key: cord-0978421-tyybg6u0
authors: Stuible, Matthew; Gervais, Christian; Lord-Dufour, Simon; Perret, Sylvie; L’Abbé, Denis; Schrag, Joseph; St-Laurent, Gilles; Durocher, Yves
title: Rapid, high-yield production of full-length SARS-CoV-2 spike ectodomain by transient gene expression in CHO cells
date: 2020-12-07
journal: J Biotechnol
DOI: 10.1016/j.jbiotec.2020.12.005
sha: 496bf3deb5aa9a799b82c521bf9d6074518945f7
doc_id: 978421
cord_uid: tyybg6u0

Recombinant forms of the spike protein of SARS-CoV-2 and related viruses have proven difficult to produce with good yields in mammalian cells. Given the panoply of potential COVID-19 diagnostic tools and therapeutic candidates that require purified spike protein and its importance for ongoing SARS-CoV-2 research, we have explored new approaches for spike production and purification. Three transient gene expression methods based on PEI-mediated transfection of CHO or HEK293 cells in suspension culture in chemically-defined media were compared for rapid production of full-length SARS-CoV-2 spike ectodomain. A high-cell-density protocol using DXB11-derived CHO(BRI/55E1) cells gave substantially better yields than the other methods. Different forms of the spike ectodomain were expressed, including the wild-type SARS-CoV-2 sequence and a mutated form (to favor expression of the full-length spike ectodomain stabilized in pre-fusion conformation), with and without fusion to putative trimerization domains. An efficient two-step affinity purification method was also developed. Ultimately, we have been able to produce highly homogenous preparations of full-length spike, both monomeric and trimeric, with yields of 100-150 mg/L in the harvested medium. The speed and productivity of this method support further development of CHO-based approaches for recombinant spike protein manufacturing.

gene expression methods based on PEI-mediated transfection of CHO or HEK293 cells in suspension culture in chemically-defined media were compared for rapid production of full-length SARS-CoV-2 spike ectodomain. A high-cell-density protocol using DXB11-derived CHO BRI/55E1 cells gave substantially better yields than the other methods. Different forms of the spike ectodomain were expressed, including the wild-type SARS-CoV-2 sequence and a mutated form (to favor expression of the full-length spike ectodomain stabilized in pre-fusion conformation), with and without fusion to putative trimerization domains. An efficient two-step affinity purification method was also developed. Ultimately, we have been able to produce highly homogenous preparations of full-length spike, both monomeric and trimeric, with yields of 100-150 mg/L in the harvested medium. The speed and productivity of this method support further development of CHO-based approaches for recombinant spike protein manufacturing.

The availability of recombinant forms of the SARS-CoV-2 spike protein is becoming increasingly important for development of novel strategies to combat the current COVID-19 pandemic. In the human immune response to the SARS coronavirus, the spike protein is a key antigen and is often the target of neutralizing antibodies found in convalescent patients [1] . Recombinant spike protein has a wide range of potential applications, including in the development of novel therapeutics (e.g. neutralizing antibodies or other spike-targeting biologics), clinical diagnostic tools (e.g. to evaluate post-infection immunity) or subunit vaccines (different forms of recombinant SARS spike protein were effective as vaccines in animal models) [2, 3] .

The SARS-CoV-2 spike protein is closely related to that of SARS-CoV-1, the virus responsible for the 2003 SARS outbreak. Both proteins are large, multi-domain glycoproteins with transmembrane domains that J o u r n a l P r e -p r o o f traverse the viral envelope and are proteolytically processed into S1 and S2 subunits. Notably, while the SARS-CoV-1 spike is only cleaved during infection of target cells, the SARS-CoV-2 spike contains a conserved furin recognition site at the S1/S2 junction, such that significant cleavage occurs during biosynthesis in host cells; this difference may impact the route of entry of the two virus types into host cells [4] . The SARS-CoV-1 spike protein was shown to assemble into homo-trimeric complexes that are found on mature viral particles [5] . When expressed in the absence of its transmembrane and Cterminal domains, the spike ectodomain (ECD) is reported to be mostly monomeric; fusion of the spike ECD C-terminus to the trimerization domain of bacteriophage T4 fibritin (T4-Fib or foldon) favors its trimerization and increases its capacity to elicit neutralizing antibodies [6] .

