Bruce A. Kerwin

Highly aggregated antibody therapeutics can enhance the in vitro innate and late-stage T-cell immune responses

Background: Aggregated biotherapeutics have the potential to induce an immune response. Results: Aggregates can enhance innate and adaptive immune responses of PBMC. Conclusion: The response depends on aggregate type, immunogenicity of the monomer, donor immune status, and high particle numbers in the in vitro assay. Significance: This is the first study showing the impact of aggregate characteristics on the potential immune response of PBMC. Aggregation of biotherapeutics has the potential to induce an immunogenic response. Here, we show that aggregated therapeutic antibodies, previously generated and determined to contain a variety of attributes (Joubert, M. K., Luo, Q., Nashed-Samuel, Y., Wypych, J., and Narhi, L. O. (2011) J. Biol. Chem. 286, 25118–25133), can enhance the in vitro innate immune response of a population of naive human peripheral blood mononuclear cells. This response depended on the aggregate type, inherent immunogenicity of the monomer, and donor responsiveness, and required a high number of particles, well above that detected in marketed drug products, at least in this in vitro system. We propose a cytokine signature as a potential biomarker of the in vitro peripheral blood mononuclear cell response to aggregates. The cytokines include IL-1β, IL-6, IL-10, MCP-1, MIP-1α, MIP-1β, MMP-2, and TNF-α. IL-6 and IL-10 might have an immunosuppressive effect on the long term immune response. Aggregates made by stirring induced the highest response compared with aggregates made by other methods. Particle size in the 2–10 μm range and the retention of some folded structure were associated with an increased response. The mechanism of aggregate activation at the innate phase was found to occur through specific cell surface receptors (the toll-like receptors TLR-2 and TLR-4, FcγRs, and the complement system). The innate signal was shown to progress to an adaptive T-cell response characterized by T-cell proliferation and secretion of T-cell cytokines. Investigating the ability of aggregates to induce cytokine signatures as biomarkers of immune responses is essential for determining their risk of immunogenicity.

Screening of monoclonal antibody formulations based on high‐throughput thermostability and viscosity measurements: Design of experiment and statistical analysis

The purpose of this study was to demonstrate the utility of combining a design of experiment (DOE) approach with high-throughput formulation screening to identify the main factors affecting protein thermostability and solution viscosity. The optimization of buffer compositions was guided by statistical analysis of the data to obtain the targeted combination of low viscosity and high thermostability. Different monoclonal antibody (mAb) formulation variables were evaluated in the study to achieve optimization of two parameters: (i) thermostability characterized by temperature of hydrophobic exposure and (ii) viscosity. High-throughput measurements were employed to characterize both parameters. The significance of each factor and the two-way interactions between them was studied by multivariable regression analysis. An experimental design was used to estimate the significance of all factors, including interaction effects. The range of optimal buffer compositions that maximized thermostability and minimized viscosity of a mAb formulation was determined. The described high-throughput methods are well suited for characterization of multiple protein formulation compositions with minimized resources such as time and material. The DOE approach can be successfully applied to the screening of mAb formulations early in the development lifecycle.

Characterization of Site-Specific Glycation During Process Development of a Human Therapeutic Monoclonal Antibody

A therapeutic recombinant monoclonal antibody (mAb1) was found to be highly susceptible to glycation during production. Up to 42% glycation was observed in mAb1, which was significantly greater than the glycation observed in 17 other monoclonal antibodies (mAbs). The majority of the glycation was localized to lysine 98 of a unique sequence in the heavy chain complementarity determining region 3. Upon incubation with 5% glucose at 37 °C for 5 days, the level of glycation rose to 80% of the total protein where the majority of the additional glycation was on the lysine 98 residue. These data suggested that the lysine 98 residue was highly susceptible to glycation. However, three other mAbs with a lysine residue in the same position did not show high rates of glycation in the forced glycation assay, suggesting that primary and perhaps secondary structural constraints could contribute to the rate of glycation at that lysine. Interestingly, a portion of the glycation in mAb1 was found to be reversible and upon incubation in phosphate buffer (pH 7) at 37 °C for 5 days, the glycation dropped from starting levels of 42% to 20%. Variation was observed in the total glycation levels between different lots of mAb1. The variability in glycation introduced charge heterogeneity in the form of an acidic peak on cation exchange chromatography and lead to product inconsistency. Mutation of lysine 98 to arginine reduced the starting level of glycation without any impact on potency.

