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

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.

Augmenting Whole Recombinant Antibody Production in HEK293E Cells: Optimizing Transfection and Culture Parameters

Optimizing recombinant antibody production is important for cost-effective therapeutics and diagnostics. With downstream impact on commercialization, higher productivity is highly sought after beyond laboratory scales, where efficient production can also accelerate antibody characterizations and investigations. Using HEK293E cells as the base model for mammalian antibody production, various transfection and culture parameters were systematically analyzed using antibody light chain production before applying them onto 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 media replacement, serum deprivation, media volume, post-transfection harvest, incubation temperature, and common nutrient supplements. Incorporating the most optimized parameters, Pertuzumab κ-chain only and whole recombinant Pertuzumab antibody production were found to increase by 452 % and 252 % respectively, that can be used to guide future cost-effective transient antibody production with the potential for further scaling up.

Tofu processing wastewater as a low-cost substrate for high activity nattokinase production using Bacillus subtilis

Background Even though tofu is a traditional Chinese food loved by Asian people the wastewater generated during the production of tofu can pollute the environment, and the treatment of this generated wastewater can increase the operating cost of the plant. In this study, the production of nattokinase could be achieved by using the nitrogen source in tofu processing wastewater (TPW) instead of using the traditional nattokinase medium. This meets the need for the low-cost fermentation of nattokinase and at the same time addresses the environmental pollution concerns caused by the wastewater. Bacillus subtilis 13,932 is, a high yielding strain of nattokinase, which is stored in our laboratory. To increase the activity of nattokinase in the tofu process wastewater fermentation medium, the medium components and culture parameters were optimized. Nattokinase with high enzymatic activity was obtained in 7 L and 100 L bioreactors when TPW was used as the sole nitrogen source catalyzed by Bacillus subtilis. Such a result demonstrates that the production of nattokinase from TPW fermentation using B. subtilis can be implemented at an industrial level. Results The peptide component in TPW is a crucial factor in the production of nattokinase. Box–Behnken design (BBD) experiments were designed to optimize various critical components, i.e., Glucose, TPW, MgSO4·7H2O, CaCl2, in nattokinase fermentation media. A maximum nattokinase activity was recorded at 37 °C, pH 7.0, 70 mL liquid medium, and 200 rpm. The highest nattokinase activities obtained from 7 to 100 L bioreactors were 8628.35 ± 113.87 IU/mL and 10,661.97 ± 72.47 IU/mL, respectively. Conclusions By replacing the nitrogen source in the original medium with TPW, there was an increase in the enzyme activity by 19.25% after optimizing the medium and culture parameters. According to the scale-up experiment from conical flasks to 100 L bioreactors, there was an increase in the activity of nattokinase by 47.89%.

Heterotypic Tumor Spheroids in Agitation-Based Cultures: A Scaffold-Free Cell Model That Sustains Long-Term Survival of Endothelial Cells

Endothelial cells (ECs) are an important component of the tumor microenvironment, playing key roles in tumor development and progression that span from angiogenesis to immune regulation and drug resistance. Heterotypic tumor spheroids are one of the most widely used in vitro tumor microenvironment models, presenting improved recapitulation of tumor microenvironments compared to 2D cultures, in a simple and low-cost setup. Heterotypic tumor spheroid models incorporating endothelial cells have been proposed but present multiple limitations, such as the short culture duration typically obtained, the use of animal-derived matrices, and poor reproducibility; the diversity of culture conditions employed hinders comparison between studies and standardization of relevant culture parameters. Herein, we developed long-term cultures of triple heterotypic spheroids composed of the HCC1954 tumor cell line, human fibroblasts, and ECs. We explored culture parameters potentially relevant for EC maintenance, such as tumor cell line, seeding cell number, cell ratio, and agitation vs. static culture. In HCC1954-based spheroids, we observed maintenance of viable EC for up to 1 month of culture in agitation, with retention of the identity markers CD31 and von Willebrand factor. At the optimized tumor cell:fibroblast:EC ratio of 1:3:10, HCC1954-based spheroids had a higher EC area/total spheroid area at 1 month of culture than the other cell ratios tested. EC maintenance was tumor cell line-dependent, and in HCC1954-based spheroids it was also dependent on the presence of fibroblasts and agitation. Moreover, vascular endothelial growth factor (VEGF) supplementation was not required for maintenance of EC, as the factor was endogenously produced. ECs co-localized with fibroblasts, which accumulated preferentially in the core of the spheroids and secreted EC-relevant extracellular matrix proteins, such as collagen I and IV. This simple model setup does not rely on artificial or animal-derived scaffolds and can serve as a useful tool to explore the culture parameters influencing heterotypic spheroids, contributing to model standardization, as well as to explore molecular cross talk of ECs within the tumor microenvironment, and potentially its effects on drug response.

PROSPECTION OF FILAMENTOUS FUNGI AND THE PRODUCTION OF AMYLASE BY ASPERGILLUS sp. M1.7.2

Amylases are used in numerous industrial applications for converting starch into products of greater value. This work aimed to prospect filamentous fungi, analyze the morphological and physiological characteristics of the isolates; as well as to select an amylase producing fungus and to optimize the parameters for the cultivation of the microorganism and biochemically characterize the amylase. Among 21 filamentous fungi isolated in Janaúba, state of Minas Gerais, Brazil, the best amylase producer was selected for standardization of culture parameters and subsequent enzymatic characterization. Maximum activity was obtained in CP medium after six days of cultivation at 30 °C. Amylases produced by this fungus are stable to variations in pH and temperature, exhibited optimum activities at 65 oC and pH 6.0, and were significantly activated in the presence of 5 and 10 mm KH2PO4.