Higher Order Structure Characterization of Two Interdomain Disulfide Bond Variants of a Bispecific Monoclonal Antibody.

Characterization and understanding of protein higher order structure (HOS) is essential at all stages of biologics development. Here, two folding variants of a bispecific monoclonal antibody, the correctly folded form and an alternative configuration with reduced potency, were characterized by several HOS characterization techniques. Specifically, differential scanning calorimetry (DSC), circular dichroism (CD), Fourier-transform infrared spectroscopy (FTIR), Raman and Raman optical activity (ROA) spectroscopy were used together to elucidate the impacts of disulfide bond scrambling in the fused scFv domains on the structure and thermal stability of the antibody.

Evaluating Clinical Safety and Analytical Impact of Subvisible Silicone Oil Particles in Biopharmaceutical Products.

Silicone oil is a commonly used lubricant in pre-filled syringes (PFSs) and can migrate over time into solution in the form of silicone oil particles (SiOPs). The presence of these SiOPs can result in elevated subvisible particle counts in PFS drug products compared to other drug presentations such as vials or cartridges. Their presence in products presents analytical challenges as they complicate quantitation and characterization of other types of subvisible particles in solution.

Rapid Quantitation of Various Therapeutic Monoclonal Antibodies Using Membranes with Fc-Specific Ligands.

Therapeutic monoclonal antibodies (mAbs) provide effective treatments for many diseases, including cancer, autoimmune disorders, and, lately, COVID-19. Monitoring the concentrations of mAbs is important during their production and subsequent processing. This work demonstrates a 5 min quantitation of most human immunoglobulin G (IgG) antibodies through capture of mAbs in membranes modified with ligands that bind to the fragment crystallizable (Fc) region.

Predicting Experimental Values and the Effects of Histidine Charge States for Monoclonal Antibodies Using Coarse-Grained Molecular Simulations.

Static light scattering (SLS) was used to characterize five monoclonal antibodies (MAbs) as a function of total ionic strength (TIS) at pH values between 5.5 and 7.0.

Best Practices for Aggregate Quantitation of Antibody Therapeutics by Sedimentation Velocity Analytical Ultracentrifugation.

Analytical ultracentrifugation (AUC) is a critical analytical tool supporting the development and manufacture of protein therapeutics. AUC is routinely used as an assay orthogonal to size exclusion chromatography for aggregate quantitation. This article distills the experimental and analysis procedures used by the authors for sedimentation velocity AUC into a series of best-practices considerations.

Revisit PEG-Induced Precipitation Assay for Protein Solubility Assessment of Monoclonal Antibody Formulations.

Purpose: Protein solubility is an important attribute of pharmaceutical monoclonal antibody (MAb) formulations, particularly at high MAb concentrations. PEG-induced protein precipitation has been routinely used to assess protein solubility. To provide insights for better understanding and implementation of PEG-induced protein precipitation assay, this work compares different solubility measures and examines their relevance to loss of protein solubility in concentrated formulations.

Quantitation of soluble aggregates by sedimentation velocity analytical ultracentrifugation using an optical alignment system - Aspects of method validation.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) is routinely used for quantitation of soluble aggregates as an orthogonal technique to size-exclusion chromatography (SEC). SV-AUC presents many advantages over the SEC, yet lower precision of aggregate quantitation by SV-AUC often complicates comparison between aggregate values generated by these techniques and subsequent decision making. In an earlier report, we described the development of an optical alignment (OA) system and evaluated the intermediate precision of aggregate quantitation offered by the OA.

An optical alignment system improves precision of soluble aggregate quantitation by sedimentation velocity analytical ultracentrifugation.

Appropriate characterization of soluble aggregates is an important aspect of biologics development and manufacturing, and sedimentation velocity analytical ultracentrifugation (SV-AUC) is often used an orthogonal technique to size-exclusion chromatography (SEC) for this purpose. Precise quantification of low levels of soluble aggregates by SV-AUC can be adversely impacted by improper cell alignment. This report describes the development of an optical system capable of quantifying cell alignment that affords a substantial improvement compared to historical approaches.

Estimating analytical variability in two-dimensional data.

Throughout the course of drug development there are many instances in which a variability assessment within a set of analytical data is required, which may be challenging for techniques that produce two-dimensional data. This note describes an interval-based approach to variability assessment and demonstrates its applicability for analysis of near-UV circular dichroism (CD) spectra. The approach is generalizable and could be applied to two-dimensional data from other analytical techniques as well.