In the literature on SARS-CoV-1 and other related coronaviruses, there are reports of various approaches for producing recombinant spike proteins. Individual domains of the spike, including the receptor-binding and hemagglutinin-esterase domains, have been produced in CHO, HEK293, Vero and insect cells [6] [7] [8] . It is also possible to express the full-length spike, including transmembrane and Cterminal domains, which can be purified following membrane solubilisation of expressing cells [9, 10] .

Finally, expression of the full-length soluble forms of the coronavirus spike ECDs has also been reported in HEK293 and insect cells [6, [11] [12] [13] [14] [15] . Importantly, however, in cases where this data is reported, yields were extremely low, ranging from 0.5-1.5 mg per litre of culture media (for expression of constructs containing the full-length ECD) [9, 14] . More recently, for the SARS-CoV-2 spike, several production and purification parameters were optimized to obtain yields up to 5 mg/L from transiently transfected HEK293 cells [16] . In addition, the tendency of the SARS-CoV-2 trimeric spike to disassemble during storage at 4°C has motivated the development of disulfide-stabilized thermostable variants, but at the cost of a 3-fold drop in volumetric yield (~400 μg/L) in Expi293 cells [17] . Higher titers have been achieved using a highly engineered, stabilized trimeric spike ECD variant, reaching up to 33 mg/L by transient gene expression (TGE) in CHO [18] . Another recent paper used optimized processes and J o u r n a l P r e -p r o o f expression vectors to obtain yields of trimeric spike ECD of up to 10 and 6 mg/L by TGE in 293 and CHO, respectively [19] . Using methionine sulfoximine (MSX) selection to establish a stable CHO pool, the same authors reported a further increase in titers of up to 53 mg/L post-IMAC purification. While significant improvements have recently been achieved, these productivities are well below desirable levels for mass production, in particular for development and manufacturing for potential diagnostic or vaccine applications at commercial-scale.

Recently, we have attempted to produce the full-length SARS-CoV-2 spike ECD using three rapid protein production platforms established previously in our group, based on polyethylenimine (PEI)-mediated plasmid transfection of CHO or HEK293 cells. Yields of soluble ECD from the two CHO-based methods were substantially higher than other reported methods, with one method yielding approximately 100-150 mg/L (depending on the form of the spike expressed) at 6-7 days post-transfection. Both the wildtype spike and a stabilized form with mutated furin cleavage site [14] were produced and purified; both forms were expressed with and without fusion to various putative trimerization domains. The CHOsecreted wild-type protein is a mix of unprocessed precursor and cleaved forms, likely including free S1, S2 and S1/S2 complexes. The stabilized protein (with furin site mutated) with or without trimerization domain fusion is secreted in a full-length, unprocessed form. Notably, for the wild-type (non-stabilized with furin site present) spike ECD constructs, we have observed dissociation of S1 from the monomeric and trimeric S1/S2 complexes during purification and storage, indicating that the furin site-deleted mutant is a better choice for ongoing production of the full-length ECD. The yield and purity we have obtained support continued method development to further improve titers and downstream processes for eventual mass production in CHO cells.

J o u r n a l P r e -p r o o f SARS-CoV-2 spike protein sequences were derived from Genbank accession number MN908947.

Protein-coding DNA sequences were codon-optimized for CHO cells, synthesized and cloned into the pTT5™ vector [20] by Genscript. The specific regions of the spike ECD contained in each construct and the sequences and locations of the stabilizing mutations in ECDm are indicated in Figure 1 . Trimerization domains were fused to the C-termini of the spike protein coding sequences followed by FLAG and 6xHis tags. Two constructs (pTT5-ECD-T4Fib and pTT5-ECDm-T4Fib) contain additional dual StrepTag-II tags between the FLAG and 6xHis tags. Amino acid sequence of the various constructs are shown in Supplementary Table 1.

Transfection and post-transfection culture of CHO-3E7 and 293-6E cells in FreeStyle F17 media were performed as described previously [21, 22] .

