Soon after the genetic sequence of SARS-CoV-2 was determined in January 2020, there was a rush to develop rapid diagnostics for the disease. The World Health Organization issued early guidance and protocols for the use of PCR to assist in diagnosing the disease. However, drawbacks like long turnaround times and invasive sampling for PCR tests led to a concerted push to develop rapid antibody (serology) tests. Serological antibody tests can quickly and cheaply screen for antibodies generated by the immune system in response to SARS-CoV-2. Their adaptability to a wide variety of formats, including  lateral flow assays, ELISA, and chemiluminescent immunoassays, all of which can be widely distributed, is another key advantage of serological tests. However, supporting the testing needs of the global population through serological testing requires a steady supply of raw materials and reagents.

Approximately 1 mg of spike protein is useful for about 3500 serological assays for COVID-19 serology testing. The spike protein of SARS-CoV-2 is a very large (~670 kDa) homotrimer with 22 N-linked glycosylation sites per monomer. As a result, the protein has been very difficult to recombinantly produce beyond low production titers (1-2 mg/L) from HEK cells. This rate of production is insufficient to meet the widescale demands of the protein needed to manufacture the serological assays. Fortunately, a team from the University of Sheffield and Sheffield Teaching Hospitals has shared their approach to boosting production of the SARS-CoV-2 spike protein using CHO cells. The group focused on vector engineering of CHO cells with optimized process conditions to boost production.

Recalling prior experiences producing difficult-to-express proteins, the researchers used mildly hypothermic culture temperatures to extend the culture longevity. For both HEK and CHO cells grown in hypothermic (32 °C) cultures, the integrated viable cell densities were lower than those grown at physiological temperature (37 °C). However, both titer and cell specific productivity (qP) were considerably higher. For HEK cells, the qP was 2.4x higher and the titer was 4.1x higher (10.2 mg/L) due to extended culture longevity in hypothermic 32 °C cultures compared with standard 37 °C cultures. The observed improvements for CHO cells were greater. CHO qP was 8.5x higher, and titer was increased by 10.9x (5.4 mg/L total) in hypothermic 32°C cultures compared with standard 37°C cultures.

To improve CHO titer relative to HEK production, synthetic promoters and the spike protein gene were electroporated into host CHO cells. Afterward, cell populations were screened for their ability to make the spike protein. As expected, the hypothermic conditions decreased viable cell densities, yet the qP for the hypothermic CHO cultures was 3.4x higher with the optimized cell line. Morevoer, the addition of certain small molecule chemical additives (e.g., valproic acid or betaine) further enhanced qP by >4.5x. This led to a final titer from the valproic acid added CHO hypothermic culture of 53 mg/L of spike protein after purification. Overall, the new CHO line and additives resulted in >5x productivity gains compared with the traditional HEK production approach.

Using the high purity (>95%) spike protein, the serological tests were validated. A panel of 234 negative samples and 26 positive samples were initially tested. The results reported an overall specificity of 100% and sensitivity of 92.3%. This test was further verified to be robust following additional inter-assay and cross-reactivity tests. The materials were finally rolled out for local assays tests of hospital employees. As of July 31, 2020, the researchers noted that the test had been run on approximately 7200 individuals with 16% positive COVID-19 antibody detection.

This study detailed how the previously reported results of 1-2 mg/L of the spike protein cultured from HEK cells could be amplified to over 50 mg/L with an engineered CHO cell line and optimized hypothermic process conditions. Therefore, over 175,000 assays could be developed from each run using the method described here. Although frequent community testing is only one piece in the scientic arsenal to combat the COVID-19 pandemic, it is an invaluable one. This improved production method brings us one step closer to slowing the spread and ending the pandemic. While 908D does not focus in the COVID arena, our products are and can be used for therapeutic mAb cocktails for COVID and for bioreactor optimization through monitoring of the extracellular environment around the CHO or HEK cells.

By, Glenn A. Harris