Technical decision-making with higher order structure data: specific binding of a nonionic detergent perturbs higher order structure of a therapeutic monoclonal antibody.

Robust higher order structure (HOS) characterization capability and strategy are critical throughout biopharmaceutical development from initial candidate selection and formulation screening to process optimization and manufacturing. This case study describes the utility of several orthogonal HOS methods as investigational tools during purification process development. An atypically high level of residual detergent in a development drug substance batch of a therapeutic monoclonal antibody triggered a root cause investigation.

Delicate balance between functionally required flexibility and aggregation risk in a β-rich protein.

Susceptibility to aggregation is general to proteins because of the potential for intermolecular interactions between hydrophobic stretches in their amino acid sequences. Protein aggregation has been implicated in several catastrophic diseases, yet we still lack in-depth understanding about how proteins are channeled to this state. Using a predominantly β-sheet protein whose folding has been explored in detail, cellular retinoic acid-binding protein 1 (CRABP1), as a model, we have tackled the challenge of understanding the links between a protein's natural tendency to fold, 'breathe', and function with its propensity to misfold and aggregate.

The Role of Aromatic-Aromatic Interactions in Strand-Strand Stabilization of β-Sheets.

Aromatic-aromatic interactions have long been believed to play key roles in protein structure, folding, and binding functions. However, we still lack full understanding of the contributions of aromatic-aromatic interactions to protein stability and the timing of their formation during folding. Here, using an aromatic ladder in the β-barrel protein, cellular retinoic acid-binding protein 1 (CRABP1), as a case study, we find that aromatic π stacking plays a greater role in the Phe65-Phe71 cross-strand pair, while in another pair, Phe50-Phe65, hydrophobic interactions are dominant.

Early folding events protect aggregation-prone regions of a β-rich protein.

Protein folding and aggregation inevitably compete with one another. This competition is even keener for proteins with frustrated landscapes, such as those rich in β structure. It is interesting that, despite their rugged energy landscapes and high β sheet content, intracellular lipid-binding proteins (iLBPs) appear to successfully avoid aggregation, as they are not implicated in aggregation diseases.

Signal relay from sensory rhodopsin I to the cognate transducer HtrI: assessing the critical change in hydrogen-bonding between Tyr-210 and Asn-53.

Sensory rhodopsin I (SRI) from Halobacterium salinarum mediates both positive and negative phototaxis in a light-dependent manner. SRI photoactivation elicits extensive structural changes which are transmitted to the cognate transducer (HtrI). The atomic structure of the SRI-HtrI complex has not been solved yet and, therefore, details on the interaction which define the binding site between receptor and transducer are missing.

Flexibility of the cytoplasmic domain of the phototaxis transducer II from Natronomonas pharaonis.

Chemo- and phototaxis systems in bacteria and archaea serve as models for more complex signal transduction mechanisms in higher eukaryotes. Previous studies of the cytoplasmic fragment of the phototaxis transducer (pHtrII-cyt) from the halophilic archaeon Natronomonas pharaonis showed that it takes the shape of a monomeric or dimeric rod under low or high salt conditions, respectively. CD spectra revealed only approximately 24% helical structure, even in 4 M KCl, leaving it an open question how the rod-like shape is achieved.

FT-IR difference spectroscopy elucidates crucial interactions of sensory rhodopsin I with the cognate transducer HtrI.

The phototaxis receptor sensory rhodopsin I (SRI) from Halobacterium salinarum interacts with its cognate transducer (HtrI) forming a transmembrane complex. After light excitation of the chromophore all-trans retinal, SRI undergoes structural changes that are ultimately transmitted to HtrI. The interaction of SRI with HtrI results in the closure of the receptor's proton pathway, which renders the photocycle recovery kinetics of SRI pH-independent.

Shape and oligomerization state of the cytoplasmic domain of the phototaxis transducer II from Natronobacterium pharaonis.

Phototaxis allows archaea to adjust flagellar motion in response to light. In the photophobic response of Natronobacterium pharaonis, light-activated sensory rhodopsin II causes conformational changes in the transducer II protein (pHtrII), initiating the two-component signaling system analogous to bacterial chemotaxis. pHtrII's cytoplasmic domain (pHtrII-cyt) is homologous to the cytoplasmic domains of eubacterial chemotaxis receptors.