The method for transient expression in CHO BRI/55E1 cells [23] is based on a published method for highdensity transfection of CHO-3E7 cells [21] , with several modifications. Cells were maintained in shake flasks in a humidified, 5% CO 2 , 37°C incubator, rotating at 120 rpm in a chemically-defined proprietary media formulation. Cells were seeded in the same media at 1x10 6 cells/ml 2 days prior to transfection to achieve a cell density of ~8x10 6 /ml at the time of transfection. Before transfection, cells were diluted with 25% fresh media and dimethylacetamide was added to 0.083% (v/v). Transfections were performed by adding PEI-DNA polyplexes (10% final culture volume) to diluted cells (90% final culture volume). To prepare polyplexes, PEI-Max (Polysciences) and plasmid DNA were diluted separately to 200 µg/ml and 28 µg/ml, respectively, in a volume of growth media equal to 5% of the final culture volume. Plasmid DNA consisted of 85% of the different pTT5™-spike constructs, 10% pTT™-Bcl-XL (anti-apoptotic effector) and 5% pTT™-GFP. The diluted PEI-Max was added to the diluted DNA and incubated for 7 minutes at room temperature before adding to cells. At 24h post-transfection, cultures were shifted to J o u r n a l P r e -p r o o f 32°C and supplemented with Anti-Clumping Supplement (1:500 dilution) and Feed 4 (2.5% v/v), both from Irvine Scientific. Additional Feed 4 (5%) was added at 5 days post-transfection. Glucose concentrations were monitored every 2-3 days to maintain >10 mM. Cell supernatants were harvested at 6-7 days post-transfection (except for time-course experiments shown in Figure 3 and Supplementary   Figure 1 ). SDS-PAGE was performed using NuPAGE 4-12% Bis-Tris gels (Invitrogen). Protein samples were heatdenatured at 70°C for 10 minutes under reducing conditions prior to separation by SDS-PAGE. Total protein staining (Coomassie Blue) and western blotting were performed using standard methods. Anti-Penta-His Alexa Fluor 488 conjugate was from Qiagen.

All protein constructs were initially purified by IMAC on Nickel Sepharose Excel resin (GE Healthcare). 0.2 µm-filtered supernatants were applied to columns by gravity flow. The column volume used for a given volume of supernatant was variable for different productions; for maximum recovery of ECD constructs from cell supernatant, a packed resin volume of 1 ml per 0.5 mg of protein is optimal.

Columns were washed with 50 mM sodium phosphate, pH 7.0, containing 300 mM NaCl and 25 mM imidazole and eluted with the same buffer containing 300 mM imidazole.

In order to achieve near homogeneity, a second step purification was required. For most constructs, ANTI-FLAG M2 affinity gel (Sigma Aldrich) was used in batch mode. IMAC-purified proteins were incubated with the gel for  2 hours with gentle overhead mixing. One ml of gel was used for every 0. Concentrations of purified proteins in DPBS were determined by spectrophotometry (A 280 ) using extinction coefficients calculated based on their amino acid composition.

UPLC-SEC-MALS analyses were conducted on a 4.6 x 150 mm BEH200 SEC column with 1.7 µm particles (Waters, Milford, MA) connected to an Acquity H-Class Bio UPLC system (Waters) with a miniDAWN™ MALS detector and Optilab ® T-rEX™ refractometer (Wyatt Technology, Santa Barbara, CA, USA). The column temperature was 30°C and the mobile phase was DPBS (HyClone SH30028.01) with 0.02% Tween 20 added. The flow rate was 0.4 mL/min. Weighted average molecular mass (M MALS ) was calculated in ASTRA 6.1 software (Wyatt) using a protein concentration determined from the refractive index signal with a dn/dc value of 0.185.

For protein expression, we used plasmid vectors containing CHO codon-optimized sequences encoding the full ECD of the SARS-CoV-2 spike protein. C-terminal affinity tags were added for purification. Two versions of the protein were expressed: one encoding an unmodified ECD protein sequence (ECD) and a second with two modifications, to block furin-mediated S1/S2 cleavage and to stabilize the pre-fusion J o u r n a l P r e -p r o o f conformation (ECDm), as recently described [14] . To attempt to mimic the native trimeric structure of the spike protein, as found on the virus surface, trimerization domains of T4-Fibritin (T4-Fib) [6] , human resistin (RSTN) [24] , or GCN4 [25] were fused to the C-terminus of the ECD in certain constructs. A fourth, proprietary trimerization sequence (T3) was also tested.

