Enhancing Biotherapeutic Protein yield through CHO Expression Optimisation

In this case study, Emma Cains and Anna Bennett conducted a series of optimisation experiments that led to a ten-fold yield increase of a Biotherapeutic protein.

Background

Peak Proteins offers CHO, (CHO-3E7) HEK293-6E and HEK Expi293 expression systems for transient mammalian protein production. The target protein is a biotherapeutic in the early stages of development and as such CHO cells were chosen for development. Through initial feasibility studies the protein was successfully produced but yields were considered inadequate, therefore a series of experiments were performed to optimise expression.

Experiment 1: DNA level and signal sequence optimisation

DNA level – It is common practise to transfect mammalian cells with carrier (or stuffer) DNA alongside the expression vector. A large amount of DNA is required for efficient transfection. The use of carrier DNA results in a reduction in the amount of vector used DNA in the transfection, meaning expression can be reduced to more physiological levels by decreasing the amount of the transgene vector to a few percent of the total DNA. Paradoxically, this can have positive effects on yields, particularly in low expressing proteins [1]. To assess whether this could be beneficial for this protein, 1%, 5%, 25% and 85% (% of coding DNA to carrier DNA) were tested.

Signal sequence – for secreted proteins, different signal sequences can also affect the level of protein expression.  In this experiment three signal sequences were tested: the native signal and two common signal sequences known to give high expression in CHO cells, IgG Kappa light chain (IgK) and Azurocidin (Azu).

These conditions were tested combinatorially, giving 12 conditions in total. For each condition, the protein was purified from culture supernatant harvested after 6 days using a single nickel affinity purification and the samples evaluated by SDS-PAGE (Fig 1).

Figure 1: SDS-PAGE gel of signal sequence and DNA level optimisation. Comparison of protein production from 250 ml CHO cultures using different signal sequences (AZU: Azurocidin, IgK and Native) and different DNA %. Samples shown were elution pools from a nickel affinity chromatography step. Arrows indicate bands identified as the correct target protein by mass spectrometry peptide mapping. The protein runs with multiple bands due to different glycosylation species being present.

They were also evaluated by the client using ELISA (Fig 4). The conclusion at this stage was that the signal sequence did not make a huge difference to the yield, but the optimum DNA proportion was seen to be 25% across all conditions.

Experiment 2: Medium and Culture Method Comparison

Our standard method uses Freestyle F17 medium, however for other protein targets we have previously increased protein expression using versions of the high-density cell expression method in BalanCD Transfectory CHO (BCDT) medium [2].

Standard conditions included the optimised 25% DNA level from experiment 1 was use in for transfection along with the with the IgK conatinin constructThese were used to compare F17 medium (control) with BCDT mediumwith 3 different feeding strategies in this experiment. Protein was purified as per the previous experiment (Fig 2).

Figure 2: SDS-PAGE gel of CHO media and feeding method optimisation. Comparison of protein production from 250ml CHO cultures using different media types; Freestyle F17 and BalanCD Transfectory medium. Three feeding regimes for the BCDT method were compared and protein was purified by a single nickel affinity chromatography step (each column had 3 elution fractions E1 – 3 collected). Arrows indicate bands identified as the correct protein by mass spectrometry peptide mapping. The protein runs with multiple bands due to different glycosylation species being present.

 The BCDT method gave increased expression over the F17 method for all feed regimes tested.  From this work feed regime 3 was selected.

Experiment 3: Consolidation and harvest time optimisation

The selected conditions from experiments 1 and 2 (25% DNA, IgK signal sequence and BCDT method, feed regime 3) were repeated to confirm the expression results and at this stage we investigated harvesting at a later date. Previous work with another protein has shown expression levels increasing to day 11, this is likely protein specific. In this experiment we compared harvesting at 8 and 11 days as before (Fig 3).

Figure 3: SDS-PAGE gel of harvest day optimisation. Comparison of protein production from 500 ml CHO cultures using 25% DNA, IgK signal sequence and BCDT feeding regime 3 harvested on day 8 and day 11. Protein was purified by a single nickel affinity chromatography step. Arrows indicate bands identified as the correct protein by mass spectrometry peptide mapping.

Day 11 harvest showed higher protein expression, suggesting that expression continues until at least day 11.  The protein sample isolated at day 11 did expectedly contain more contaminants, whichhas been witnessed previously in long cultures.  Since yield was the priority for this particular client, day 11 harvest was selected.

The progress of these experiments can be seen in Fig 4.

Figure 4: Protein quantification by ELISA assay. Protein samples collected from each optimisation stage were quantified using an ELISA assay. Data shows total μg from a 250 mL culture from signal sequence and DNA % optimisation feasibility (experiment 1) and follow up harvest time comparison when using BCDT feeding regime 3 (experiment 3).

Scale Up

Utilising the original conditions resulted in protein yields of 0.5 mg/L.  Using these optimised conditions, a 22 L final volume wavebag grow was undertaken  using the Sartorius Biostat RM bioreactor system. The scale up showed protein purity to be comparable to small scale expressions by SDS-PAGE and a final yield of 105 mg was obtained.(as determined by ELISA) This equated to a much improved 4.8 mg/L, approximately a 10 fold increase.

Conclusion

This biotherapeutic protein of interest was low expressing and through optimisation of DNA level for transfection, growth media and conditions, and signal sequences here at Peak Proteins, we successfully produced 105 mg from a single 22 L wavebag grow, a purified yield of 4.8 mg/L.  This represents close to a 10-fold yield improvement in yield, to the satisfaction of the client.