Size-exclusion HPLC, coupled with ICP/MS (SEC-ICP/MS), was applied to assess metal binding to Immunoglobulin G (IgG) mAbs. IgG1s and IgG2 drugs were investigated. Cu(II) was selected as the metal of interest due to its known ability to bind and enhance the degradation of mAbs. In conjunction with other techniques, this method may provide in-depth knowledge to probe the mechanisms of metal-induced mAb degradation and biophysical properties in biologics process development.

Previous work has demonstrated that 20 mM peracetic acid is a highly effective sporicidal solution that is compatible with 316L stainless steel and can be utilized to mitigate bioburden contamination. Perasan® A (Enviro Tech), a stabilized peracetic acid solution, was studied as an alternative sporicidal cleaning solution that could be implemented in manufacturing. Work presented herein demonstrated Perasan® A is a suitable replacement for peracetic acid.

Applied Molecular Transport (symbol: AMTI) utilizes a naturally occurring bacterial toxin transport system to deliver oral therapeutics payloads in a targeted fashion. This targeted transport mechanism effectively crosses the protective barrier of the intestinal epithelium (IE), to facilitates direct contact with the immune system. This direct access to the immune system suggests an effective treatment of immune dysregulation and the restoration of immune homeostasis. To that end, AMT platform constructed a pipeline of therapeutic proteins, consisting of a therapeutic protein such as interleukin, covalently attached through an amino acid short sequence linker to a bacterial carrier domain. These proteins are then expressed in E. coli and isolated as inclusion bodies. AMT has developed a proprietary downstream process to solubilize the tight knot inclusion bodies, subsequently refolding the protein, by which restoring both, its transport activity and therapeutic effectiveness. Subsequently, AMT built a downstream purification platform employing unique chromatography resins and classical preparative protein chromatography protocols excluding the need for affinity-type purification such as Protein A. These processes were scaled up and are successfully executed by the company internal CMO. This DSP development work has yielded several patent applications.

This talk will cover computational modelling that can be used to simulate chromatographic process performance, including economic analysis utilizing process modeling, as well as presenting case studies and lessons learned from modeling vs. experimentation.

Design of Experiments (DoE) is commonly used during process development, however the type of design that can be utilized depends on the objective of the study. This presentation discusses the establishment of appropriate designs and subsequent analysis for the optimization of individual chromatography steps or establishing the robustness of a platform purification process containing affinity, cation exchange (CEX) and anion exchange (AEX) chromatography steps, using a combination of risk assessment and rational design selection.

Hydrophobic interaction chromatography (HIC) provides a facile method of biomacromolecule separation, relying more on the global characteristics of solvated proteins rather than the charge-state specificity of ion exchange methods. HIC separations on polyester C-CP fibers provide for high-velocity separations without mass transfer limitations. We demonstrate these concepts in terms of analytical and preparative protein separations. The same protocols allow for rapid purification of therapeutically-relevant exosomes and virus particles.

Laterally-fed membrane chromatography (LFMC) combine high-speed with high resolution in separation. In this presentation we discuss the use of the latest version of LFMC devices, the i.e., the z²LFMC device, for purification of PEGylated proteins. The purification of these protein-polymer conjugate molecules, which are increasingly being used as biopharmaceuticals is challenging as the molecules being separated differ only very slightly in terms of their physicochemical properties. Our results show that an z²LFMC device could be used to separate a specific PEGylated protein from impurities, including very closely related molecules such as positional isomers, that are very difficult to separate using other devices.   

Affinity tags have been widely applied to purification of G protein-coupled receptors (GPCRs) for structural studies by X-ray crystallography and other biophysical techniques. However, the binding affinity and elution conditions used for commercially available resin/tag combinations don't always meet the requirements of downstream applications. We developed a novel calcium-dependent, EF-based affinity system that allows capture and high recovery of GPCR from dilute solutions containing detergents, salts, and glycerol. The binding of the EF1 tag to the resin at these conditions is very strong (high picomolar to low nanomolar range) that allows efficient purification without any loss of the target protein. The elution of the captured receptor is achieved by the addition of EDTA, at very mild conditions that do not hinder the activity of this labile protein. The small (4.5 kDa) EF1 tag was successfully applied for purification of GPCR expressed in E. coli, mammalian, as well as insect cells. EF1 tag can be attached either to the N- or C-termini of the target receptor and did not interfere with its biological activity as demonstrated by ligand binding and G protein activation. Furthermore, surface plasmon resonance studies were performed on EF-tagged receptor and demonstrated the utility of this tag for surface capture of the protein for subsequent biophysical characterization.

Proteins are extraordinarily diverse, so generic approaches are needed to harness their potential. The SpyTag peptide spontaneously reacts with the protein SpyCatcher. SpyTag-linked proteins can be purified by Spy&Go, detected sensitively, anchored irreversibly, or linked to an effector or multimerization toolbox. Applications include SARS-CoV-2 and influenza vaccination, diagnostics and antibodies.

Membrane proteins are prime targets for drug discovery, yet challenging to purify and manipulate due to their facile aggregation. Our laboratory develops methods to streamline their isolation by direct transfer from crude membrane to water-soluble Peptidiscs in the presence of native lipids. This workflow increases stability and facilitates downstream exploitation in biochemical, structural and pharmacologic laboratory settings.

Enzymes in multi-step metabolic pathways utilize an array of regulatory mechanisms to maintain a delicate homeostasis. Carrier proteins, in particular, play an essential role in shuttling substrates between appropriate enzymes in metabolic pathways. Though hypothesized, allosteric regulation of enzyme activity has never before been demonstrated for any carrier protein-dependent pathway, studying these mechanisms has remained challenging due to the transient and dynamic nature of protein-protein interactions, the vast diversity substrates, and substrate instability. Here, we demonstrate a unique communication mechanism between the acyl carrier protein (ACP) and partner enzymes using solution NMR spectroscopy and molecular dynamics to elucidate allostery that is dependent on fatty-acid chain length. We demonstrate, for the first time, that partner enzymes can allosterically distinguish between chain lengths via protein-protein interactions via structural features of substrate-sequestered ACP, without the need for stochastic chain flipping. These results illuminate details of cargo communication by ACP that can serve as a foundation for engineering carrier protein dependent pathways for specific, desired products.