Fibroblast growth factor 21 (FGF21) is a promising drug candidate for the treatment of type 2 diabetes. However, the use of wild type native FGF21 is challenging due to several limitations. Among these are its short half-life, its susceptibility to in vivo proteolytic degradation and its propensity to in vitro aggregation. We here describe a rationale-based protein engineering approach to generate a potent long-acting FGF21 analog with improved resistance to proteolysis and aggregation. A recombinant Fc-FGF21 fusion protein was constructed by fusing the Fc domain of human IgG1 to the N-terminus of human mature FGF21 via a linker peptide. The Fc positioned at the N-terminus was determined to be superior to the C-terminus as the N-terminal Fc fusion retained the βKlotho binding affinity and the in vitro and in vivo potency similar to native FGF21. Two specific point mutations were introduced into FGF21. The leucine to arginine substitution at position 98 (L98R) suppressed FGF21 aggregation at high concentrations and elevated temperatures. The proline to glycine replacement at position 171 (P171G) eliminated a site-specific proteolytic cleavage of FGF21 identified in mice and cynomolgus monkeys. The derived Fc-FGF21(RG) molecule demonstrated a significantly improved circulating half-life while maintaining the in vitro activity similar to that of wild type protein. The half-life of Fc-FGF21(RG) was 11 h in mice and 30 h in monkeys as compared to 1-2 h for native FGF21 or Fc-FGF21 wild type. A single administration of Fc-FGF21(RG) in diabetic mice resulted in a sustained reduction in blood glucose levels and body weight gains up to 5-7 days, whereas the efficacy of FGF21 or Fc-FGF21 lasted only for 1 day. In summary, we engineered a potent and efficacious long-acting FGF21 analog with a favorable pharmaceutical property for potential clinical development.

Tumor necrosis factor (TNF) antagonists are efficacious in the treatment of various autoimmune diseases. Two classes of TNF antagonists are currently commercially available: soluble TNF receptor-Fc fusion proteins (etanercept) and anti-TNF mAbs (adalimumab and infliximab). The classes differ in molecular structures and mechanisms of action. The interactions between TNF antagonists with TNF molecules were characterized. The anti-TNF mAbs, but not the soluble TNF receptor, formed visible lines of precipitation in Ouchterlony assays. The molecular weights of complexes formed by TNF (52 kDa) with either etanercept (130 kDa), adalimumab (150 kDa), or infliximab (average 165 kDa) were determined by size exclusion chromatography-light-scattering assays. Etanercept and TNF formed complexes of 180 and 300 kDa, representing one and two etanercept monomers bound to a TNF trimer, respectively. Adalimumab and infliximab formed a variety of complexes with TNF with molecular weights as high as 4,000 and 14,000 kDa, respectively, suggesting the presence of complexes with a wide range of sizes and stoichiometries. The absence of large complex formation with the binding of soluble receptor-fusion proteins to TNF may account for the different clinical efficacy and safety profiles of the two classes of TNF antagonists.

