Advances and drawbacks of the adaptation to serum-free culture of CHO-K1 cells for monoclonal antibody production

Currently, mammalian cell technology has become the focus of biopharmaceutical production, with strict regulatory scrutiny of the techniques employed. Major concerns about the presence of animal-derived components in the culture media led to the development of serum-free (SF) culture processes. However, cell adaptation to SF conditions is still a major challenge and limiting step of process development. Thus, this study aims to assess the impact of SF adaptation on monoclonal antibody production (mAb), identify the most critical steps of cell adaptation to the SF EX-CELL medium, and create basic process guidelines.. The success of SF adaptation was dependent on critical steps that included: accentuated cell sensitivity to common culture procedures (centrifugation, trypsinization); initial cell concentration; time given at each step of serum-reduction; and, most importantly, medium supplements used to support adaptation. Indeed, only one of the five supplement combinations assessed (rhinsulin, ammonium metavanadate, nickel chloride and stannous chloride) succeeded for the CHO-K1 cell line used. This work also revealed that the chemically-defined EX-CELL medium benefits mAb production in comparison with the general purpose Dulbecco's Modified Eagle's Medium, but the complete removal of serum attenuates these positive effects.The authors acknowledge funding and support from the Portuguese Foundation for Science and Technology (FCT), namely grant ref SFRH/BD/46661/2008 for Maria Elisa Rodrigues and SFRH/BD/46660/2008 for Ana Rita Costa

Epigenetic effects of metformin: From molecular mechanisms to clinical implications

There is a growing body of evidence that links epigenetic modifications to type 2 diabetes. Researchers have more recently investigated effects of commonly used medications, including those prescribed for diabetes, on epigenetic processes. This work reviews the influence of the widely used antidiabetic drug metformin on epigenomics, microRNA levels and subsequent gene expression, and potential clinical implications. Metformin may influence the activity of numerous epigenetic modifying enzymes, mostly by modulating the activation of AMP-activated protein kinase (AMPK). Activated AMPK can phosphorylate numerous substrates, including epigenetic enzymes such as histone acetyltransferases (HATs), class II histone deacetylases (HDACs) and DNA methyltransferases (DNMTs), usually resulting in their inhibition; however, HAT1 activity may be increased. Metformin has also been reported to decrease expression of multiple histone methyltransferases, to increase the activity of the class III HDAC SIRT1 and to decrease the influence of DNMT inhibitors. There is evidence that these alterations influence the epigenome and gene expression, and may contribute to the antidiabetic properties of metformin and, potentially, may protect against cancer, cardiovascular disease, cognitive decline and aging. The expression levels of numerous microRNAs are also reportedly influenced by metformin treatment and may confer antidiabetic and anticancer activities. However, as the reported effects of metformin on epigenetic enzymes act to both increase and decrease histone acetylation, histone and DNA methylation, and gene expression, a significant degree of uncertainty exists concerning the overall effect of metformin on the epigenome, on gene expression, and on the subsequent effect on the health of metformin users

Topical Application of an Irreversible Small Molecule Inhibitor of Lysyl Oxidases Ameliorates Skin Scarring and Fibrosis

Scarring is a lifelong consequence of skin injury, with scar stiffness and poor appearance presenting physical and psychological barriers to a return to normal life. Lysyl oxidases are a family of enzymes that play a critical role in scar formation and maintenance. Lysyl oxidases stabilize the main component of scar tissue, collagen, and drive scar stiffness and appearance. Here we describe the development and characterisation of an irreversible lysyl oxidase inhibitor, PXS-6302. PXS-6302 is ideally suited for skin treatment, readily penetrating the skin when applied as a cream and abolishing lysyl oxidase activity. In murine models of injury and fibrosis, topical application reduces collagen deposition and cross-linking. Topical application of PXS-6302 after injury also significantly improves scar appearance without reducing tissue strength in porcine injury models. PXS-6302 therefore represents a promising therapeutic to ameliorate scar formation, with potentially broader applications in other fibrotic diseases

DNA methylation and body mass index from birth to adolescence : meta-analyses of epigenome-wide association studies

