Despite improvements in volumetric titer for monoclonal antibody (MAb) production processes using Chinese hamster ovary (CHO) cells, some "difficult-to-express" (DTE) MAbs inexplicably reach much lower process titers. These DTE MAbs require intensive cell line and process development activity, rendering them more costly or even unsuitable to manufacture. To rapidly and rationally identify an optimal strategy to improve production of DTE MAbs, we have developed an engineering design platform combining high-yielding transient production, empirical modeling of MAb synthesis incorporating an unfolded protein response (UPR) regulatory loop with directed expression and cell engineering approaches. Utilizing a panel of eight IgG1 λ MAbs varying >4-fold in volumetric titer, we showed that MAb-specific limitations on folding and assembly rate functioned to induce a proportionate UPR in host CHO cells with a corresponding reduction in cell growth rate. Derived from comparative empirical modeling of cellular constraints on the production of each MAb we employed two strategies to increase production of DTE MAbs designed to avoid UPR induction through an improvement in the rate/cellular capacity for MAb folding and assembly reactions. Firstly, we altered the transfected LC:HC gene ratio and secondly, we co-expressed a variety of molecular chaperones, foldases or UPR transactivators (BiP, CypB, PDI, and active forms of ATF6 and XBP1) with recombinant MAbs. DTE MAb production was significantly improved by both strategies, although the mode of action was dependent upon the approach employed. Increased LC:HC ratio or CypB co-expression improved cell growth with no effect on qP. In contrast, BiP, ATF6c and XBP1s co-expression increased qP and reduced cell growth. This study demonstrates that expression-engineering strategies to improve production of DTE proteins in mammalian cells should be product specific, and based on rapid predictive tools to assess the relative impact of different engineering interventions.

Single nucleotide polymorphisms (SNPs) in the fat mass and obesity-associated (FTO) locus are associated with obesity, but lifestyle factors may modulate the obesity risk related to FTO. This study examined the physical activity and dietary patterns of 528 physically active white men and women (mean (SD): 34.9 (9.5) years, 26.6 (4.3) kg·m−2) carrying different risk variants of FTO SNP rs9939609. Sex, age, and anthropometric measurements (stature, body mass, and waist circumference) were self-reported using an online questionnaire, and body mass index and waist-to-height ratio were calculated. Physical activity and eating behaviour were assessed using the International Physical Activity Questionnaire (IPAQ) and Three-Factor Eating Questionnaire (TFEQ), respectively. Body mass, body mass index (BMI), waist circumference, and waist-to-height ratio were not significantly different between individuals expressing different FTO rs9939609 risk variants (all P ≥ 0.66). The cohort was physically active (4516 (3043) total MET min·week−1), although homozygous risk allele carriers (AA) displayed higher TFEQ cognitive restraint compared with nonrisk allele carriers (TT) (ES = 0.33 and P=0.03). In conclusion, obesity-related parameters were not different in physically active individuals expressing different risk variants of FTO rs9939609, although homozygous risk allele carriers exhibited higher cognitive restraint.

Running title: FTO and obesity riskWord count: 2,621 Disclosure: The authors declared no conflict of interest Abstract Objective: To examine physical activity and dietary patterns in physically active individuals carrying different risk variants of the fat mass and obesity-associated gene (FTO) rs9939609 single nucleotide polymorphism (SNP).Methods: A total of 528 white men and women (mean (SD): 34.9(9.5) years, 26.6(4.3) kg·m -2 ) were genotyped for FTO rs9939609 SNP. Sex, age and anthropometric measurements (stature, body mass, waist circumference) were self-reported using an online questionnaire, and body mass index and waist-to-height ratio were calculated. Physical activity level and eating behaviour were assessed using the International Physical Activity Questionnaire and Three-Factor Eating Questionnaire (TFEQ), respectively.Results: Body mass, body mass index, waist circumference and waist-to-height ratio were not significantly different between individuals carrying different FTO rs9939609 risk variants (all P≥0.66). The cohort was physically active (4516(3043) total MET min·week -1 ), although risk allele carriers (AT/AA) reported higher total physical activity (effect size = 0.22, P=0.03), and homozygous risk allele carriers (AA) displayed higher TFEQ cognitive restraint (effect size = 0.33, P=0.03) compared with non-risk allele carriers (TT). Conclusions:Obesity-related parameters were not different in physically active individuals carrying different risk variants of FTO rs9939609, but higher physical activity and cognitive restraint in risk allele carriers may reduce genetic predisposition to weight gain.