Trimeric SARS-CoV-2 Spike Proteins Produced from CHO Cells in Bioreactors Are High-Quality Antigens

: The spike protein of SARS-CoV-2 is essential for virus entry into human cells and is also 26 the main antigenic determinant of the virus, being therefor essential to induce and detect antibodies. As a consequence, the global demand for spike proteins has rapidly increased and could exceed 28 hundreds of grams to kilograms annually. Coronavirus spikes are large, heavily glycosylated, homo- trimeric complexes, with inherent instability. Their poor manufacturability now threatens the availability of these proteins for large scale manufacture of vaccines and diagnostic tests. Here, we outline a scalable, GMP-ready, chemically defined process for production of a stabilized form of the trimeric spike protein. The process is chemically defined and based on a clonal, suspension-CHO cell line and purification of the protein via a two-step, scalable down-stream process. The trimeric conformation was confirmed using Cryo-EM and HPLC analysis. Binding to SARS-CoV-2 host cells was shown using a virus-inhibition assay. The diagnostic sensitivity and specificity for detection of serum SARS-CoV-2 specific IgG1 was investigated and found to exceed that of spike fragments (S1 37 and RBD).

meet the unprecedented global demand for ingredients for these. We believe that the SARS-CoV-2 48 trimeric Spike complex could be a useful ingredient for vaccine and diagnostic applications. efficacy of these vaccines is hoped or predicted to be between 50 and 70%. To boost the immune 52 response, it is likely that follow-up vaccinations will be required. An adjuvated subunit vaccine based 53 on the stabilized trimeric spike protein would be ideal for this purpose. Subunit vaccines can be 54 produced in a cost-effective way and after lyophilization they can be transported and stored at 55 ambient temperature. Also, whether any vaccination approach has elicited a sufficiently protective 56 immune response against SARS-CoV-2 and its perpetuity needs to be verified. To address this, the 57 quantification of antibody levels against SARS-CoV-2 in serum is the most practical approach. For 58 this reason, there will be a high demand for diagnostic tests for quantification of SARS-CoV-2-specific 59 antibodies. The antigen which provides the best sensitivity and specificity for detection of SARS-60 CoV-2 antibodies is the trimeric form of the spike protein [3].

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The viral spike (S) protein complex is a surface-exposed homo-trimeric structure, that mediates

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Hamster Ovary (CHO) cells. We wished a process that can be used to produce high quality trimeric 73 S protein for diagnostic tests as well as for vaccine applications. We used the fully characterized and 74 CMC-compliant CHOExpress™ cell host, single use equipment, chemically defined media and 75 additives. Also, regulatory requirements from DNA construction to production in bioreactors were 76 strictly followed. The clonally derived cell line and the scalable production process outlined here, 77 should allow manufacture of trimeric S protein in grams and even kilograms, should the demand 78 rise to such a level.

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A bead-based serological assay [15] was used. Briefly, patient sera were collected and reacted 127 with commercially available RBD and monomeric S1 domain protein, as well as S trimer and S RBD.

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Relative optical readings were taken for each protein specific assay using the control sera and the

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HEK293-produced monomeric S1 protein was used as positive control. We used the test to analyze a domain; ROC, receiver operator characteristic; S1, spike protein subunit 1; S, full-length spike protein.

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Within the control and within the COVID-19 sample sets, for RBD, S1 monomer and trimeric S 231 no significant differences were found (P > 0.05) in average signal-intensity (Figure 5 a). The mean 232 values for control and COVID samples however showed a near 100 fold difference, while the 233 individual data for reactivity had a large range, as has been reported by other groups as well. With S trimer compared to HEK293-produced S1 monomer and CHO-produced RBD (Figure 4 b)

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Using an optimized CHO expression system and corresponding scalable production approach 241 with chemically defined ingredients from transfection to purification, near wildtype trimeric SARS

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CoV-2 spike protein preparations could be provided in sufficient quantities. The S trimer was purified 243 to high purity and proved to be efficient in blocking virus infectivity in an in-vitro model and highly 244 specific and sensitive towards sera of COVID-19 patients.