Steric Repulsion Forces Contributed by PEGylation of Interleukin-1 Receptor Antagonist Reduce Gelation and Aggregation at the Silicone Oil-Water Interface

Abstract

Silicone oil, used as a lubricating coating in pharmaceutical containers, has been implicated as a cause of therapeutic protein aggregation. After adsorbing to silicone oil-water interfaces, proteins may form interfacial gels, which can be transported into solution as insoluble aggregates if the interfaces are perturbed. Mechanical interfacial perturbation of both monomeric recombinant human interleukin-1 receptor antagonist (rhIL-1ra) and PEGylated rhIL-1ra (PEG rhIL-1ra) in siliconized syringes resulted in losses of soluble monomeric protein. However, the loss of rhIL-1ra was twice that for PEG rhIL-1ra, even though in solution, PEG rhIL-1ra had a lower ΔGunf and exhibited a more perturbed tertiary structure at the interface. Net proteinprotein interactions in solution for rhIL-1ra were attractive, but increased steric repulsion due to PEGylation led to net repulsive interactions for PEG rhIL-1ra. Attractive interactions for rhIL-1ra were associated with increases in intermolecular β-sheet content at the interface, whereas no intermolecular β-sheet structures were observed for adsorbed PEG rhIL-1ra. rhIL-1ra formed interfacial gels that were five times stronger than those formed by PEG rhIL-1ra. Thus, the steric repulsion contributed by the PEGylation resulted in decreased interfacial gelation and in the reduction of aggregation, in spite of the destabilizing effects of PEGylation on the protein’s conformational stability.

Domain I of β2GPI is capable of blocking serum IgA antiphospholipid antibodies binding in vitro: an effect enhanced by PEGylation

“…The potential for aggregation is also higher in the non-PEGylated protein as PEGylation has been shown to reduce aggregation. 16,17 These results are in contrast to our previous research using IgG ab2GPI antibodies where significant inhibitory potential of DI was retained but not enhanced after PEGylation. 13 The strongest inhibition was seen in those patients with both high IgA aDI/ab2GPI and high IgG aDI/ab2GPI.…”

DEHP Nanodroplets Leached From Polyvinyl Chloride IV Bags Promote Aggregation of IVIG and Activate Complement in Human Serum

“…71,72 Biophysical characterization methods such as intrinsic fluorescence quenching and circular dichroism have shown that perturbations in protein structure may occur after protein adsorption to silicone oilewater interfaces presented by these droplets. 40,43,65 Owing to interference caused by the aromatic and chiral nature of DEHP, we could not use these same biophysical characterization methods to probe possible structural perturbations in proteins at the DEHP-water interface. Instead, we showed that IVIG adsorbed strongly to DEHP nanodroplets, resulting in surface coverages similar to those previously reported for monoclonal antibodies at the silicone oilewater interface.…”

“…Furthermore, IL-1ra gels at DEHP-water interfaces exhibited more variability in strength compared with those at the silicone oilewater interface. 43,53 A slower rate of gelation combined with weaker gels could explain reduced rates of IVIG aggregation in formulations containing DEHP nanodroplets compared to rates in formulations containing silicone oil nanodroplets.…”

“…The formation of intermolecular protein networks at the DEHP-water interface was investigated using interleukin-1 receptor antagonist (IL-1ra) using interfacial shear rheology and compared with corresponding results for silicone oil-water interfaces obtained from the literature. 43 Finally, as an indicator of the potential immunogenic properties of DEHP suspensions, we evaluated complement activation in human serum by IVIG formulations containing DEHP and silicone oil nanodroplets.…”

The Molecular Interaction Process

“…1,2,[5][6][7][8][9][10] Over several years, John Carpenter and Ted Randolph have published hundreds of valuable and fundamental insights into the many ways solution conditions and solution handling can lead to high viscosity, which impairs manufacturing, as well as to aggregation, that can compromise safety and efficacy of biopharmaceutics. [11][12][13][14][15][16][17][18][19][20][21][22][23] A common feature of their research is the underlying presence of protein-protein interactions that lead to unwanted outcomes.…”