Advances in structural biology have yielded an abundance of high-resolution descriptions of folded states of proteins. However, atomic-level understanding of the structure and dynamics of unfolded proteins under folding conditions has remained elusive due to the enormous conformational heterogeneity of the unfolded ensemble and the very low population of unfolded states under folding conditions. We have recently combined a series of time-resolved biophysical techniques (time-resolved FRET and small-angle x-ray scattering) with computational simulations to obtain a high-resolution description of an unfolded protein ensemble under folding conditions. These unfolded states are characterized by discernible sequence-specific conformational preferences. These preferences are averaged over by conformational fluctuations, giving rise to ensemble-averaged properties consistent with those of random coils. These findings increase the understanding of functional and pathological interactions involving unfolded forms of proteins, which can be relevant to human pathology and biotechnology applications.

Enzymes are ubiquitous in nature and play a central role in medicine as drug targets and also as therapeutics in their own right. Thus simple and accurate enzyme activity assays are critical to the success of pharmaceutical science. Isothermal titration calorimetry (ITC) is emerging as a powerful tool for measuring the kinetics of challenging enzyme systems. Rather than tracking the concentrations of substrates and products over the course of a reaction, it detects the heat that is released or absorbed during catalysis in real time. Since almost all chemical reactions release or absorb heat, ITC can be applied nearly universally. The technique does not require customized reporter molecules, additional coupled enzymes, or post-reaction separation, as do many traditional enzyme assays. Furthermore, kinetic ITC experiments can be performed with conventional dilute enzymatic reaction mixtures, even with opaque samples, and require far less enzyme than ITC binding studies.

We have developed a suite of new ITC-based methods that rapidly yield the affinity and the mode of inhibitor binding, the rates of association and dissociation of inhibitors and allosteric modulators, and the full kinetic profiles of Bi-substrate enzymes. We have validated these approaches using trypsin/benzamidine, a panel of covalent and non-covalent inhibitors of prolyl oligopeptidase (POP), and M-type pyruvate kinase, illustrating the versatility of ITC-based enzyme kinetic assays.

Brightness is a unique fluctuation parameter of fluorescence correlation spectroscopy and provides a powerful tool for the study of protein interactions in living cells. This talk introduces the basics of brightness measurements along with a discussion of practical considerations in the choice of fluorescent label and the calibration measurements necessary for quantitative interpretation of brightness data. Protocols to identify and avoid common pitfalls, such as photobleaching and saturation, are addressed as well. The analysis of brightness data provides information about the average stoichiometry of fluorescently labeled protein complexes within the cell. While this presentation mainly focuses on the application of fluorescence correlation spectroscopy to determine the average stoichiometry of homo oligomers via brightness titration experiments, we also touch upon the characterization of hetero-protein complexes by extending brightness experiments to two-color fluorescence correlation spectroscopy.

Protein higher order structure (HOS) is an important product quality attribute that governs the structure function characteristics, safety, and efficacy of therapeutic proteins. Infrared (IR) spectroscopy has long been recognized as a powerful biophysical tool in determining protein secondary structure and monitoring the dynamic structural changes. Such biophysics analyses help establish process and product knowledge, understand the impact of upstream (cell culture) and downstream (purification) process conditions, create stable formulations, monitor product stability, and assess product comparability when process improvements are implemented (or establish biosimilarity to originator products). This paper provides an overview of a novel automated mid-IR spectroscopic technique called microfluidic modulation IR spectroscopy (MMS or MM-IR) for the characterization of protein secondary structure. The study demonstrates that MM-IR secondary structure analysis of therapeutic monoclonal antibodies (mAb) is comparable with a conventional Fourier transform IR (FTIR) method. More importantly the study shows MM-IR exhibits higher sensitivity and repeatability for low concentration samples over FTIR, as well as provides automated operation and superior robustness with simplified data analysis, increasing the utility of the instrument in determination of mAb secondary structure. Therefore, we propose that the MM-IR method can be widely applied in characterization and comparability /biosimilarity studies for biopharmaceutical process and product development.

We are developing micro- and nanofluidic devices to study virus assembly and disassembly at the single-particle level. Analysis of single particles provides unprecedented insight into biological processes that is often missed when a population is studied as an ensemble. To characterize capsid assembly of hepatitis B virus (HBV), we are using resistive-pulse sensing as a label-free, nondestructive technique. This single-particle method permits real-time detection and has sufficient sensitivity to monitor assembly at biologically relevant concentrations and over a range of reaction conditions. With these nanofluidic devices, we are evaluating how assembly effectors (or potential antivirals) modulate the assembly process and produce a variety of particle morphologies, including normal capsids, kinetically trapped intermediates, and aberrant structures. We are also studying how the presence of chaotropes, e.g., guanidine hydrochloride (GuHCl), leads to either assembly or disassembly of virus-like particles, depending on chaotrope concentration.

