Biotherapeutics have grown to become one of the more promising classes of drug compounds. During the period 2004–2012, the market share of biotherapeutics versus conventional small molecules grew from 12% to 20%, with worldwide sales estimated at US$153bn in 2012.1 By 2018, sales will increase to $215bn, with an estimated CAGR of 6.4% over the next decade.2
Some of this growth is driven by the advantages offered by biotherapeutics, which include greater specificity, potentially higher efficacy and lower toxicity. However, their development is not without its challenges. For example, as biotherapeutics are produced inside living cells, they must be carefully purified before they can be used in research or the clinic. Undetected contamination with host cell proteins (HCPs) can have very serious implications, triggering undesirable immune reactions in patients.
In one recent high profile example, Inspiration Biopharmaceuticals had to put a hold on the clinical testing of IB1001, an intravenous recombinant factor IX (rFIX) for preventing bleeding in hemophiliacs. During a Phase III clinical trial, a group of patients started to exhibit an immune reaction against proteins from the host cell type used to produce the therapeutic. As would be expected, such hold-ups are costly and time-consuming, often requiring further pre-clinical R&D and an adjustment to the production pipeline.
Detecting HCP impurities
Given the considerations around drug efficacy and patient safety, regulators worldwide, including the FDA,3 require careful monitoring of residual HCP levels in manufactured biotherapeutics. The most widely used approach for HCP monitoring is the HCP ELISA – an immunoassay that relies on a polyclonal antibody reagent that must be ‘capable of detecting a wide range of potential HCP impurities (ICH Q6B)’. The accuracy of the immunoassay depends on the range of HCPs detectable by the antibody reagent and increasingly, regulators are requiring more stringent evaluations of the range of HCPs detectable by the antibody reagent.
Among the various methods to evaluate the antibody reagent (polyclonal anti-HCP antibodies), 2-D electrophoresis (2-DE) followed by western blotting is the current gold standard.3,4 The standard workflow for this evaluation consists of four steps.
First, HCP mixtures are resolved in replicate by 2-DE. Second, one of the 2-DE gels is processed for sensitive total protein detection. Third, proteins from the replicate 2-DE gel are transferred to a solid support membrane and processed for western blotting with the antibody reagent being evaluated. Finally, images from total protein detection and western blotting are overlaid to obtain a match rate for determining the percentage of host cell proteins that are detectable by the anti-HCP antibodies.
Unfortunately, the laborious nature of the standard workflow and experimental variation inherent to replicate 2-DE analyses affects overall reliability of the entire evaluation. Evaluation reliability is a critical component of the overall HCP monitoring strategy since it not only serves as the ‘readout’ of antibody coverage (and hence determines the accuracy of the HCP ELISA), but also provides a means to identify those proteins that must be monitored using orthogonal methods.
Lack of reproducibility
To evaluate anti-HCP antibodies by 2-DE and western blotting, perhaps the biggest technical issue that must be overcome is the lack of reproducibility observed between replicate 2-DE gels. This problem is further compounded if the protein transfer from the 2-DE to the western blot membrane is not efficient, with both issues leading to erroneous match rate estimates. Additionally, imaging and software options that simplify overlay analyses (between total protein staining post-2-DE and western blotting) and standardise match rate determinations would be helpful.
Tools that simplify, standardise and accelerate 2-DE and western blotting have recently been developed and validated. Integrating these into a workflow with optimised imaging systems and automated image analysis software can be used to perform reliable evaluations of anti-HCP antibodies. In particular, the workflow developed by Bio-Rad Laboratories uses optimised reagents, techniques and hardware, enabling anti-HCP antibodies to be reliably evaluated in less than two days (see Figure 1).
Working from the same membrane
A key component of the new workflow is the ability to assess total protein staining and anti-HCP antibody reactivity using the same membrane post 2-DE gel separation. Doing so avoids the variability and error introduced by running and analysing multiple gels for the same antibody mixture. In order to use the same membrane, total protein detection is carried out via non-covalent labelling with the SYPRO Ruby reagent.
Unlike dye-labelling, SYPRO does not covalently modify the HCPs or affect their immunogenicity, limiting any potential impact on subsequent western blot analysis. 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.
As a single membrane will be relied upon for the entire analysis, it is essential to confirm that all of the HCPs are efficiently transferred from the 2-DE gel prior to the total protein stain and western blot. To ensure this happens, the new workflow takes advantage of specialised SDS-PAGE chemistry (TGX) and protein transfer systems (Trans-Blot Turbo) that combine to leave no detectable protein on post-transfer gels (see Figure 2). This provides users with confidence that low amounts of immunoreaction on the blot are due to the limited coverage offered by the antibodies being tested, rather than the absence of immobilised protein.
Figure 2: The new protocol ensures that all the protein is successfully transferred from the 2-DE gel to the western blot membrane
The last stage in the process uses dedicated image analysis software to overlay the total protein and antibody binding signal distributions (see Figure 3). As both procedures utilise the same membrane, the overlays match and antibody coverage can be calculated with high accuracy.
The speed and reliability of the Bio-Rad workflow not only ensures effective evaluations of the final anti-HCP antibodies prior to their use in ELISAs, but is also very useful during development of the same antibodies. Rockland Immunochemicals recently used the 2-DE and western blotting workflow described above to discriminate between various immunisation and purification strategies for development of their anti-HCP antibodies raised against E.coli HCP mixtures.
Figure 3: Image analysis is used to detect individual spots on the SYPRO Ruby and western blots, with the overlay demonstrating the fraction of total protein effectively bound (detected) by the antibody. This is then used to calculate a coverage estimate (%)
Greater resolving power
Rockland’s researchers found that evaluation by 2-DE and western blotting demonstrated greater resolving power (as compared with conventional SDS-PAGE/1-DE and western blotting) and this increased resolution was critical for effective monitoring of the immune response, and the determination of a better final anti-HCP antibody reagent.
Using Bio-Rad’s workflow, the company was able to develop a validated E.coli HCP Western Blot kit that contains 2-D validated antibodies. They are also in the process of developing additional HCP antibodies and ELISA kits against other species using this method.
In conclusion, biotherapeutic proteins are a rapidly increasing focus of pharmaceutical R&D subject to unique regulatory and technical requirements. One of these requirements is centred on the accurate monitoring and effective removal of host cell derived protein impurities. While removal of HCPs is achieved by orthogonal chromatographic purification schemes, an HCP ELISA/immunoassay is most often used to monitor their residual levels.
The HCP ELISA uses a polyclonal antibody reagent that must be capable of detecting a wide range of potential host cell derived impurities. To ensure that the antibody reagent used in an ELISA is capable of detecting a large proportion of HCPs, there is increasing regulatory expectation that it must be evaluated via a 2-DE and western blotting workflow. However, in its current guise the workflow can be unreliable and slow, thus inhibiting its wider adoption.
By integrating unique instruments and consumables, the new Bio-Rad workflow offers highly confident evaluation of anti-HCP antibody coverage for ELISA development
References
1. EvaluatePharma (2012), World Preview 2018: Embracing the Patent Cliff.
2. VisionGain (2012), Biosimilars and Follow-on Biologics: World Market 2012–2022.
3. Rellahan (2013). Process Related Impurities and their Impact on Product Quality – An FDA Perspective and Recommendations (With Emphasis on Host Cell Protein (HCP) Impurities). CASSS.org. www.casss.org/associations/9165/files/Barbara%20Rellahan%20%20%20Impurities%204.pdf
4. Savino et al (2011). Development of an in-house process-specific ELISA for detecting HCP in a therapeutic antibody. BioProcess International, 9 (3): 38–47.