Improvement of recombinant protein production by an anti-apoptotic protein from hemolymph of Lonomia obliqua

Apoptosis is a major problem in animal cell culture during production of biopharmaceuticals, such as recombinant proteins or viral particles. In the present work baculovirus-insect cell expression system (BEVS/IC) is used as model to produce rotavirus like-particles, composed by three layers of three different viral proteins (VP2, VP6 and VP7). In this model baculovirus infection also induces host cell death. Herein a new strategy to enhance cell life span and to increase recombinant rotavirus protein production of BEVS/IC system was developed. This strategy relies on hemolymph from Lonomia oblique (total extracts or a semi-purified fraction) medium supplementation. The total extract and a purified fraction from hemolymph of Lonomia obliqua were able to protect Sf-9 cell culture against apoptosis triggered by oxidative stress (using the pro-oxidant agents tert butylhydroperoxide and hydrogen peroxide) and by baculovirus infection. Furthermore, hemolymph enhance final recombinant protein production, as it was observed by the increased amounts of VP6 and VP7, which were measured by the semi-quantitative western blot method. In conclusion, hemolymph medium supplementation can be a promising strategy to improve cell viability and productivity of recombinant protein in BEVS/IC system

Formulation of a protein-free medium based on IPL-41 for the sustained growth of Drosophila melanogaster S2 cells

An animal protein-free medium was developed for Drosophila melanogaster S2 (S2AcGPV2) cells genetically modified to produce the rabies virus G glycoprotein (GPV). IPL-41, used as a basal medium, was supplemented with yeastolate, carbohydrates, amino acids and lipids aiming initially to reduce and further to eliminate the need of fetal bovine serum. The S2AcGPV2 cells were fully capable of growing in serum-free supplemented IPL-41 medium containing 6 g L−1 yeastolate ultrafiltrate, 10 g L−1 glucose, 3.5 g L−1 glutamine, 0.5 g L−1 fructose, 2 g L−1 lactose, 0.6 g L−1 tyrosine, 1.48 g L−1 methionine and 1% (v/v) lipid emulsion, reaching 19 × 106 cells mL−1. Maximum specific growth rate and cell productivity were 0.025 h−1 and 0.57 × 105 cells mL−1 h−1, respectively. Glucose and lactose were consumed during cell culture, but not fructose. Lactate concentration generally decreased during cell culture, while ammonium concentration reached 167 mg L−1, however, without noticeable deleterious effects on cell growth. GPV concentration values achieved were, however, modest in the proposed medium formulation