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.
Polystyrene (PS) is one of the major plastics contributing to environmental pollution with its durability and resistance to natural biodegradation. Recent research showed that mealworms (Tenebrio molitor) and superworms (Zophobas morio) are naturally able to consume PS as a carbon food source and degrade them without observable toxic effects. In this study, we explored the effects of possible food additives and use of worm frass as potential plant fertilizers. We found that small amounts of sucrose and bran increased PS consumption and that the worm frass alone could support dragon fruit cacti (Hylocereus undatus) growth, with superworm frass in particular, supporting better growth and rooting than mealworm frass and control media over a fortnight. As known fish and poultry feed, these findings present worms as a natural solution to simultaneously tackle both the global plastic problem and urban farming issue in a zero-waste sustainable bioremediation cycle.
Many molecular biology applications require fast plasmid DNA extraction, spurring multiple studies on how to speed up the process. It is regularly instructed in standard laboratory protocols to plate out frozen glycerol bacterial stocks prior to bacteria incubation in liquid media and subsequent plasmid extraction, although the rationale for this is often unexplained (other than for the isolation of single colonies). Given the commonality and importance of this laboratory operation, such a practice is time-consuming and laborious. To study the impact of this practice and the alternative direct culturing method, we investigated the association between bacterial cell mass and its potential influence on plasmid yields from the 2 methods. Our results showed no difference with preplating for 7 out of 8 plasmid constructs used in the study, suggesting that direct glycerol recovery would not lead to poorer plasmid yields. The findings support the rationale for direct glycerol recovery for plasmid extraction, without the need of an intermediate preplating step.
Polystyrene or PS is one of the plastics contributing to environmental pollution with its durability and resistance to biodegradation. Recent research has found that mealworms (Tenebrio molitor) and superworms (Zophobas morio) are able to utilize polystyrene as a carbon food source and degrade them without toxic effects. In this study, we studied food additives to augment the plastic consumption and found that small additions of sucrose and bran were able to encourage PS consumption. To close the plastic carbon cycle, we also evaluated the use of the worm frass for dragon-fruit cacti (Hylocereus undatus) growth and found that superworm frass supported rooting and growth better than mealworm frass and control media over a fortnight. From the results, superworms are shown to be a suitable natural solution to the PS plastic problem that can also support plant growth towards a zero-waste sustainable bioremediation cycle.
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