Muppalla Sukumar

Opalescent Appearance of an IgG1 Antibody at High Concentrations and Its Relationship to Noncovalent Association

AbstractPurpose. Therapeutic antibodies are often formulated at a high concentration where they may have an opalescent appearance. The aim of this study is to understand the origin of this opalescence, especially its relationship to noncovalent association and physical stability. Methods. The turbidity and the association state of an IgG1 antibody were investigated as a function of concentration and temperature using static and dynamic light scattering, nephelometric turbidity, and analytical ultracentrifugation. Results. The antibody had increasingly opalescent appearance in the concentration range 5-50 mg/ml. The opalescence was greater at refrigerated temperature but was readily reversible upon warming to room temperature. Turbidity measured at 25°C was linear with concentration, as expected for Rayleigh scatter in the absence of association. In the concentration range 1-50 mg/ml, the weight average molecular weights were close to that expected for a monomer. Zimm plot analysis of the data yielded a negative second virial coefficient, indicative of attractive solute-solute interactions. The hydrodynamic diameter was independent of concentration and remained unchanged as a function of aging at room temperature. Conclusions. The results indicate that opalescent appearance is not due to self-association but is a simple consequence of Rayleigh scatter. Opalescent appearance did not result in physical instability.

Phage passage after extended processing in small‐virus‐retentive filters

Retention of a two small phages (ΦX‐174 and pp7) by direct‐flow small‐virus‐retentive filters [Viresolve NFP (normal‐flow parvovirus), Virosart CPV (canine parvovirus), Ultipor DV20 and Planova 20N] was studied using a commercial‐process fluid. Phage passage occurred in each filter type, particularly when overloaded with phage. Clearances of pp7 and ΦX‐174 were similar for any given filter brand, arguing that the two phages are equivalent for testing small‐virus‐retentive filters. The patterns of flux under constant pressure and instantaneous LRV (log reduction value) in relationship to cumulative phage load differed between brands, consistent with the current industry understanding that each brand possesses specific performance attributes. Phages are a powerful and universal tool for evaluating filter performance. Validation of filter performance with phages such as pp7 or ΦX‐174 as models for small mammalian viruses represents an attractive alternative to the current practice.

Hybrid insulin cocrystals for controlled release delivery

The ability to tailor the release profile of a drug by manipulating its formulation matrix offers important therapeutic advantages. We show here that human insulin can be cocrystallized at preselected ratios with the fully active lipophilically modified insulin derivative octanoyl-Nε-LysB29–human insulin (C8-HI). The cocrystal is analogous to the NPH (neutral protamine Hagedorn) crystalline complex formed with human insulin, which is commonly used as the long-acting insulin component of diabetes therapy. The in vitro and in vivo release rates of the cocrystal can be controlled by adjusting the relative proportions of the two insulin components. We identified a cocrystal composition comprising 75% C8-HI and 25% human insulin that exhibits near-ideal basal pharmacodynamics in somatostatin-treated beagle dogs. The dependence of release rate on cocrystal ratio provides a robust mechanism for modulating insulin pharmacodynamics. These findings show that a crystalline protein matrix may accommodate a chemical modification that alters the dissolution rate of the crystal in a therapeutically useful way, yet that is structurally innocuous enough to preserve the pharmaceutical integrity of the original microcrystalline entity and the pharmacological activity of the parent molecule.

Quantification and characterization of subvisible proteinaceous particles in opalescent mAb formulations using micro-flow imaging.

Micro-flow imaging (MFI) has been shown to be more sensitive than light obscuration (LO) methods for measuring subvisible proteinaceous particles in protein formulations. Given the potential challenges in detecting particulates in opalescent mAb formulations, the accuracy of MFI to size and count particles in opalescent solutions was investigated and compared to LO and membrane microscopy methods. Proteinaceous monoclonal antibody (mAb) particles, generated either by chemical denaturation or agitation stress, polystyrene and glass particles were used as model systems for measurements in opalescent mAb solutions. The sizing and counting accuracies of MFI were unaffected by the opalescence of the medium. Using glass particles as a model system for proteinaceous particles, MFI was able to detect relatively low particle concentrations (approximately 10/mL) in opalescent solutions. MFI showed excellent linearity (R(2) = 0.9969) for quantifying proteinaceous particles in opalescent solutions over a wide range of particle concentrations (approximately 20-160,000/mL). Analyses of MFI particle image intensities revealed significant differences in the transparency of proteinaceous particles as a function of their size and mode of generation. LO method significantly underestimated proteinaceous particles, particularly those in the 2-10 microm size range. The less opaque proteinaceous particles were relatively more underestimated by the LO method in opalescent solutions.

