The technique of gel electrophoresis in protein analysis is one of the oldest and simplest procedures for investigating sample structure in a gross molecular sense and for assessing subunit composition, as well as for getting a feel for sample purity. Over the years, it has been refined and shown to be used for other areas of structural investigation such as Western blotting for protein identification work, 2D gel electrophoresis to assess sample make-up and for host cell protein studies.
CE-SDS Gel electrophoresis is therefore, despite the fact that it is decades old, a technique that still has significant applicability in structural analysis. Indeed, the generation of electrophoretic profiles is still a requirement of the ICH Q6B guidelines (1) when examining the structure of a biological therapeutic. This extends into the arena of biosimilar structural characterization, since this document is referenced in both the EU and FDA biosimilars analytical guidelines (2,3).
Since this technique is still important in providing key structural data, companies have looked at the procedure itself in order to find ways to improve the methodology, as well as associated instrumentation, with the aim of giving improved reliability, reproducibility of data generation and eliminating issues associated with traditional slab gel set-ups. As a result, the technique of capillary gel electrophoresis has been developed and shown to be applicable to the structural analysis of a wide range of biological products across a broad molecular weight range (approx. 10-270kDa).
The procedure itself, as the name suggests, uses a reusable, preassembled SDS polyacrylamide gel-filled capillary which provides reproducible sample runs. Direct UV detection of the separated protein components eliminates the need for separate staining procedures and the issues of reproducibility associated with them. The UV detection and data processing systems also provide a means to readily generate reliable relative quantitation of detected components. Moreover, CE-SDS analysis is quick, easy and the instrumentation is straightforward to use and, of course, the less steps there are to perform, the less chance there is for divergence between runs and the greater the precision will be.
At BioPharmaSpec, we use a ProteinSimple Maurice S system for CE-SDS analysis, which complements our ProteinSimple icIEF system. We have performed tests on the Maurice S system using a range of different biomolecules reflecting the types of biopharmaceuticals most commonly encountered, namely monoclonal antibodies (mAbs), heavily glycosylated glycoproteins and PEGylated proteins. You can find an article we’ve written on our findings here.
We conducted a range of studies across these molecular types and we found good reproducibility for multiple injections of each of the samples tested. For mAbs, the data showed that the system can be used to identify partial antibody species (e.g. light-heavy chain dimers, confirmed in subsequent runs with reducing agent added and observing the loss of the light-heavy chain dimer peak and corresponding increase in the response for individual light and heavy chain peaks) as well as low levels of non-glycosylated antibody in the sample preparations (confirmed in subsequent runs with the addition of PNGase F showing a reduction in the glycosylated peak and corresponding increase in the non-glycosylated peak).
Stacked traces for three reduced Denosumab samples. (A) Peaks for the heavy, light, and non-glycosylated heavy chains are observed. (B) Treatment of the same three reduced samples with the PNGase F enzyme results in the de-glycosylation of the heavy chain, which shifts to the position typically seen for non-glycosylated heavy chains.
Analysis of heavily glycosylated glycoproteins (using Etanercept as the model product), demonstrated that structural dissection of the molecule through judicious use of reduction, de-N-glycosylation and/or neuraminidase treatments in various combinations was able to pull apart variance in the structures through the way different batches responded to the treatments. This is useful for demonstrating the range of response for batches of biosimilar and innovator in side-by-side comparative studies and assessing the degree of overlap in the peak profile distributions.
The study we performed also included assessment of PEGylated proteins, which is a commonly used chemical modification of proteins and glycoproteins via the attachment of a large polyethylene glycol (PEG) unit to the protein. The attached PEG unit is commonly anywhere between 5kDa and 20kDa but doesn’t have an absolute size limit. The studies we performed with PEGylated proteins showed that they can be analyzed readily using this system. Samples can be screened for the presence of non-PEGylated species or species that are over PEGylated, thus CE-SDS is a useful tool for the detection of impurities and these can be monitored much as with mAbs, which can lead to process refinements at the production or purification stages.
The improvements to the traditional gel electrophoresis process and development of CE-SDS have moved this technique very much into an area where it can be exploited as a quick and relatively simple procedure that can provide meaningful data, in a qualitative and relative quantitation sense. The applications for CE-SDS in our labs are for investigative, structural analysis for new drugs, as well as for biosimilar investigations and assessments of manufacturing processes. Critically, the data generated can be considered to be orthogonal to other mass determining techniques (notably mass spectrometry itself). This idea of orthogonality is a key concept for regulatory agencies who expect it to be built in to any structural analysis investigations submitted to them. We have previously covered the idea of orthogonality in this article and also in our blog.