Background DNA methylation has been shown to be associated with adiposity in adulthood. However, whether similar DNA methylation patterns are associated with childhood and adolescent body mass index (BMI) is largely unknown. More insight into this relationship at younger ages may have implications for future prevention of obesity and its related traits. Methods We examined whether DNA methylation in cord blood and whole blood in childhood and adolescence was associated with BMI in the age range from 2 to 18 years using both cross-sectional and longitudinal models. We performed meta-analyses of epigenome-wide association studies including up to 4133 children from 23 studies. We examined the overlap of findings reported in previous studies in children and adults with those in our analyses and calculated enrichment. Results DNA methylation at three CpGs (cg05937453, cg25212453, and cg10040131), each in a different age range, was associated with BMI at Bonferroni significance, P <1.06 x 10(-7), with a 0.96 standard deviation score (SDS) (standard error (SE) 0.17), 0.32 SDS (SE 0.06), and 0.32 BMI SDS (SE 0.06) higher BMI per 10% increase in methylation, respectively. DNA methylation at nine additional CpGs in the cross-sectional childhood model was associated with BMI at false discovery rate significance. The strength of the associations of DNA methylation at the 187 CpGs previously identified to be associated with adult BMI, increased with advancing age across childhood and adolescence in our analyses. In addition, correlation coefficients between effect estimates for those CpGs in adults and in children and adolescents also increased. Among the top findings for each age range, we observed increasing enrichment for the CpGs that were previously identified in adults (birth P-enrichment = 1; childhood P-enrichment = 2.00 x 10(-4); adolescence P-enrichment = 2.10 x 10(-7)). Conclusions There were only minimal associations of DNA methylation with childhood and adolescent BMI. With the advancing age of the participants across childhood and adolescence, we observed increasing overlap with altered DNA methylation loci reported in association with adult BMI. These findings may be compatible with the hypothesis that DNA methylation differences are mostly a consequence rather than a cause of obesity.Peer reviewe

DNA methylation and body mass index from birth to adolescence: meta-analyses of epigenome-wide association studies

Background DNA methylation has been shown to be associated with adiposity in adulthood. However, whether similar DNA methylation patterns are associated with childhood and adolescent body mass index (BMI) is largely unknown. More insight into this relationship at younger ages may have implications for future prevention of obesity and its related traits. Methods We examined whether DNA methylation in cord blood and whole blood in childhood and adolescence was associated with BMI in the age range from 2 to 18 years using both cross-sectional and longitudinal models. We performed meta-analyses of epigenome-wide association studies including up to 4133 children from 23 studies. We examined the overlap of findings reported in previous studies in children and adults with those in our analyses and calculated enrichment. Results DNA methylation at three CpGs (cg05937453, cg25212453, and cg10040131), each in a different age range, was associated with BMI at Bonferroni significance, P < 1.06 x 10(-7), with a 0.96 standard deviation score (SDS) (standard error (SE) 0.17), 0.32 SDS (SE 0.06), and 0.32 BMI SDS (SE 0.06) higher BMI per 10% increase in methylation, respectively. DNA methylation at nine additional CpGs in the cross-sectional childhood model was associated with BMI at false discovery rate significance. The strength of the associations of DNA methylation at the 187 CpGs previously identified to be associated with adult BMI, increased with advancing age across childhood and adolescence in our analyses. In addition, correlation coefficients between effect estimates for those CpGs in adults and in children and adolescents also increased. Among the top findings for each age range, we observed increasing enrichment for the CpGs that were previously identified in adults (birth P-enrichment = 1; childhood P-enrichment = 2.00 x 10(-4); adolescence P-enrichment = 2.10 x 10(-7)). Conclusions There were only minimal associations of DNA methylation with childhood and adolescent BMI. With the advancing age of the participants across childhood and adolescence, we observed increasing overlap with altered DNA methylation loci reported in association with adult BMI. These findings may be compatible with the hypothesis that DNA methylation differences are mostly a consequence rather than a cause of obesity

Evaluation of the OSCAR (TM) system for the production of monoclonal antibodies by CHO-K1 cells

Biopharmaceutical production of complex recombinant protein therapeutics currently relies on mammalian cells. The development of high-yielding stable cell lines requires processes of transfection, selection and adaptation. With several technologies available, selection has been most frequently based on dihydrofolate reductase or glutamine synthetase systems, which can be very time-consuming. Due to the pressure to reduce development costs and speed up time to market, new technologies are emerging, as the promising OSCAR™ expression system that could provide more rapid development of high-yielding stable cell lines than the traditional systems. However, further evaluation of its application in a wider range of cell types and media is still necessary. In this study, application of OSCAR™ for the transfection of a CHO-K1 cell line with a monoclonal antibody was evaluated. OSCAR™ was reasonably fast and simple, without negative impact on cell growth characteristics. However, minigene selection was critical, with only pDWM128 working for the cell line assessed. Initial relatively high levels of production decreased significantly in the first few weeks of passing, remaining relatively stable although with low yield thereafter. The results suggest that more work is required to develop methodologies and prove that OSCAR™ has significant value to the bioproduction industryFundação para a Ciência e a Tecnologia (FCT

