The use of catechins in Chinese hamster ovary cell media for the improvement of monoclonal antibody yields and a reduction of acidic species

Catechin compounds have potential benefits for recombinant monoclonal antibody (Mab) production as chemical additives in cell culture media. In this study, four catechin compounds catechin (Cat), epicatechin (EC), gallocatechin-gallate (GCG), and epigallocatechin-gallate (EGCG) were added to cell culture media (at 50 μM) and their effects on the recombinant Chinese hamster ovary (CHO) cell culture, specific productivity, and Mab quality were assessed. The results indicate that the improvement of specific productivity was linked to cell growth inhibition. All catechins caused cell phase growth arrest by lowering the number of cells in the G1/G0 phase and increasing the cells in the S and G2/M phases. Late addition of the catechin resulted in a significantly higher final IgG concentration. Cat and EC caused an improvement in the final antibody titer of 1.5 ± 0.1 and 1.3 ± 0.1 fold, respectively. Catechins with a galloyl group (GCG and EGCG) arrested cell growth and reduced cell specific productivity at the concentrations tested. The Cat-treated IgG was found to have reduced acidic species with a corresponding increase in the main peak.     

The effect of exenatide re-exposure on safety and efficacy

Exenatide, a synthetic peptide originally isolated from salivary secretions of Heloderma suspectum, like other subcutaneously injected peptides, can cause antibody formation. Despite that antibody formation has been observed in some patients, results from previous clinical trials have not shown safety and efficacy concerns in exenatide-naïve patients. The objective of this multicenter, open-label study was to investigate the response of anti-exenatide antibody formation and the incidence of immune-related and hypersensitivity reactions after exenatide re-exposure. Fifty-eight patients (57% male; 59 ± 10 years; weight 85 ± 19 kg; HbA1c 8.1 ± 0.9%; duration of diabetes 10 ± 5 years) were enrolled. At study initiation, 98.3% of patients were taking 1 or more antidiabetes drugs, including oral medication and various types of insulin. Treatment-emergent adverse events (TEAEs) at any time during the study were observed in 40 and 47% of patients with positive and negative treatment-emergent antibodies, respectively. Immune-related AEs were observed in 6 patients (4 were antibody positive). These AEs had not been reported in their previous exposure to exenatide. Re-exposure to exenatide did not result in increased hypersensitivity reactions. Overall, 72% of patients had a baseline to endpoint reduction in HbA1c (range −0.1 to −2.8%), and 87% of antibody negative versus 62% of antibody positive patients had an HbA1c endpoint reduction. The study design and the patients’ baseline characteristics, including diabetes treatment at study initiation, are confounding factors limiting clinical conclusions on exenatide's glycemic effect in this patient population. The study results indicate that anti-exenatide antibody formation did not increase the incidence of TEAEs in patients re-exposed to exenatide.     

Using spectrophotometry and spectral mapping technique for the study of the production of manganese-dependent and manganese-independent peroxidases by Pleurotus ostreatus

The activity of manganese-dependent and manganese-independent peroxidases produced by Pleurotus ostreatus in culture media composed of agro-residues was measured by visible spectrophotometry. The overall enzyme activity and its selectivity were separated by using spectral mapping technique followed by nonlinear mapping. The relationships between the parameters of enzyme production and the composition of culture media and fermentation time was assessed by stepwise regression analysis. Calculations proved that the addition of extract of straw to the culture media significantly decreased the overall production of both enzymes, whereas the selectivity of enzyme production was influenced by amount of potato extract and the concentration of total sugar in the culture media. Enzyme activity depended quadratically on the fermentation time.     

Interactions between Carbon and Nitrogen Metabolism in Fibrobacter succinogenes S85: a 1H and 13C Nuclear Magnetic Resonance and Enzymatic Study

