ABSTRACT
BACKGROUND: Recombinant monoclonal antibodies have been marketed in last three decades as the major therapeutic proteins against different cancers. However choosing a proper medium and supplements to reach the high expression is a challenging step. Despite of commercial serum free and chemically defined media, there are still numerous researches seeking the optimum media to gain higher expression titer. Selecting the best basal media followed by proper supplementation to increase the cell density and expression titer needs proper and accurate investigation. METHODS: In this study, we have determined the expression titer of monoclonal antibody against human CD20 using soy extract, Essential Amino Acid, Non-Essential Amino Acid, Panexin NTS, Peptone, Yeast extract, Insulin-transferrin selenite, Human Serum Albumin, Bovine Serum Albumin, Lipid, and two commercially available supplements, Power and Xtreme feed. In each experiment, the expression level was compared with a well defined media, ProCHO5, RPMI 1640 and DMEM-F12. RESULTS: It has been shown that supplementing the ProCHO5 basal medium with 10% power feed or combination of 5% PanexinNTS,1.5 g/L yeast and 1.5g/L peptone results in the best production levels with 450 and 425 mg/L of anti CD20 mAb expression level, respectively. CONCLUSION: Panexin NTS, yeast and peptone cane be proper supplement for fed-batch cell culture instead of commercial Power feed supplement which is a cost effective way to increase expression level. And thereby ProCHO5 may be replaced with common media such as RPMI 1640 and DMEM-F12.
ABSTRACT
Langerhans islet transplantation is a much less invasive approach compared with the pancreas transplantation to 'cure' diabetes. However, destruction of transplanted islets by the immune system is an impediment for a successful treatment. Chemical grafting of monomethoxy poly(ethylene glycol) onto pancreatic islet capsule is a novel approach in islet immunoisolation. The aim of this study was to determine an optimized condition for grafting of monomethoxy poly(ethylene glycol) succinimidyl propionate (mPEG-SPA) on islets capsule. Independent variables such as reaction time, the percentage of longer mPEG in the mixture, and polymer concentration were optimized using a three-factor, three-level Box-Behnken statistical design. The dependent variable was IL-2 (interleukin-2) secretion of lymphocytes co-cultured with PEGylated or uncoated control islets for 7 days co-culturing. A mathematical relationship is obtained which explained the main and quadratic effects and the interaction of factors which affected IL-2 secretion. Response surface methodology predicted the optimized values of reaction time, the percentage of longer mPEG in the mixture, and polymer concentration of 60 min to be 63.7% mPEG10 and 22 mg/mL, respectively, for the minimization of the secreted IL-2 as response. Islets which were PEGylated at this condition were transplanted to diabetic rats. The modified islets could survive for 24 days without the aid of any immunosuppressive drugs and it is the longest survival date reported so far. However, free islets (unmodified islets as control) are completely destroyed within 7 days. These results strongly suggest that this new protocol provides an effective clinical means of decreasing transplanted islet immunogenicity.
ABSTRACT
Islet transplantation is one of the promising ways to treat diabetes. To reduce the immune system response, several methods have been developed, a novel one being the grafting of methoxy polyethylene glycol (mPEG) derivatives onto collagen capsules of islets. In this study, the effects of the first and second generations of activated mPEG on the immunological response of polyethylene glycol (PEG) grafted pancreatic islets were studied. mPEG-Succinimidyl carbonate (mPEG-SC) and mPEG-succinimidyl propionic acid (mPEG-SPA) (with nominal molecular weight 5 kDa), typical of the first and second generations of activated mPEG, were selected, respectively. Both activated mPEGs did not affect the morphology, viability, or functionality of PEGylated islets compared to free islets (naked islets). The amount of IL-2 secreted from lymphocytes co-cultured with mPEG-SPA grafted islets (131.83 ± 15.28 pg/ml) was not significantly different from that with mPEG-SC grafted islets (156.09 ± 27.94 pg/ml). These results indicated that both mPEG-SC and mPEG-SPA had the same effect for camouflaging Langerhans islets, but the former is more suitable due to its easier synthesis process.