Evaluation of macroporous and microporous carriers for CHO-K1 cell growth and monoclonal antibody production

The emergence of microcarrier technology has brought a renewed interest in anchorage-dependent cell culture for high-yield processes. Well-known in vaccine production, microcarrier culture also has potential for application in other fields. In this work, two types of microcarriers were evaluated for small-scale monoclonal antibody (mAb) production by CHO-K1 cells. Cultures (5 mL) of microporous Cytodex 3 and macroporous CultiSpher-S carriers were performed in vented conical tubes and subsequently scaled-up (20 mL) to shake-flasks, testing combinations of different culture conditions (cell concentration, microcarrier concentration, rocking methodology, rocking speed and initial culture volume). Culture performance was evaluated considering mAb production and cell growth at the phases of initial adhesion and proliferation. The best culture performances were obtained with Cytodex 3, regarding cell proliferation (average 1.85 ± 0.11 x 106 cells/mL against 0.60 ± 0.08 x 106 cells/mL for CultiSpher-S), mAb production (2.04 ± 0.41 µg/mL against 0.99 ± 0.35 µg/mL for CultiSpher-S) and culture longevity (30 days against 10-15 days for CultiSpher-S), probably due to the collagen-coated dextran matrix that potentiates adhesion and prevents detachment. The culture conditions of greater influence were rocking mechanism (Cytodex 3, pulse followed by continuous) and initial cell concentration (CultiSpher-S, 4x105 cells/mL). Microcarriers proved to be a viable and favorable alternative to standard adherent and suspended cultures for mAb production by CHO-K1 cells, with simple operation, easy scale-up and significantly higher levels of mAb production. However, variations of microcarrier culture performance in different vessels reiterate the need for optimization at each step of the scale-up process.The authors acknowledge funding and support from the Portuguese Foundation for Science and Technology (FCT), namely grant ref SFRH/BD/46661/2008 for Maria Elisa Rodrigues and SFRH/BD/46660/2008 for Ana Rita Costa

Hydroalumination of Alkenes and Alkynes by Primary Aluminum Hydrides under Mild Conditions

The reactions of the sterically crowded primary alane (Ar^{Pr<i>i</i>₈}AlH₂)₂ (Ar^{Pr<i>i</i>₈} = C₆H-2,6­(C₆H₂-2,4,6-Pr^<i>i</i>₃)₂-3,5-Pr^<i>i</i>₂) with alkynes and alkenes are described. It is shown that hydroalumination of the terminal alkynes HCCSiMe₃ and HCCPh readily occurs under mild conditions via the <i>cis</i>-addition of the Al–H moiety across the CC triple bond with no evidence of hydrogen elimination. Hydroalumination was observed also with a range of terminal olefins, but no reactivity was observed with internal alkenes or alkynes. The relatively high reactivity of (Ar^{Pr<i>i</i>₈}AlH₂)₂ was attributed to the steric crowding of the large terphenyl substituent, which favors dissociation of the alane and increases the availability of the more reactive three-coordinate aluminum site in the monomer. In keeping with this view, studies of the reactions of the three primary alanes (Ar^{Pr<i>i</i>₈}AlH₂)₂, (Ar^{Pr<i>i</i>₄}AlH₂)₂ (Ar^{Pr<i>i</i>₄} = C₆H₃-2,6­(C₆H₃-2,6-Prⁱ₂)₂), and (Ar^Me₆AlH₂)₂ (Ar^Me₆ = C₆H₃-2,6­(C₆H₂-2,4,6-Me₃)₂) with alkenes showed that the reaction rates are inversely proportional to the size of the terphenyl substituent, consistent with higher reactivity of the aluminum monomer. The structures of the alkenyl insertion products, Ar^{Pr<i>i</i>₈}Al­(CHCHPh)₂ and Ar^{Pr<i>i</i>₈}Al­(CHCHSiMe₃)₂, the alkylated derivative, Ar^{Pr<i>i</i>₈}Al­(CH₂CH₂SiMe₃)₂, and the precursor aluminates {Li­(OEt₂)­H₃AlAr^{Pr<i>i</i>₈}·Li­(OEt₂)₂H₃AlAr^{Pr<i>i</i>₈}}, (LiH₃AlAr^{Pr<i>i</i>₈})₂, and alanes (Ar^{Pr<i>i</i>₈}AlH₂)₂, and (Ar^{Pr<i>i</i>₄}AlH₂)₂ were determined by X-ray crystallography