The endocrine fibroblast growth factor 21 (FGF21) requires both fibroblast growth factor receptor (FGFR) and b-Klotho for signaling. In this study, we sought to understand the inter-molecular physical interactions in the FGF21 ⁄ FGFR ⁄ b-Klotho complex by deleting key regions in FGFR1c or FGF21. Deletion of the D1 and the D1-D2 linker (the D1 ⁄ linker region) from FGFR1c led to b-Klotho-independent receptor activation by FGF21, suggesting that there may be a direct interaction between FGF21 and the D1 ⁄ linker region-deficient FGFR1c. Consistent with this, the extracellular portion of FGFR1c lacking the D1 ⁄ linker region blocked FGF21 action in a reporter assay, presumably by binding to and sequestering FGF21 from acting on cell surface receptor complex. In addition, the D1 ⁄ linker region-deficient FGFR1c had enhanced interaction with b-Klotho. Further, we demonstrated that deletion of the D1 ⁄ linker region enhanced the formation of the FGF21 ⁄ b-Klotho ⁄ FGFR1c ternary complex in both Biacore and asymmetrical flow field flow fraction-ation studies. Finally, we found that the N-terminus of FGF21 is involved in the interaction with FGFR1c and FGF21 ⁄ b-Klotho ⁄ FGFR1c ternary complex formation. Taken together, our data suggest that the D1 ⁄ linker region regulates both the FGF21 ⁄ FGFR1c and FGFR1c ⁄ b-Klotho interaction, and a direct interaction of FGF21 with FGFR1c may be an important step in receptor-mediated FGF21 signaling. Key words: D1 and the D1-D2 linker, FGF21, FGFR1c, b-Klotho Abbreviations: AF4, asymmetrical flow field flow fractionation; D1, immunoglobin domain 1; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; HSPG, heparin sulfate proteoglycan; mIMCD3, murine renal inner medullary collecting duct; the D1 ⁄ linker region, D1 and the D1-D2 linker. Fibroblast growth factor 21 (FGF21) and its two other subfamily members, FGF19 and FGF23, have emerged as potent endocrine hormones involved in the regulation of diverse physiological functions (1-3). In particular, FGF21 has generated a lot of excitement for its potential of becoming a therapeutic for type 2 diabetes and dyslipi-demia (4). Noticeably, administration of recombinant FGF21 lowered plasma glucose, insulin, triglycerides, and cholesterol levels and improved insulin sensitivity, hepatic steatosis, and obesity in a range of diabetes ⁄ obesity animal models (3,5-7). The key differences that distinguish the FGF19 subfamily members from paracrine FGFs are their lack of or weak heparin-binding properties (8) and their dependence on Klotho family transmembrane proteins as co-receptor for the activation of FGF receptor (FGFR) sig-naling (9). a-Klotho is required for FGF23 to regulate renal phosphate metabolism (10), whereas b-Klotho is required for FGF21 to regulate glucose and lipid metabolism (11-14) and FGF19 to regulate bile acid metabolism (15,16). Interestingly, FGF19 can also induce signaling with a-Klotho as a co-receptor, (16) although the physiological implication is still unclear. In contrast, paracrine FGFs signal...

in Wiley Online Library (wileyonlinelibrary.com) Mammalian expression systems are used routinely for the production of recombinant proteins as therapeutic molecules as well as research tools. Transient expression has become increasingly popular in recent years due to its rapid timeline and improvements in expression level. While improvements to transient expression systems have focused mainly on the level of protein expression, the aspect of protein quality has received little attention. The removal of undesirable products, such as aggregation, depends primarily on purification, requiring additional cumbersome steps, which can lead to a lower product yield and longer timelines. In this study, we show that reducing the level of transcription by transfecting at a lower gene dose improves the quality of secreted molecules prone to aggregation. For gene dosing to have this effect, it is critical for the carrier DNA to be an empty vector containing the same elements as the gene containing plasmid. This approach can be used in combination with a temperature shift to hypothermic conditions during production to enhance the effect. The observed improvements not only minimized aggregation levels, but also generated products with overall superior quality, including more homogeneous signal peptide cleavage and N-linked glycosylation profiles. These techniques have produced a similar improvement in product quality with a variety of other molecules, suggesting that this may be a general approach to enhance product quality from transient expression systems.

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Strong ion-exchange protein chromatography is one of the most powerful and most common steps for protein purification in both discovery research and manufacturing. However, the demands on protein purification of early drug discovery and later stage manufacturing are quite different. In order to shorten the time of developing a purification process for new protein drug candidates, there is a need for a strong ion-exchange resin that will be optimum for both stages. This article details a novel anion-exchange resin suitable for research, as well as for clinical manufacturing. In this study, a novel Q resin anion-exchange prototype was evaluated and compared to the GE Healthcare Q Sepharose® Fast Flow (QFF) and Q Sepharose® High Performance (QHP) resins. This study specifically focused on the following: resolution, dynamic binding capacity, flow rate, back pressure, and scale up. The evaluation was performed in both small- and large-scale experiments. From all the comparable data, the prototype resin is adaptable for both discovery research and manufacturing. Its wide-range operation suitability could potentially shorten the time required to develop conventional purification protocols for clinical manufacturing.

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