A new baseline (30 s) was then established and then the association (180 s) and dissociation (360 s) of FcRIIIa-H6 was measured by dipping the biosensor into solutions of FcRIIIa-H6 and PBS respectively

A new baseline (30 s) was then established and then the association (180 s) and dissociation (360 s) of FcRIIIa-H6 was measured by dipping the biosensor into solutions of FcRIIIa-H6 and PBS respectively. immobilized FcRIIIa, were developed to assess FcRIIIa affinity in kinetic binding studies. The HM-Fc and Man5-Fc were highly similar to one another with high affinity for FcRIIIa, while GlcNAc-Fc had weak affinity, and the non-glycosylated N297Q-Fc had no measurable affinity for FcRIIIa. These four IgG1 Fc glycoforms were also evaluated in terms of physical and chemical stability profiles, and then used as model system to mathematically assess overall biosimilarity, as described in a series of companion GSK3368715 dihydrochloride papers. analytical tests can be utilized to determine similarity in biosimilar studies and analysis assessments, we have developed a series of well-defined IgG1 Fc glycoforms as a model system. The use of a series of well-defined glycoproteins in these studies should enable identification of important structural and biological features for TIL4 comparability and biosimilarity analyses. A series of IgG1 Fc glycoforms were chosen as the protein model system for biosimilarity analysis because as a fragment of full-length IgG1 it is a simpler system to study but contains the CH2 and CH3 constant domains which are present in all human IgG1 based mAb therapeutics. The Fc region is critical to antibody function in that it mediates effector functions such as antibody dependent cellular cytotoxicity (ADCC)15C24 and complement dependent cytotoxicity (CDC)25C27. The Fc region of an IgG1 is also important in antibody clearance because binding of the Fc region to the neonatal Fc receptor (FcRn) increases half-life28C31. In addition, N-linked glycosylation at asparagine 297 (N297) in the Fc region is known to modulate the biological activity19,32C39 and physical properties of IgG1 Fc,1,32,40C48 and this can be used to establish similarities and differences between the members of this model system. A series of sequentially truncated glycoforms of IgG1 Fc (Figure 1), which differ only in the size of the N-linked glycan at N297 or a single conservative amino acid mutation (N297Q), were chosen as members for the model system. Previous studies have indicated that these glycoforms would display a range of biological activities and physical properties1,32,45 that would be advantageous for our biosimilarity studies. Open in a separate window Figure 1 Production of homogenous IgG1 Fc glycoforms. HM-Fc was expressed in glycoengineered and utilized as a precursor for generation of GlcNAc-Fc and Man5-Fc. N297Q-Fc was recombinantly expressed in a different yeast strain. It was first necessary to develop laboratory production methods to produce sufficient quantities (100 mg each) of the well-defined IgG1 Fc glycoforms to conduct the wide range of analytical tests necessary for biosimilarity analysis. Presented here are the methods established to produce the glycoforms shown in Figure 1 through yeast expression, purification and enzymatic synthesis. Also presented in this work is the initial biochemical characterization of these glycoforms, the development of binding assays for IgG1 Fc binding to an Fc receptor using biolayer interferometry (BLI), and determination of the affinity of the different IgG1 Fc glycoforms for that Fc receptor as an initial evaluation of biological activity. Subsequent companion papers examine the physical and chemical stability profiles of these IgG1 Fc glycoforms, and the use of the resulting physicochemical data sets, to develop a mathematical algorithm for biosimilarity assessments.49C51 Materials and Methods Materials GSK3368715 dihydrochloride Yeast nitrogen base (YNB) was obtained from Sunrise Biosciences, and Bacto? Tryptone and Yeast Extract was purchased from Becton Dickinson and Company (Franklin Lakes, NJ). Antifoam 204 was obtained from Sigma-Aldrich (St. Louis, MO). Certified ACS grade crystalline sucrose was purchased from Fisher Scientific (Pittsburg, PA). Boc-triglycine was supplied by Bachem Americas, Inc. (Torrance, CA). The enzymes PNGase F, Sortase, and -1,2-mannosidase (BT3990, -1,2-mannosidase) were produced inhouse.52C55 Endoglycosidase H and restriction enzymes were obtained from New England Biolabs (Ipswich, MA). Protein G resin was produced by coupling protein G (recombinantly expressed in E. coli)56 with Sepharose? CL-4B (Sigma-Aldrich, St Louis, MO) using divinyl sulfone as a coupling reagent.57 General chemicals were purchased from Sigma-Aldrich and Fisher Scientific unless otherwise noted. Production and initial characterization of IgG1 Fc Glycoforms Expression of High Mannose IgG1 Fc (HM-Fc) HM-Fc was expressed in a glycosylationdeficient strain of (an OCH1 and PNO1 deleted, IgG1 Fc expressing, SMD1168 strain of produced by Xiao et.al.58 was utilized) using glycerol (growth phase) and methanol (induction phase) as carbon sources in a NBS BioFlo 415 GSK3368715 dihydrochloride fermenter (Eppendorf). The starter culture (2 mL) was allowed to grow at 25C for about 72 hrs in YPD media (1% yeast extract, 2% peptone, 5% glucose and zeocin 100 g/ml). This starter GSK3368715 dihydrochloride culture was then inoculated into 250 mL of YPD.