We rapidly assessed the potential of three expression platforms for production of SARS-CoV-2 spike: two methods, based on EBNA1-expressing CHO (CHO-3E7) [21] and HEK293 (293-6E) [22] have served as core platforms for recombinant protein production in our group for several years, and generally perform can improve yields [22] . As shown in Figure 2A , western blotting of bulk supernatants with an antipenta-His antibody shows two clear bands in the CHO-3E7 supernatants transfected with 25-85% coding DNA, but little signal in the 293-6E supernatants transfected with 1-95% coding DNA. Two bands are detected in the anti-penta-His blots, at ~80 kDa and ~180 kDa, which likely correspond to the S2 subunit (after cleavage at the S1/S2 site) and the unprocessed precursor, respectively. These two proteins can J o u r n a l P r e -p r o o f be observed as faint bands by Ponceau S staining of membranes prior to western blotting. Based on amino acid sequence, the predicted molecular weight of the unprocessed spike is 143 kDa. The higher observed molecular weight is very likely due to glycosylation: the SARS-CoV-2 spike protein contains 22 confirmed N-linked glycosylation sites [26] . In addition, for the SARS-CoV-1 spike, enzymatic removal of glycans decreases apparent molecular weight by SDS-PAGE by 30-40 kDa [27] . Similar expression tests were performed for the stabilized spike protein (ECDm-RSTN) with the CHO-3E7 and CHO BRI/55E1 platforms, using a range of coding DNA ratios. As shown in Figure 2B , an anti-penta-His western blot using unpurified supernatants gave a single band at ~180 kDa, which corresponds to the size of the unprocessed precursor protein observed with expression of the non-stabilized spike ( Figure   2A ). The CHO BRI/55E1 method gives substantially higher yields, based on band intensity on the anti-penta-His blot and Ponceau S stained membrane. For both cell lines, there was little difference in expression between 25 and 85% of coding DNA.

To estimate the concentration of spike protein in the culture supernatants, we performed western blots with unpurified supernatants from HEK293, CHO-3E7 and CHO BRI/55E1 cells transfected with ECDm-RSTN along with a series of dilutions of highly purified ECDm protein ( Figure 3 ). Samples were taken up to 7 days post-transfection for CHO BRI/55E1 cells and up to 5 days post-transfection for CHO-3E7 and HEK293 cells (CHO-3E7 and HEK293 cell viability dropped to <70% on Day 5). As shown in Figure 3B , densitometry analysis of this blot allows us to estimate that yields in the transfected CHO BRI/55E1 supernatants reach >150 µg/ml at 7 days post-transfection. As expected, concentrations in CHO-3E7 supernatants were lower (~25 µg/ml at Day 5) while they were nearly undetectable in 293-6E supernatants. A similar comparison was performed for the ECDm protein without trimerization domain in CHO BRI/55E1 and 293-6E cells (Supplementary Figure 1) ; in this case CHO BRI/55E1 yields were ~100 µg/ml and 293-6E yields were ~20 µg/ml.

The differences in yields from the three cell lines were reproducible and were not due to differences transfection efficiency: for all transfections, we either co-transfected 5% of a GFP expression vector or included a GFP-only transfected external control, and we monitored GFP expression at 24-48 hours posttransfection (data not shown). Also, we did not observe any obvious negative effect of spike protein expression on cell growth or viability.

The differences in the amounts of spike protein secreted by the three cell lines are not reflected by intracellular spike protein levels. As shown in Figure 3A and Supplementary Figure 1C , recombinant spike protein is easily detectable in total protein extracts from all three cell lines by western blotting.

Intracellular protein levels are slightly lower in CHO-3E7 and 293-6E cells compared to CHO BRI/55E1 in Figure 3A , but certainly, these differences do not reflect the substantial differences in concentrations of secreted protein. These results confirm that all three cell lines were transfected and appear to be able to produce intracellular spike protein at similar levels; however, an explanation for the low level of protein secretion, especially in 293-6E cells, is still elusive.

