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.

High Catalytic Activity of Lipase from Yarrowia lipolytica Immobilized by Microencapsulation

Microencapsulation of lipase from Yarrowia lipolytica IMUFRJ 50682 was performed by ionotropic gelation with sodium alginate. Sodium alginate, calcium chloride and chitosan concentrations as well as complexation time were evaluated through experimental designs to increase immobilization yield (IY) and immobilized lipase activity (ImLipA) using p-nitrophenyl laurate as substrate. To adjust both parameters (IY and ImLipA), the desirability function showed that microcapsule formation with 3.1%(w/v) sodium alginate, 0.19%(w/v) chitosan, 0.14 M calcium chloride, and 1 min complexation time are ideal for maximal immobilization yield and immobilized lipase activity. A nearly twofold enhancement in Immobilization yield and an increase up to 280 U/g of the lipase activity of the microcapsules were achieved using the experimental design optimization tool. Chitosan was vital for the high activity of this new biocatalyst, which could be reused a second time with about 50% of initial activity and for four more times with about 20% of activity.

Stripping Voltammetry on a new Modified Glassy Carbon Electrode for Lead Content Determination in Soft Water

This study develops a new electrochemical method for quantification of Pb in soft water using stripping voltammetry with a glassy carbon electrode modified with the functionalized azulene: (2E)-2-(azulen-1-ylmethylidene) hydrazine carbothioamide (L) as a complexing polymer film. The optimized steps of the proposed method consisted in experimental establishing of four parameters, namely: reduction time; reduction potential; complexation time; pH in acetate buffer and phosphate buffer. The modified electrodes were prepared in L solutions (1mM) in acetonitrile (CH3CN) containing tetra n-butyl ammonium perchlorate (0.1 M TBAP) as supporting electrolyte. The electrochemical method includes four main operations: 1) polymeric film formation by controlled potential electrolysis (CPE) at 1.7V using a charge of 1 mC; 2) film conditioning in 0.1 M buffer acetate solution using cyclic voltammetry; 3) 25 min complexation time in a standard solution prepared from lead nitrate (II) using as matrix ultrapure water or a real sample; 4) stripping by differential pulse voltammetry method (DPV) in acetate buffer at pH 4.0, after 120 s reduction time at a reduction potential of -1.0 V. Determination time for a single standard solution or real sample was around 45 min. Evaluation of the method performance parameters (linearity, working range, detection limit, quantification limit, repeatability, and recovery) was done. It confirms that the proposed method is suitable to detect and quantify lead content over 1.2 �g/L in soft water samples.

DEVELOPMENT AND VALIDATION OF A U.V SPECTROPHOTOMETRIC METHOD FOR ESTIMATION OF CISPLATIN IN PHARMACEUTICAL FORMULATIONS

A simple, accurate, specific and reproducible colorimetric method was developed for cisplatin in pharmaceutical formulations. The analytical method was based on the formation of a stable, light blue coloured complex upon reaction between cisplatin and ortho-phenylene diamine at pH 7.4. The λmax of the solution was obtained at 702 nm in phosphate buffer saline 7.4. Optimized OPDA concentration and complexation time for the procedure were 1.2 mg/mL and 10 minutes respectively. Developed method in PBS 7.4 was found to be linear in the range of 1-10μg/mL with a regression coefficient of 0.999 and exhibiting line equation y = 0.102x – 0.004. Recovery of marketed cisplatin injection was found to be in the range of 98.8-99.1%. Drug content for the developed nanoformulation was in the range of 98-101% indicating no interference of excipients. These results illustrate that the developed method could be successfully employed for quantitative estimation of cisplatin in pharmaceutical formulations.

Lability of Cd, Cr, Cu, Mn and Pb complexed by aquatic humic substances

The lability of Cd(II), Cr(III), Cu(II), Mn(II) and Pb(II) complexed by humic substances (HSs) was investigated by means of ion exchange on cellulose modified with p-aminobenzoic groups (Cell-PAB), using a batch procedure. The HSs were extracted from water samples using adsorption in a column packed with XAD 8 resin. The metal-HS complexes were prepared by adding solutions containing all the aforementioned metal ions ( Cd(II), Cr(III), Cu(II), Mn(II) and Pb(II) ). The results indicated that the distribution coefficients (Kd) of Cell-PAB decreased with the presence of HSs, and that the lability of metal fractions complexed by HSs decreases in pH values > 4.0, complexation time > 10 h and HS concentration > 500 mg L-1. The metal exchange between HSs and Cell-PAB exhibited the following order of metal ion lability: Cd < Pb < Mn <FONT FACE=Symbol>@</FONT> Cr < Cu.