Uncovering methods for the prevention of protein aggregation and improvement of product quality in a transient expression system.

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.

Rationale-Based Engineering of a Potent Long-Acting FGF21 Analog for the Treatment of Type 2 Diabetes.

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 ßKlotho 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.

A novel anion-exchange resin suitable for both discovery research and clinical manufacturing purposes.

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.

Understanding the physical interactions in the FGF21/FGFR/ß-Klotho complex: structural requirements and implications in FGF21 signaling.

The endocrine fibroblast growth factor 21 (FGF21) requires both fibroblast growth factor receptor (FGFR) and ß-Klotho for signaling. In this study, we sought to understand the inter-molecular physical interactions in the FGF21/FGFR/ß-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 ß-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 ß-Klotho. Further, we demonstrated that deletion of the D1/linker region enhanced the formation of the FGF21/ß-Klotho/FGFR1c ternary complex in both Biacore and asymmetrical flow field flow fractionation studies. Finally, we found that the N-terminus of FGF21 is involved in the interaction with FGFR1c and FGF21/ß-Klotho/FGFR1c ternary complex formation. Taken together, our data suggest that the D1/linker region regulates both the FGF21/FGFR1c and FGFR1c/ß-Klotho interaction, and a direct interaction of FGF21 with FGFR1c may be an important step in receptor-mediated FGF21 signaling.

A fluorescent-based HPLC assay for quantification of cysteine and cysteamine adducts in Escherichia coli-derived proteins.

Recombinant proteins expressed in Escherichia coli are often produced as unfolded, inactive forms accumulated in inclusion bodies. Redox-coupled thiols are typically employed in the refolding process in order to catalyze the formation of correct disulfide bonds at maximal folding efficiency. These thiols and the recombinant proteins can form mixed disulfide bonds to generate thiol-protein adducts. In this work, we apply a fluorescent-based assay for the quantification of cysteine and cysteamine adducts as observed in E. coli-derived proteins. The thiols are released by reduction of the adducted protein, collected and labeled with a fluorescent reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The derivatized thiols are separated by reversed-phase HPLC and can be accurately quantified after method optimization. The estimated thiol content represents total amount of adducted forms present in the analyzed samples. The limit of quantification (LOQ) was established; specifically, the lowest amount of quantifiable cysteine adduction is 30 picograms and the lowest amount of quantifiable cysteamine adduction is 60 picograms. The assay is useful for quantification of adducts in final purified products as well as in-process samples from various purification steps. The assay indicates that the purification process accomplishes a decrease in cysteine adduction from 0.19 nmol adduct/nmol protein to 0.03 nmol adduct/nmol protein as well as a decrease in cysteamine adduction from 0.24 nmol adduct/nmol protein to 0.14 nmol adduct/nmol protein.

Binding characteristics of tumor necrosis factor receptor-Fc fusion proteins vs anti-tumor necrosis factor mAbs.

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.