Predictive In Vitro Vitreous and Serum Models and Methods to Assess Thiol-Related Quality Attributes in Protein Therapeutics.

In vitro models that mimic the in vivo environment can greatly facilitate and support criticality assessment of product quality attributes for therapeutic drugs to ensure product quality. An in vitro model is established to study and predict the impact of thiol-related attributes on safety or efficacy of intraocular antibody products. This model simulates the physiological redox environment of rabbit vitreous and maintains a steady-state redox potential using reduced and oxidized forms of glutathione. A similar in vitro model that mimics the thiol redox conditions of human blood has been previously established and has become a predictive tool to study intravenous (IV) therapeutic proteins. We utilized both vitreous and serum models to study the potential impact of antibody variants (trisulfides and free-thiols) on product qualities of different antibodies. The studies demonstrate that both models are effective tools to monitor changes of thiol-related attributes under physiological conditions, providing insights on these thiol-related attributes and allowing for more informed assessment of biological relevance and criticality of the attributes. Furthermore, we propose that the approach using an in vitro study for the product quality attribute assessment can be used to predict in vivo effects for future molecules during the development of biopharmaceuticals, reducing the need for live subject studies.

Rates and impact of human antibody glycation in vivo.

Glycation of immunoglobulin G (IgG) can result from incubation with a reducing sugar in vitro or during circulation in vivo. Upon injection of a recombinantly produced human therapeutic IgG into humans, changes in the glycation levels could be observed as a function of circulation time. Mass changes on the individual IgG polypeptide chains as the results of glycation were determined using reversed-phase liquid chromatography/mass spectrometry. Changes to the light and heavy chains were low but easily detectable at 0.00092 and 0.0021 glucose (Glc) additions per chain per day, respectively. Levels of glycation found on the Fc portion of IgG isolated from healthy subjects, using a similar analytical approach, were on average 0.045 Glc molecules per fragment. In vivo glycation rates could be approximated in vitro by modeling the physiological glycation reaction with a simplified incubation containing physiological Glc concentrations, pH and temperature but with a high concentration of a single purified IgG. To test the impact of glycation on IgG function, highly glycated IgG1 and IgG2 were prepared containing on average 42-49 Glc molecules per IgG. Binding to FcγIIIa receptors, neonatal Fc receptor or protein A was similar or identical to the non-glycated IgG controls. Although the modifications were well distributed throughout the protein sequence, and at high enough levels to affect the elution position by size-exclusion chromatography, no changes in the tested Fc functions were observed.

N-terminal glutamate to pyroglutamate conversion in vivo for human IgG2 antibodies.

Therapeutic proteins contain a large number of post-translational modifications, some of which could potentially impact their safety or efficacy. In one of these changes, pyroglutamate can form on the N terminus of the polypeptide chain. Both glutamine and glutamate at the N termini of recombinant monoclonal antibodies can cyclize spontaneously to pyroglutamate (pE) in vitro. Glutamate conversion to pyroglutamate occurs more slowly than from glutamine but has been observed under near physiological conditions. Here we investigated to what extent human IgG2 N-terminal glutamate converts to pE in vivo. Pyroglutamate levels increased over time after injection into humans, with the rate of formation differing between polypeptide chains. These changes were replicated for the same antibodies in vitro under physiological pH and temperature conditions, indicating that the changes observed in vivo were due to chemical conversion not differential clearance. Differences in the conversion rates between the light chain and heavy chain on an antibody were eliminated by denaturing the protein, revealing that structural elements affect pE formation rates. By enzymatically releasing pE from endogenous antibodies isolated from human serum, we could estimate the naturally occurring levels of this post-translational modification. Together, these techniques and results can be used to predict the exposure of pE for therapeutic antibodies and to guide criticality assessments for this attribute.

Human IgG2 antibody disulfide rearrangement in vivo.

Proteins destined to circulate in the blood are first folded and assembled in the endoplasmic reticulum of secretory cells. For antibodies, like many other serum proteins, the folding and assembly steps involve the formation of disulfide bonds. Such bonds have been thought to be static features of proteins, stabilizing domains, and linking polypeptide chains, although some cases of extracellular disulfide bond cleavage have been noted. Recently, the human IgG2 antibody subclass was found to possess multiple structures differing in specific disulfide linkages. These structures are naturally occurring and can, in some cases, affect the activity of the antibody. Here we show that these IgG2 disulfide linkages interconvert while circulating in humans. Secretory cells initially produce primarily one form (IgG2-A), which is rapidly converted to a second form (IgG2-A/B) while circulating in the blood, followed by a slower conversion to a third form (IgG2-B). This work demonstrates that the disulfide structure of the IgG2 antibody is dynamic in vivo, on a time scale similar to that of the protein's lifetime. Thus, changes to the IgG2 disulfide structure provide a marker of the protein's age and may alter its activity over its lifetime.