Based on these results, we decided to pursue the CHO BRI/55E1 TGE platform for ECD expression, and we proceeded to develop a purification method for supernatants from these cells. Bulk supernatants were first passed on an IMAC (nickel-Sepharose excel) column; as shown in Figure 4 for the ECD-T3 construct, the spike protein is prominent in the eluate from this column; as expected, three forms of the protein are observed: full-length S1/S2 as well as the processed S1 and S2 subunits. Several other non-specific proteins are also present. Following a second-step purification of this eluate on an anti-FLAG column, these contaminating proteins were effectively removed. Importantly, we observed a significant amount of S1 subunit in the flow-through and washes from the anti-FLAG column for the ECD-T3 construct as well as all other constructs with intact protease cleavage sites. We believe that this represents slow dissociation of S1 from S2 (the FLAG/His tag is fused to S2); S1 does not immediately dissociate from S2 after S1/S2 processing, but it is evident over time with multiple steps of purification/storage. Certainly, J o u r n a l P r e -p r o o f this result supports the use of the mutated form of the spike ECD (ECDm), lacking the S1/S2 cleavage site, for production of full-length recombinant spike protein.

Our purification method has evolved with our growing experience purifying different spike ECD constructs. In particular, in our initial IMAC purification runs, a large proportion (~50%) of the protein was found in the column flow-through. The standard binding capacity for the Ni-Sepharose excel IMAC column is >10 mg/ml, but after considerable effort troubleshooting this step, we have concluded that the capacity is much lower for the spike ECD proteins (~0.5 mg/ml). We do not believe that this low binding capacity is due to interfering substances or low pH, because supernatant buffer exchange by tangential flow filtration prior to purification did not improve binding (data not shown); rather, we suspect the size and/or shape of the spike constructs may prevent diffusion into the pores of the Ni-Sepharose excel resin. With an appropriate amount of supernatant loaded on the IMAC column, we are now able to capture the expected amount of ECD protein based on its abundance in the bulk supernatant.

Several different SARS-CoV-2 spike ECD constructs were purified in this manner, including full-length, S1, and S2 forms of the wild-type spike (ECD, S1 and S2), the stabilized full-length spike (ECDm) and ECD and ECDm fused to the different trimerization domains. The constructs with the T4-Fib trimerization domain contain an additional Twin-Strep-Tag® between the FLAG and 6xHis sequences and were purified by IMAC followed by a Streptrap™ HP column. As shown in Figure 5 , the purified proteins were analyzed by UPLC-SEC ( Figure 4A and 4B) with a MALS detector for molecular weight determination ( Figure 5C ), as well as by SDS-PAGE/Coomassie staining ( Figure 5D ). As expected, for all of the non-mutated ECD constructs (with and without trimerization domains), the purified protein consists of the S2 subunit with lower levels of S1 and uncleaved full-length protein. By SDS-PAGE, the sizes of S1 and S2 that result from processing of the full-length ECD match well the sizes of S1 and S2 when expressed individually. By UPLC-SEC, purified ECD, S1 and S2 show single predominant peaks of UV absorbance, with lower-J o u r n a l P r e -p r o o f abundance, fast-eluting peaks which are likely multimers or aggregates (or uncleaved full-length protein, in the case of ECD). Notably, the MALS molecular weight estimate for the principal peak in the ECD preparation is 75 kDa, which matches the MALS estimate for S2 expressed by itself, supporting the assertion that after purification, the ECD preparation is composed mostly of S2. The mutated ECD (ECDm) is estimated to have a molecular weight of 174 kDa, which is approximately equal to that of the sum of S1 and S2 expressed alone.

Purified constructs containing trimerization domains were also analyzed. By SDS-PAGE ( Figure 5D , lower panel), they are quite similar, although the amount of S1 lost during purification of the ECD constructs is variable. The mutated (ECDm) trimerization domain fusions all appear to be highly pure by SDS-PAGE.

