A critical review of recent trends, and a future perspective of optical spectroscopy as PAT in biopharmaceutical downstream processing

As competition in the biopharmaceutical market gets keener due to the market entry of biosimilars, process analytical technologies (PATs) play an important role for process automation and cost reduction. This article will give a general overview and address the recent innovations and applications of spectroscopic methods as PAT tools in the downstream processing of biologics. As data analysis strategies are a crucial part of PAT, the review discusses frequently used data analysis techniques and addresses data fusion methodologies as the combination of several sensors is moving forward in the field. The last chapter will give an outlook on the application of spectroscopic methods in combination with chemometrics and model predictive control (MPC) for downstream processes

Process monitoring of virus-like particle reassembly by diafiltration with UV/Vis spectroscopy and light scattering

Virus‐like particles (VLPs) have shown great potential as biopharmaceuticals in the marketand in clinics. Nonenveloped, in vivo assembled VLPs are typically disassembled andreassembled in vitro to improve particle stability, homogeneity, and immunogenicity. At theindustrial scale, cross‐flow filtration (CFF) is the method of choice for performingreassembly by diafiltration. Here, we developed an experimental CFF setup with an on‐linemeasurement loop for the implementation of process analytical technology (PAT). Themeasurement loop included an ultraviolet and visible (UV/Vis) spectrometer as well as alight scattering photometer. These sensors allowed for monitoring protein concentration,protein tertiary structure, and protein quaternary structure. The experimental setup wastested with three Hepatitis B core Antigen (HBcAg) variants. With each variant, threereassembly processes were performed at different transmembrane pressures (TMPs).While light scattering provided information on the assembly progress, UV/Vis allowed formonitoring the protein concentration and the rate of VLP assembly based on themicroenvironment of Tyrosine‐132. VLP formation was verified by off‐line dynamic lightscattering (DLS) and transmission electron microscopy (TEM). Furthermore, the experi-mental results provided evidence of aggregate‐related assembly inhibition and showedthat off‐line size‐exclusion chromatography does not provide a complete picture of theparticle content. Finally, a Partial‐Least Squares (PLS) model was calibrated to predict VLPconcentrations in the process solution.Q²values of 0.947–0.984 were reached for thethree HBcAg variants. In summary, the proposed experimental setup provides a powerfulplatform for developing and monitoring VLP reassembly steps by CFF

Modeling the impact of amino acid substitution in a monoclonal antibody on cation exchange chromatography

A vital part of biopharmaceutical research is decision making around which lead candidate should be progressed in early-phase development. When multiple antibody candidates show similar biological activity, developability aspects are taken into account to ease the challenges of manufacturing the potential drug candidate. While current strategies for developability assessment mainly focus on drug product stability, only limited information is available on how antibody candidates with minimal differences in their primary structure behave during downstream processing. With increasing time-to-market pressure and an abundance of monoclonal antibodies (mAbs) in development pipelines, developability assessments should also consider the ability of mAbs to integrate into the downstream platform. This study investigates the influence of amino acid substitutions in the complementarity-determining region (CDR) of a full-length IgG1 mAb on the elution behavior in preparative cation exchange chromatography. Single amino acid substitutions within the investigated mAb resulted in an additional positive charge in the light chain (L) and heavy chain (H) CDR, respectively. The mAb variants showed an increased retention volume in linear gradient elution compared with the wild-type antibody. Furthermore, the substitution of tryptophan with lysine in the H-CDR3 increased charge heterogeneity of the product. A multiscale in silico analysis, consisting of homology modeling, protein surface analysis, and mechanistic chromatography modeling increased understanding of the adsorption mechanism. The results reveal the potential effects of lead optimization during antibody drug discovery on downstream processing