High-throughput analysis of concentration-dependent antibody self-association

Monoclonal antibodies are typically monomeric and nonviscous at low concentrations, yet they display highly variable associative and viscous behavior at elevated concentrations. Although measurements of antibody self-association are critical for understanding this complex behavior, traditional biophysical methods are not capable of characterizing such concentration-dependent self-association in a high-throughput manner. Here we describe a nanoparticle-based method, termed self-interaction nanoparticle spectroscopy, that is capable of rapidly measuring concentration-dependent self-interactions for three human monoclonal antibodies with unique solution behaviors. We demonstrate that gold nanoparticles conjugated with antibodies at low protein concentrations (<40 μg/mL) display self-association behavior (as measured by the interparticle distance-dependent plasmon wavelength) that is well correlated with static light-scattering measurements obtained at three orders of magnitude higher antibody concentrations. Using this methodology, we find that the antibodies display a complex pH-dependent self-association behavior that is strongly influenced by the solution ionic strength. Importantly, we find that a polyclonal human antibody is nonassociative for all solution conditions evaluated in this work, suggesting that antibody self-association is more specific than previously realized. We expect that our findings will guide rational manipulation of antibody phase behavior, and enable studies that elucidate sequence and structural determinants of antibody self-association.

Local interactions in a Schellman motif dictate interhelical arrangement in a protein fragment

BACKGROUND As an approach to understanding the role of local sequence in determining protein tertiary structure, we have examined the conformation of a 23-residue peptide fragment corresponding to the structurally conserved helix-Schellman motif-helix (H-Sm-H) domain (residue 10-32) of cellular retinoic acid binding protein, along with variants designed to probe the contributions of the helix-terminating Gly23 and the hydrophobic interactions between Leu 19 and Val24 in stabilizing the Schellman motif and hence helix termination. RESULTS In aqueous solution, NMR data for the H-Sm-H peptide show that it samples a largely helical conformation with a break in the helix at the point of the turn in the protein. The data also establish the presence of local hydrophobic interactions and intramolecular hydrogen bonds characteristic of a Schellman motif. Absence of helix termination in trifluoroethanol, a solvent known to disrupt hydrophobic interactions, along with an analysis of H alpha chemical shifts and NOEs in the variant peptides, suggest a major role for glycine in terminating the helix, with local hydrophobic interactions further stabilizing the Schellman motif. CONCLUSIONS The presence of a Schellman motif in this isolated fragment in water is governed by local interactions and specifies the interspatial arrangement of the helices. This observation underlines the structure predictive value of folding motifs. As proposed for a Schellman motif, helix termination in this fragment is dictated by the local distribution of polar/apolar residues, which is reminiscent of the binary code for protein folding.

Non-native intermediate conformational states of human growth hormone in the presence of organic solvents

Purpose.Manufacturing processes expose protein pharmaceuticals to organic solvents that may perturb the native folded state, increasing the potential for irreversible aggregation or surface adsorption. The aim of this study was to characterize the conformational states of human growth hormone (hGH) in aqueous ethanolic solutions.Methods.The higher order structure of hGH was investigated using far- and near-UV circular dichroism (CD) and fluorescence spectroscopy as orthogonal techniques, and the hydrodynamic size was monitored using dynamic light scattering.Results.CD data suggested that the secondary structure of hGH remained unchanged up to 50\% (v/v) ethanol, but the tertiary structure was perturbed at ã20% ethanol. Fluorescence anisotropy, however, showed that the mobility of the buried Trp residue was restricted even at 30% ethanol, suggesting a differently packed structural core in 30% ethanol relative to the native structure. Consistent with this result, thermal unfolding of hGH in 30% ethanol was more facile compared to that in 0% and 20% ethanol. At >40% ethanol, fluorescence data were consistent with increased solvent exposure of the tryptophan.Conclusions.The results point to progressive unfolding of hGH that increases solvent exposure of the hydrophobic core as a function of ethanol concentration and suggest that non-native intermediate states are populated in 30–60% ethanol.

Surface Interactions of Monoclonal Antibodies Characterized by Quartz Crystal Microbalance with Dissipation: Impact of Hydrophobicity and Protein Self-Interactions

Surface adsorption of two monoclonal antibodies (mAb1 and mAb2), with widely different hydrophobicity and self-association behavior in solution, was examined by quartz crystal microbalance with dissipation monitoring to understand how adsorption and protein self-interactions near the surface are impacted by their intrinsic properties. The dependence of mass and viscoelastic properties of the adsorbed protein layer on the type of surface, presence of a surfactant, protein concentration, and pH were examined. Adsorption was significantly reduced in the presence of surfactant for both proteins, but for the more hydrophobic mAb2, residual adsorption remained on polystyrene (PS) and Teflon surfaces. Protein concentration had little impact on the adsorbed protein mass for silicon dioxide surface but had a significant impact for PS and Teflon surfaces. At high protein concentrations, an irreversible layer formed first upon which a reversible layer builds. Reversible adsorption was significantly greater at higher protein concentrations and significantly higher for mAb2, consistent with its higher propensity to reversibly self-associate in solution. The viscoelastic properties suggest that adsorbed protein layer at high protein concentrations is more hydrated. The adsorbed protein layer at lower pH was more hydrated, and possibly more unfolded, consistent with the behavior of the antibody in bulk solution.