Notably, for the UPLC-SEC analysis, due to varying degrees of loss of S1 from the trimeric ECD constructs, the UPLC-SEC profiles and MALS data are very difficult to interpret (data not shown). For the mutated constructs, the RSTN and T3 fusions give highly uniform purified preparations by UPLC-SEC that are estimated by MALS to have molecular weights of 503 and 517 kDa, respectively. This is consistent with the expected molecular weight of a trimer of ECDm (174 kDa). For the T4-Fib and GCN4 fusions, the principal peaks by UPLC have similar retention times as the other trimerization domains; however, the presence of shoulders on the main peak indicates that it could be composed of more than one species, and therefore, the MALS molecular weight estimates for the trimeric protein are likely inaccurate.

Notably, T4-Fib is the most commonly used trimerization domain used in the literature for coronavirus spike proteins. Together, these results suggest that RSTN and T3 fusions could be better for preparing uniform preparations of SARS-CoV-2 spike ECD with trimeric stoichiometry. Very recently, an ELISA based assay using ECDm-RSTN was developed for sensitive detection of anti-spike antibodies in COVID-19 patients [28] , as has already been reported for the T4-Fib fusion [29] . Additional analyses will be required to evaluate how well the different trimerization domain fusions mimic the conformation of the transmembrane spike protein in its native state.

In summary, we have developed a method for transient production in CHO cells of full-length SARS-CoV-2 spike ECD, yielding 100-150 µg/ml within 7 days of plasmid transfection. This is significantly higher than yields reported previously for production of recombinant coronavirus spike proteins. It is important to note that our estimates of spike ECD yields are based on concentrations in harvested supernatants evaluated by western blotting/densitometry while other have reported yields of purified protein (eg. [19] ), so our values are not directly comparable. Our results demonstrate that full-length recombinant spike protein is not inherently difficult to produce at high levels in mammalian cells, but that productivity is greatly dependent on host cell line selection: consistent with literature reports, we obtained low yields from transfected HEK293 cells. Between the two CHO host cell lines that were tested, CHO BRI/55E1 performed better than CHO-3E7; we cannot conclude whether this is due to differences in protocols (transfection cell density, media and feed are different), cell background (CHO BRI/55E1 and CHO-3E7 are derived from different parental cells) or other factors.

The mutated construct (ECDm) is likely the best starting point for ongoing development of recombinant protein for therapeutic or diagnostic applications: it can be readily expressed in CHO cells and can be purified to near homogeneity, as assessed by SEC and SDS-PAGE. To mimic the conformation of the spike protein on the virus surface, the RSTN and T3 fusions were effective at promoting uniform, trimeric stoichiometry. It is notable that the stabilized trimeric spike was recently shown as the most potent spike construct for generating neutralizing antibodies in mice [30] .

The challenge of manufacturing sufficient amounts of spike protein is likely an important reason why alternative methods, such as mRNA-, adenovirus-, and AAV-based approaches to induce spike protein expression in vaccinated individuals, have gained prominence with COVID-19 vaccine developers.

However, production of vaccine antigen in the form of recombinant spike protein in CHO cells has an intrinsic advantage over these newer technologies: the extensive, decades-long safety record of CHO- Based on past experience with other recombinant proteins, we expected that TGE yields could be further improved in stably transfected CHO pools or clones [23] ; indeed, more recently, we have generated CHO pools that can reach yields >800 mg/L for ECDm-RSTN (manuscript in preparation).

Nonetheless, the speed at which we were able to generate substantial amounts of spike protein by CHO TGE highlights how this approach could complement other methods, in particular for rapid response to health emergencies. As detailed previously [31] , we believe that development of alternatives to clonebased CHO production methods, including stable pools and TGE, should be prioritized as potential novel methods for rapid, large-scale manufacturing of biologics. 

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 

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A and B) Proteins purified by IMAC and anti-FLAG (or IMAC and StrepTrap for T4-Fib constructs) were separated by UPLC on a BEH200 SEC column. Protein elution was monitored by measuring UV absorbance of eluate (vertical axis). The position of the FLAG peptide elution peak is marked with an asterisk. (C) The molecular weight of the major elution peak for each sample was estimated by MALS. Values marked with asterisks are likely inaccurate due to peak shouldering

We thank the Quality Attributes Characterization team at the National Research Council of Canada for performing UPLC-SEC/MALS analyses. This is NRC publication #53486.J o u r n a l P r e -p r o o f