Simple, rapidly electroassembled thiolated PEG‐based sensor interfaces enable rapid interrogation of antibody titer and glycosylation

Abstract

Process conditions established during the development and manufacture of recombinant protein therapeutics dramatically impacts their quality and clinical efficacy. Technologies that enable rapid assessment of product quality are critically important. Here, we describe the development of sensor interfaces that directly connect to electronics and enable near real-time assessment of antibody titer and N-linked galactosylation. We make use of a spatially resolved electroassembled thiolated polyethylene glycol hydrogel that enables electroactivated disulfide linkages. For titer assessment, we constructed a cysteinylated protein G that can be linked to the thiolated hydrogel allowing for robust capture and assessment of antibody concentration. For detecting galactosylation, the hydrogel is linked with thiolated sugars and their corresponding lectins, which enables antibody capture based on glycan pattern. Importantly, we demonstrate linear assessment of total antibody concentration over an industrially relevant range and the selective capture and quantification of antibodies with terminal β-galactose glycans. We also show that the interfaces can be reused after surface regeneration using a low pH buffer.Our functionalized interfaces offer advantages in their simplicity, rapid assembly, connectivity to electronics, and reusability. As they assemble directly onto electrodes that also serve as I/O registers, we envision incorporation into diagnostic platforms including those in manufacturing settings. K E Y W O R D S antibody titer, critical quality attributes, near real time monitoring, N-linked glycosylation 1 | INTRODUCTION Recombinant antibody therapeutics constitute a large segment of the biologics market as they have shown clinical and commercial success in many therapeutic areas, including cancer, respiratory and autoimmune diseases, among others (Ecker et al., 2015; Kaplon & Reichert, 2019; Nelson et al., 2010). The development process for antibody therapeutics is costly and time-intensive due to the complexities associated with optimizing host cell lines, bioreactors, and processing conditions, along with the post-synthesis product

Protein G: β‐galactosidase fusion protein for multi‐modal bioanalytical applications

“…Lastly, we wanted to demonstrate the capability of protein G: β‐gal when used in an electroassembled sensor format. We first created a sensor interface directly assembled onto sensing electrodes via electronic signals, as described above, 3 and then used the same electrode to assess IgG level in a complex sample. This electroassembly methodology enables spatially defined functionalization of electronic surfaces 24,25 .…”

“…The electroassembled hydrogel is then overlayed with the recognition element (cys‐tagged protein G 31 ) whose cysteine tag engineered onto its C‐terminus allows it to covalently bond via disulfide bonds under an additional oxidative charge to the electrode (some thiol groups of the PEG are electrochemically oxidized to sulfenic acid and this covalently couples to the thiols of the terminal cysteines engineered onto protein G). This hydrogel film thus forms the detection interface 3,26,31 . To measure IgG, the detection interface was then incubated with IgG (serving as the analyte) and protein G: β‐gal of this work (serving as the reporter).…”

“…A thiolated polyethylene glycol hydrogel interface was electroassembled as described previously 3 . Briefly, the interfaces were constructed by immersing the surface of a 2 mm diameter gold standard electrode (working electrode) in a solution containing Fc (5 mM) and PEG thiol (50 mg/ml) along with a platinum wire (counter electrode) and an Ag/AgCl reference electrode.…”

“…This hydrogel film thus forms the detection interface. 3,26,31 To measure IgG, the detection interface was then incubated with IgG (serving as the analyte) and protein G: β-gal of this work (serving as the reporter).…”

“…β‐gal provides a significant benefit compared to other reporter enzymes (e.g., horseradish peroxide, HRP) which involve more complicated electron transfer schemes as they often employ both a substrate and a redox mediator in order to enable an electrochemical response 16,17 . While β‐gal activity is associated with its tetrameric form, 18,19 chimeric proteins expressed from E. coli are free to associate into active conformations and importantly, since the protein when expressed in E. coli is not glycosylated, its use in evaluating an analyte's glycosylation state is facilitated 3 . Overall, our protein G: β‐gal may serve as a versatile reporter in a variety of analytical applications.…”