Augmenting recombinant antibody production in HEK293E cells: optimizing transfection and culture parameters

Abstract

Background Optimising recombinant antibody production is important for cost-effective therapeutics and diagnostics. With impact on commercialisation, higher productivity beyond laboratory scales is highly sought, where efficient production can also accelerate antibody characterisations and investigations. Methods Investigating HEK293E cells for mammalian antibody production, various transfection and culture parameters were systematically analysed for antibody light chain production before evaluating them for whole antibody production. Transfection parameters investigated include seeding cell density, the concentration of the transfection reagent and DNA, complexation time, temperature, and volume, as well as culture parameters such as medium replacement, serum deprivation, use of cell maintenance antibiotic, incubation temperature, medium volume, post-transfection harvest day and common nutrient supplements. Results Using 2 mL adherent HEK293E cell culture transfections with 25 kDa linear Polyethylenimine in the most optimised parameters, we demonstrated a ~ 2-fold production increase for light chain alone and for whole antibody production reaching 536 and 49 μg respectively in a cost-effective manner. With the addition of peptone, κ light chain increased by ~ 4-fold to 1032 μg while whole antibody increased to a lesser extent by ~ 2.5-fold to 51 μg, with benefits potentially for antibodies limited by their light chains in production. Conclusions Our optimised findings show promise for a more efficient and convenient antibody production method through transfection and culture optimisations that can be incorporated to scale up processes and with potential transferability to other mammalian-based recombinant protein production using HEK293E cells. Statement of Significance Recombinant antibody production is crucial for antibody research and development. Systematically investigating transfection and culture parameters such as PEI/DNA concentrations, complexation time, volume, and temperature, supplements, etc., we demonstrated a ~ 4-fold light chain alone production increase to 1032 μg and a 2.5-fold whole antibody production increase to 51 μg from 2 mL transfections.

Variable-heavy (VH) families influencing IgA1&2 engagement to the antigen, FcαRI and superantigen proteins G, A, and L

“…Transfection, production, and purification and were performed as described previously 49 , 57 , 80 .…”

More Than Meets the Kappa for Antibody Superantigen Protein L (PpL)

“…The plasmids were transformed into competent E. coli (DH5α) strains [ 25 ] followed by plasmid extraction (Biobasic Pte Ltd, Singapore, Singapore, Cat: BS614). Transfection, production, and purification were done using HEK293E with homemade transfection agent based on linear PEI [ 26 ] (PolyScience, Singpaore, Singapore, Cat: 23966-1), followed by 14 days post-transfection supernatant harvest that were subjected to AKTA Pure system equipped with Protein G column for affinity purification and size exclusion column for collection of monomeric fractions as characterised previously [ 21 , 22 ].…”

The synergistic effects of the VH families and CH regions of multimeric IgM on its interaction with FcμR, and antigen

“…The production supernatant was harvested af-ter 14 days post-transfection and purified using AKTA equipped with a PpL affinity col-umn (Cat: 17547815, Cytiva). as previously optimized [20] and [38]. The purified recom-binant IgMs were then subjected to HiLoad Superdex 200 pg preparative SEC columns (Cat: 28989335, Cytiva) for the collection of the spontaneously formed hexamer fractions as previously described work [4] where only the multimeric fraction was collected for analysis.…”

“…With recent evidence showing that the antibody regions can cross-influence one an-other such as in the variable (V-) regions, particularly the framework regions (FWR), can influence the FcR binding sites (and thus FcR binding) at the heavy chain constant regions (CH) for IgA [14,15], IgE [16], and IgG1 [17], a holistic [18] investigation for multimeric IgMs can elucidate the contribution of oligomerization. Considering how the non-antigen interacting regions such as the signal peptide [19] impact antibody production (apart from the obvious VH-VL pairing on production [20]) and how the different regions such as within the V-and C-regions can come together to form amino acid stretches and pockets that can bind non-antigens [21,22], there remains much to investigate at the whole anti-body level and the effects of such oligomerization. With the further complication of antibody superantigens that bind to antibody re-gions [27,28] to modulate antigen and receptor engagement [28], there is a further need to determine how multimeric IgM can be influenced by superantigen binding (particularly Protein L or PpL which binds the Vκ light chain).…”