO HCP Antibody Evaluations

To test the application of same membrane  2DE electrophoresis and Western blotting analyses for evaluations of anti-CHO HCP antibodies, the workflow was used to evaluate a commercially available anti-CHO antibody reagent against two CHO-derived samples (CHO total protein and secreted protein).

A single 2DE analysis of each sample was performed using optimized conditions for 1st dimension IEF (11 cm pH 3–10 NL ReadyStrip IPG strips) and 2nd dimension electrophoresis (Criterion TGX Any kD gels). After 2DE, proteins were transferred to PVDF membranes using the Trans-Blot Turbo in less than 10 minutes. To monitor efficiency of transfer, the post-transfer gel was stained using Oriole fluorescent gel stain and compared against a replicate 2DE gel of each sample stained identically.

Proteins transferred to PVDF were visualized by SYPRO Ruby fluorescent protein staining and subsequently processed for chemiluminescent Western blotting with anti-CHO antibodies and HRP-conjugated secondary antibodies. Images were obtained on the multipurpose ChemiDoc MP imager; spot matching and image alignment were done via PDQuest 2DE analysis software.


Results demonstrated that same membrane analysis not only overcomes reproducibility issues between 2DE replicates, but also enables high-confidence spot matching and image alignment via 2DE analysis software. Second, fast highly efficient protein transfer with no detectable protein left on post-transfer gels increases thoroughness of downstream analyses (Figure 2).


Figure 2. Visualization of post-2DE protein transfer efficiency using the new protocol.

Third, fluorescent noncovalent protein staining and chemiluminescent Western blotting analyses achieves high sensitivity protein detection without the loss of 2DE resolution or the potential for erroneous match rate estimates (Figure 3). Fourth, the use of same imager for both analyses generates directly comparable high-resolution images. Fifth, the entire workflow generates high-confidence results in less than two days.


Figure 3. HCP antibody coverage (%) calculated using overlay image analysis.

Conclusions

For monitoring of host cell proteins across the production process or in the purified final drug substance, most manufacturers rely on an HCP ELISA/immunoassay. U.S. regulators now require that the HCP ELISA be evaluated to detect a wide range of potential HCPs using 2DE and Western blotting. Bio-Rad Laboratories now offers a streamlined, accelerated 2DE and Western blotting workflow for highly reliable, accelerated evaluations of anti-HCP antibodies in less than two days.

The enhanced reliability and speed achieved in this workflow stems from the use of SDS-PAGE gels using TGX chemistry, fast and highly efficient protein transfer post-2DE, and performing highly sensitive (fluorescent) total protein staining and chemiluminescent Western blotting on same membrane for reliability of spot matching.

In addition, since the proteins remain unmodified chemically there is no loss in immunogenicity of proteins towards the anti-HCP antibodies, nor is there any loss in sample resolution associated with minimal covalent labelling strategies. The lack of resolution loss also facilitates the use of medium-sized 2DE gels that further accelerates the entire workflow.


























Anton Posch (Anton_Posch@bio-rad.com) is senior scientist and Tom Berkleman (Thomas_Berkelman@bio-rad.com) is senior staff scientist at Bio-Rad Laboratories.