Clarity in cell line development

GSK pioneer a single-step cloning strategy to generate novel recombinant sub unit vaccine candidates.

GSK Vaccines’ team shows the benefits of a new single-step cloning strategy to reduce effort and timelines to generate CHO cell lines expressing novel vaccine candidates.

Currently we are living in unprecedented times with a global health threat from a novel respiratory disease that has spread within weeks.  We are trying to subvert a large percentage of the population succumbing to this virus through early detection and isolation strategy.  There is the potential need for a long-term treatment if variants of COVID-19 persist in the human population and to reduce the fatality rate.  New vaccines and more rapid development timelines are needed to provide immunity to these new threats.

A fantastic effort has started with science working hard to abate this crisis.  The rapid response of vaccine manufacturers with close to 50 candidates listed by the WHO1 ranging from novel RNA-based vaccines to re-purposing of already marketed drugs.  A successful drug can kerb the transmission and reduce the respiratory burden on people carrying COVID-19.  Interestingly, we can look at the antibody repertoire of people who have recovered from the infection, or use B-cell screening to find an active antibody, or generate protein subunits to prime the immune system.  To scale this therapy, it will require a fast and effective way to create a cell line producing the recombinant subunit vaccine protein(s), which is effective, shows appropriate antigenicity and high yield.

GlaxoSmithKline (GSK) has had a strong track record in developing vaccines to 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 host as the cell factory shows that the analysis of clones is limited in the early stages with the focus on the growth of clones.  In part, this is determined by 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 comments that new instruments have come to the market in the last two years. Still, 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 a flexible and multifaceted workflows which leverage the capabilities of the manufacturing groups and discovery groups to assess the performance of the cell lines during primary screening.  The workflow devised by the team at GSK still uses traditional FACS to deposit single cells into 96-well multi-well plates.  The presence of single cells is confirmed by the Solentim Cell Metric® CLD, according to regulatory recommendations to confirm a cell line is derived from a single cell.

Then the growth of the single cells was 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 required throughput.  Also, the Luminex assay can 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, allowing the team to estimate specific productivity values to rank the clones.  The team could quickly narrow the candidate clones down to 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.

Resources

  1. WHO novel coronavirus candidate vaccine landscape, https://www.who.int/blueprint/priority-diseases/key-action/novel-coronavirus-landscape-ncov.pdf?ua=1
  2. Li, X, Zhang, Y, Jing, L, et al. “Integration of high‐throughput analytics and cell imaging enables direct early productivity and product quality assessment during Chinese Hamster ovary cell line development for a complex multi‐subunit vaccine antigen.” Biotechnol Progress., published 30 September 2019. https://doi.org/10.1002/btpr.2914

Figures

Solentim GSK recombinant subunit vaccine 1

Figure 1 Cell counting with the Solentim Cell Metric CLD. (a,b) Thumbnail views from Cell Metric CLD of colony outgrowth and cell count for individual clones representative 96-well plate at Day 12 after single cell cloning; (c) growth curves of selected clones (N = 10) with distinct growth rates; (d) a representative monoclonality report generated by Cell Metric CLD.  (courtesy of Li et al2)

Solentim GSK recombinant subunit vaccine 2

Figure 2 The productivity and product quality comparison of top clones selected by conventional and direct early clone screening processes. Volumetric productivity (VP) and specific productivity (qP) were determined by Octet assay using anti-CMV Pentamer mAb 4I22 for fed-batch cell culture supernatants. Top 10 clones were ranked based on the Day 14 fed-batch VP. Product quality of the clones was evaluated by SDS-PAGE with Coomassie blue staining and Western blot. (a, c and e) VP, qP, and SDS-PAGE/Western blot analysis for top 10 clones derived from direct early clone screening process; (b, d and f) VP, qP, and SDS-PAGE/Western blot analysis for top 10 clones derived from conventional clone screening process. *Clone 40 (CL40) was from the low VP/qP cluster defined by multivariant VP and qP analysis. WB, Western blot; L, protein ladder. (courtesy of Li et al2)

Solentim GSK recombinant subunit vaccine 3

Figure 3   Proposed single-step early clone screening process with the integration of Luminex/Cell Metric CLD platform. (courtesy of Li et al2)

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