When issues arise in drug manufacturing facilities, drug shortages may follow. In fact, more than half of all drug shortages are caused by manufacturing and quality issues. This underscores the need for a robust and reliable drug supply chain. Modernizing pharmaceutical manufacturing technology is a promising approach to help reduce and prevent drug quality and shortage problems. In fact, other industrial sectors (such as the electronics, chemicals, and automobile industries) have embraced the use of advanced manufacturing technologies and have demonstrated improved quality, increased efficiency, and a reduced number of product failures. By adopting similar technological advances, the pharmaceutical industry can establish a more robust and flexible drug manufacturing process with fewer interruptions. This will minimize product failures and provide greater assurance that the product will consistently deliver the expected clinical performance.

FDA is undertaking a new approach by working closely with drug makers and other relevant stakeholders to ensure that cutting-edge, scientifically sound methods are used in drug manufacturing (including both biotechnology and small-molecule products). The new FDA approach aims to help the pharmaceutical industry adopt novel technologies in producing medicines that are consistently safe and effective. The new approach emphasizes the utilization of (1) FDA’s Emerging Technology Program to provide opportunities for early FDA-industry interactions during technology development, (2) integrated quality assessment to facilitate an effective regulatory evaluation of emerging technology in a drug application, (3) regulatory science and research to enhance scientific understanding of novel technologies and support risk assessments, and (4) close collaborations and coordination with other regulatory agencies to support harmonization of scientific and regulatory approaches or standards. This presentation will discuss FDA’s approach and experience with emerging technologies.

NMR spectroscopy is increasingly being recognized as an important tool for the characterization of protein therapeutics including monoclonal antibodies (mAbs). Owing to their large size and lack of stable isotope enrichments, traditional NMR structural approaches are not practical on large mAb therapeutics and therefore a number of alternative techniques have been proposed, including the 1D 1H PROFILE method as well as the 2D 1H-13C methyl correlation method. Both approaches have their relative strengths and weakness owing to the inherent sensitivity and resolution of the respective 1D and 2D modalities. In order to aid the adoption of NMR by the biopharmaceutical industry, establishing fit-for-purpose of the two methods for specific applications to mAb characterization is warranted.

Here, we present a comparative study of the 1D-PROFILE and 2D-methyl methods on a panel of mAb samples to determine the degree to which each method is suited to detect spectral difference between the samples and the degree of complementarity between the two methods. Results of the study illustrate how employing NMR as a multi-modal platform provides a more complete pictures of HOS. Furthermore, we will demonstrate how the combination of 1D 1H NMR with principal component analysis allows for detection of relevant spectral variation even with low experimental signal-to-noise. This allows for the potential characterization of protein therapeutics at low concentrations, low volumes, or at lower field strengths than commonly employed for protein characterization, thus greatly extending the applicability of NMR for HOS characterization of biotherapeutics.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is gaining more traction in biopharma for characterization and comparability between biosimilars and innovator biotherapeutics. Glycosylation is an important attribute that can significantly affect efficacy and safety. HDX-MS distinguished effects in structural dynamics caused by changes in terminal glycan structure within IgG1, and stronger effects were observed for interactions between IgG1 glycoforms and a soluble FcγR1a receptor. FcγR1a receptor recognizes the Fc portion of IgG1, and aIL8-Fc is a humanized IgG1κ that targets IL8. aIL8-hFc was expressed in CHO cells with restriction of glycotransferases, and three aIL8-hFc glycoforms (G0F, G2F, and SAF) were made using solid phase enzymatic remodeling. A robotic HDX-MS instrument measured deuterium uptake versus time for apo-FcγR1a, apo-aIL8-Fc, and holo-FcγR1a--aIL8-Fc complexes. Deuterium uptake rates were calculated for peptic peptides, and apo-mAb-glycoforms differing by as little as one terminal sugar group displayed distinct deuterium uptake patterns, even in Fab domain. Deuterium uptake by FcγR1a receptor also varied with contacting mAb-glycoform, indicating that glycan structure affects the contacting residues.

The SARS-CoV-2 spike (S) glycoprotein contains an immunodominant receptor-binding domain (RBD) targeted by most neutralizing antibodies (Abs) in COVID-19 patient plasma. Little is known about neutralizing Abs binding to epitopes outside the RBD and their contribution to protection. We discovered human monoclonal Abs (mAbs) which recognize the SARS-CoV-2 S N-terminal domain (NTD) and show that a subset of them potently neutralize SARS-CoV-2. We define an antigenic map of the SARS-CoV-2 NTD and identify a ‘supersite’ recognized by all known NTD-specific neutralizing mAbs. These mAbs protect hamsters from SARS-CoV-2 challenge, albeit selecting escape mutants in some animals. Indeed, all variants of concern harbor frequent escape mutations within the NTD supersite. The CAL.20C variant (B.1.427/B.1.429) was originally detected in California and is currently spreading throughout the US and 29 additional countries. We show that while the variant can escape some RBD-targeting neutralizing mAbs, it evades every tested NTD-targeting neutralizing mAb. This is owing the S13I mutation shifting signal peptide cleavage site, and the W152C mutation creating new disulfide bond at the NTD antigenic supersite. Likewise, plasma from vaccinated or convalescent individuals exhibited neutralizing titers that were reduced 3-6 fold against the CAL.20C variant. These data demonstrate ongoing selective pressure from NTD-specific neutralizing mAbs and the importance of NTD-specific neutralizing mAbs for protective immunity and vaccine design.