GSK pioneer a single-step cloning strategy to generate novel recombinant sub unit vaccine candidates.
A Guest Blog by Mark Stockdale, Solentim
GSK Vaccines’ team demonstrate the benefits of a new, single-step cloning strategy to generate CHO cell lines expressing novel vaccine candidates, reducing effort and shortening timelines. Currently we are living in unprecedented times with a global health threat from a novel, highly infectious respiratory disease. We are trying to prevent a large percentage of the population succumbing to this virus through early detection and isolation strategies. There is the potential need for a long-term treatment to reduce fatality rates if variants of COVID-19 persist in the human population. New vaccines and more rapid development timelines are needed to provide immunity to this new threat, and others like it. A fantastic effort has started, with scientific researchers and vaccine manufacturers working hard to abate this crisis; close to 150 COVID-19 vaccine candidates are now listed by the WHO1 ranging from novel RNA-based vaccines to the re-purposing of already marketed drugs. A successful drug could curb transmission and reduce the respiratory burden on those infected with COVID-19. One route to vaccine discovery is to analyse the antibody repertoire of people who have recovered from the infection, use B-cell screening to find an active antibody, or generate protein subunits to prime the immune system. In order to effectively scale therapies of this nature, we will require a fast and effective way to create cell lines producing the recombinant subunit vaccine protein(s), which shows appropriate antigenicity and high yield. GlaxoSmithKline (GSK) has a strong track record in developing vaccines for some of the most prominent threats to humankind. Recently Xiangming Li2 in Marcin Bugno’s team at GSK Rockville looked at their capabilities to generate multi-subunit recombinant vaccine proteins against human CMV (cytomegalovirus), and the technology that could help reduce the response time and effort required. Assessment of the classic cell line development process using CHO hosts as the cell factory shows that the analysis of clones is limited in the early stages, focusing instead on clone outgrowth to determine performance. In part, this is due to the lack of sensitive assays for productivity assessment and accurate methods to determine cell number, allowing specific productivity to be determined, a strong predictor for lead clones. Li et al2 comment that, although new instruments have reached the market in the last two years, these systems are focussed predominately on classical antibody workflows, with assays limited for productivity assessment of monoclonal IgG antibodies in a semi-quantitative manner: ‘Beacon (Berkeley Lights), utilizes nanofluidics and OptoElectro position technology to achieve single-cell cloning and high cell density culture conditions, potentially providing an advantage for selecting clones compatible with upstream bioreactor processes. However, similarly to ClonePix technology, the productivity is estimated indirectly, based on diffusion-based staining of the secreted antibody with a fluorophore-tagged small molecule binding human IgG Fc’. With the expanding need to express antibody fragments and novel proteins, this calls for flexible and multifaceted workflows which leverage the capabilities of manufacturing groups and discovery groups to assess the performance of the cell lines during primary screening. The workflow devised by the team at GSK uses traditional FACS to deposit single cells into 96-well multi-well plates. The presence of a single cell in each well is confirmed by the Solentim Cell Metric® CLD, according to regulatory recommendations, to confirm a cell line is derived from a single cell. The growth of each single cell was then monitored using the Cell Metric CLD to accurately determine the cell number per well in a 10-14 day window (see figure 1). The outgrowth reported from single cells was approximately 20%, which is typical for the CHO platform used in the experiment. At day 14, half of the supernatant was harvested from the single-cell isolation plates for quantification of the multi-subunit protein. The volumetric titre was anticipated to be in the ng/mL range, far below the sensitivity of many high-throughput quantitative assays, so this required the adoption of a multiplex immunoassay with sensitivity in the ng/mL range.
The team adapted a Luminex bead-based sandwich immunoassay to give the required sensitivity and throughput. Helpfully, the Luminex assay can also be used for epitope profiling of the secreted recombinant protein. Using the combination of the Cell Metric CLD and the Luminex assay the team could reliably determine cell counts and volumetric productivity, and therefore estimate specific productivity values to rank the clones. This enabled the rapid selection of the lead clones, with comparable results in the scale-up to bioreactors compared with the conventional screening approach (see figure 2). The GSK team successfully devised what they termed a ‘single-step clone selection strategy’ for a non-antibody format recombinant protein (see figure 3), which reduced the effort required to obtain lead clones, eliminated a 2-3 week expansion step, and de-risked the workflow earlier. This workflow could be applicable to other CHO platforms being used widely and may have utility as we target current and future epidemics.