Biochemical assessment of erythropoietin products from Asia versus US Epoetin alfa manufactured by Amgen

We compared the physical and chemical properties of purported copies of recombinant human erythropoietin (rHuEPO) purchased from Korea, China, and India with the innovator product, Epoetin alfa, manufactured by Amgen Inc. The products were characterized for similarity in the types of glycoforms present, the relative degree of unfolding, in vitro potency, presence of covalent aggregates, and presence of cleavage products using established analytical methods. All products were different from Epoetin alfa (Epogen). The purported copies of rHuEPO from Korea, India, and China contained more glycoforms and other impurities. The in vitro relative potency varied for each product when based on the labeled concentration, while the concentration based on ELISA analysis brought the relative potency, for most products closer to 100%. These data emphasize potential biochemical discrepancies resulting from different cell lines and manufacturing processes. Concentrations varied within products and did not always match the information provided on the product label. As it is not possible to reliably correlate such biochemical discrepancies to clinical consequences, or the lack thereof, these data support the need for extensive preclinical testing and clinical testing of all investigational products as not all safety and efficacy aspects can be assessed during preclinical evaluation.

Effect of Sugar Molecules on the Viscosity of High Concentration Monoclonal Antibody Solutions

ABSTRACTPurposeTo assess the effect of sugar molecules on solution viscosity at high protein concentrations.MethodsA high throughput dynamic light scattering method was used to measure the viscosity of monoclonal antibody solutions. The effects of protein concentration, type of sugar molecule (trehalose, sucrose, sorbitol, glucose, fructose, xylose and galactose), temperature and ionic strength were evaluated. Differential scanning fluorimetry was used to reveal the effect of the same sugars on protein stability and to provide insight into the mechanism by which sugars increase viscosity.ResultsThe addition of all seven types of sugar molecules studied result in a significant increase in viscosity of high concentration monoclonal antibody solutions. Similar effects of sugars were observed in the two mAbs examined; viscosity could be reduced by increasing the ionic strength or temperature. The effect by sugars was enhanced at higher protein concentrations.ConclusionsDisaccharides have a greater effect on the solution viscosity at high protein concentrations compared to monosaccharides. The effect may be explained by commonly accepted mechanisms of interactions between sugar and protein molecules in solution.

Comparison of high-throughput biophysical methods to identify stabilizing excipients for a model IgG2 monoclonal antibody: Conformational stability and kinetic aggregation measurements

The overall conformational stability of a model IgG2 monoclonal antibody (mAb) was examined as a function of temperature and pH using an empirical phase diagram approach. Stabilizing excipients were then identified based on high-throughput methods including (1) kinetic studies measuring aggregation via increases in optical density and (2) thermally induced structural transitions as measured by differential scanning calorimetry (DSC) and fluorescence spectroscopy. The kinetic profiles of antibody aggregation at 65 °C were pH dependent and correlated well with pH effects on secondary and tertiary structural transitions due to heat stress. For the screening of stabilizing excipients, the inhibition of the rate of protein aggregation at pH 4.5 at 65°C, as represented by changes in optical density, was shown to have a clear trend with a modest correlation coefficient compared with the stabilizing effect of the same excipients on the conformational stability of the antibody as measured by DSC and tryptophan fluorescence spectroscopy. These results demonstrate the utility of combining high-throughput data from protein aggregation kinetic experiments and conformational stability studies to identify stabilizing excipients that minimize the physical degradation of an IgG2 mAb.