The effect of the presence of ammonia on [1-¹³C]glucose metabolism in the rumen fibrolytic bacterium Fibrobacter succinogenes S85 was studied by ¹³C and ¹H nuclear magnetic resonance (NMR). Ammonia halved the level of glycogen storage and increased the rate of glucose conversion into acetate and succinate 2.2-fold and 1.4-fold, respectively, reducing the succinate-to-acetate ratio. The ¹³C enrichment of succinate and acetate was precisely quantified by ¹³C-filtered spin-echo difference ¹H-NMR spectroscopy. The presence of ammonia did not modify the ¹³C enrichment of succinate C-2 (without ammonia, 20.8%, and with ammonia, 21.6%), indicating that the isotopic dilution of metabolites due to utilization of endogenous glycogen was not affected. In contrast, the presence of ammonia markedly decreased the ¹³C enrichment of acetate C-2 (from 40 to 31%), reflecting enhanced reversal of the succinate synthesis pathway. The reversal of glycolysis was unaffected by the presence of ammonia as shown by ¹³C-NMR analysis. Study of cell extracts showed that the main pathways of ammonia assimilation in F. succinogenes were glutamate dehydrogenase and alanine dehydrogenase. Glutamine synthetase activity was not detected. Glutamate dehydrogenase was active with both NAD and NADP as cofactors and was not repressed under ammonia limitation in the culture. Glutamate-pyruvate and glutamate-oxaloacetate transaminase activities were evidenced by spectrophotometry and ¹H NMR. When cells were incubated in vivo with [1-¹³C]glucose, only ¹³C-labeled aspartate, glutamate, alanine, and valine were detected. Their labelings were consistent with the proposed amino acid synthesis pathway and with the reversal of the succinate synthesis pathway.     

Scavenger-driven fish carcass decomposition and phosphorus recycling: Laboratory experiments with freshwater fish and crayfish

1. Fish carcass decomposition can generate fluxes of nutrients to the water column at levels comparable to other major nutrient sources. However, relatively little is known about the biological processes modulating fish decay or the rates at which carcass-bound nutrients are made available to the biota.
2. This study focused on quantifying scavenger-mediated phosphorus (P) recycling, because the availability of this essential element can regulate the trophic state of aquatic ecosystems.
3. To explore the role of some important macroscopic aquatic scavengers in P recycling, laboratory experiments were conducted where carcasses of two fish species (common bleak Alburnus alburnus; pumpkinseed Lepomis gibbosus) were offered to two size classes of black bullhead (Ameiurus melas) and two crustacean species (spiny-cheek crayfish Faxonius limosus; narrow-clawed crayfish Astacus leptodactylus).
4. Our results show that the black bullhead and the two crayfish species are highly efficient macroscopic decomposers as the P contents of scavenged carcasses were reduced at significantly higher rates compared to those of microbially decomposed (control) carcasses.
5. Pumpkinseed carcasses proved to be more resistant to rapid decomposition, as they typically lost lower proportions of their P content than bleak carcasses during the course of the experiments.
6. Scavengers sequestered a relatively large fraction (up to 33% in black bullhead and 36% in crayfish) of total carcass P in their bodies. This suggests that the consumer species used in this study can transfer/return considerable quantities of carcass-derived nutrients directly to higher trophic levels incorporated into their own tissues, and may serve as an additional, short-term sink of these nutrients.

     

Resveratrol addition to Chinese hamster ovary cell culture media: The effect on cell growth,monoclonal antibody synthesis,and its chemical modification

The effect of the addition of resveratrol to cell culture media during the production of monoclonal antibodies was investigated. Treatments of Chinese hamster ovary (CHO) cells expressing immunoglobulin G (IgG) with 25 and 50 μM resveratrol showed that resveratrol was capable of slowing cell growth while almost doubling cell-specific productivity to 4.7 ± 0.6 pg IgG/cell·day, resulting in up to a 1.37-fold increase of the final IgG titer. A resveratrol concentration of 50 μM slowed the progression through the cell cycle temporarily by trapping cells in the S-phase. Cation exchange chromatography showed no significant difference in the composition of acidic or basic IgG species and size exclusion chromatography indicated no change in fragmentation or aggregation of the recombinant IgG in the treatment groups. Resveratrol could be used as a chemical additive to CHO media where it would enhance IgG productivity and provide a degree of protection against hydroxyl and superoxide free radicals, expanding the range of options for process improvement available to monoclonal antibody manufacturers.     

Functional diversity: a review of methodology and current knowledge in freshwater macroinvertebrate research

Although several studies have examined the functional diversity of freshwater macroinvertebrates, the variety of methodologies combined with the absence of a synthetic review make our understanding of this field incomplete. Therefore, we reviewed the current methodology for assessing functional diversity in freshwater macroinvertebrate research. Our review showed that most papers quantified functional diversity using biological traits, among which feeding habits were the most common traits probably due to the assumed links between feeding and ecosystem functions. A large number of diversity measures have been applied for quantifying functional diversity of freshwater macroinvertebrate assemblages, among which Rao’s quadratic entropy looks like the most frequent. In most papers, functional diversity was positively related to taxon richness, and functional redundancy was a key concept in explaining this correlation. Most studies detected strong influence of the environmental factors as well as human impact on functional diversity. Finally, our review revealed that functional diversity research is biased towards European running waters and is hindered by yet insufficient information on the autecology of macroinvertebrates.