Peptide Cysteine Thiyl Radicals Abstract Hydrogen Atoms from Surrounding Amino Acids : The Photolysis of a Cystine Containing Model Peptide

Peptide cysteine thiyl radicals were generated through UV-photolysis of disulfide precursors, in order to follow intramolecular reactions of those radicals with neighboring amino acids. When reactions were carried out in D(2)O, there was a significant incorporation of deuterium specifically into the C(alpha)-H bonds of glycine residues in positions i+1 and i-1 to the Cys residue, indicating a fast reversible H-atom transfer. This H-atom transfer occurred prior to the formation of final, nonradical products including free thiol, thioaldehyde, and aldehyde. Such fast H-atom transfer is relevant to biologic conditions of oxidative stress and to the stabilization of proteins against oxidation, where the formation of carbon-centered radicals in proteins may lead to fragmentation, intramolecular cross-linking, aggregation and/or epimerization.

Physical and biophysical effects of polysorbate 20 and 80 on darbepoetin alfa.

We studied the physical and biophysical affects of the nonionic surfactants polysorbate 20 and 80 and their mechanism of interaction using darbepoetin alfa, a 4-helix bundle protein, as the exemplary protein. Differences were observed between the abilities of the polysorbates to prevent surface loss/aggregation and correlated with each polysorbates initiation of micelle formation prior to the critical micelle concentration (CMC). The biophysical properties monitored by far-UV circular dichroism (CD) and tryptophan (Trp) fluorescence showed effects due to polysorbates, but were not correlated with their CMC. At a constant protein concentration PS-80 induced alpha-helix in the protein with a maximal effect at 15:1 molar ratio of PS-80/protein. PS-20 initially induced alpha-helix with a maximal effect at 1.5:1 ratio followed by a decrease in the alpha-helix content. PS-80 had no effect on near-UV CD but increased Trp fluorescence only at the 150:1 polysorbate/protein ratio. PS-20 decreased the near-UV CD and Trp fluorescence. Thermodynamic studies by isothermal titration calorimetry (ITC) demonstrated that the protein interacts with monomeric polysorbate, but not with polysorbate micelles. The data suggest that the polysorbates differentially interact with the protein and that the biophysical effects are dependent on the structure of the polysorbate and the polysorbate to protein ratio.

Interactions between PEG and type I soluble tumor necrosis factor receptor: Modulation by pH and by PEGylation at the N terminus

The effects of polyethylene glycol (PEG) on protein structure and the molecular details that regulate its association to polypeptides are largely unknown. These issues were addressed using type I soluble tumor necrosis factor receptor (sTNF‐RI) as a model system. Changes in solution viscosity established that a truncated form of sTNF‐RI bound free PEG in a pH‐dependent manner. Above pH 5.3, the viscosity escalated as the pH increased, while no effect occurred below pH 5.0. Conjugation of 2 kD, 5 kD, or 20 kD PEG to the N terminus attenuated the viscosity at the higher pH values. Tryptophan phosphorescence spectroscopy correlated changes in the protein structure about Trp‐107, at the C terminus, with the pH‐dependent and PEGylation‐dependent attenuation of the viscosity. The results indicate that specific interactions between PEG and the truncated form of sTNF‐RI are elicited by an increased flexibility of the truncated protein combined perhaps with removal of steric or charge barriers. Covalently bound PEG at the N terminus reduced the protein affinity for the free polymer and induced a more rigid and polar configuration around Trp‐107. Deprotonation of His‐105, which is perpendicular to Trp‐107, was integral to the binding mechanism producing a pH‐dependent switching mechanism. These findings stress the importance of surface charge and structural plasticity in determining macromolecular binding affinities and demonstrate the ability of conjugated PEG to modify the localized surface structure in proteins away from the site of conjugation.

Photolysis of an intrachain peptide disulfide bond: primary and secondary processes, formation of H2S, and hydrogen transfer reactions.

The photodissociation of intrachain disulfide bonds in a model peptide and salmon calcitonin generates a series of cyclic peptide products following the generation of a CysS(*) thiyl radical pair. Key to the formation of these cyclic products are disproportionation and reversible hydrogen atom transfer reactions as well as secondary photoreactions, which lead to C-S bond breakage of primary photoproducts. Depending on the wavelength of the incident light, disulfides ultimately convert into cyclic thioethers. An important photolytic product is H(2)S, which is highly relevant for the production and storage of protein pharmaceuticals, where H(2)S can catalyze disulfide scrambling and protein degradation.