(527e) Adsorption of Recombinant Human Interleukin-1 Receptor Antagonist to Silicone Oil-Water Interfaces Leads to Gel Formation and Subsequent Surface-Induced Aggregation

Therapeutic
proteins formulated in prefilled syringes (PFS) lubricated with silicone oil
contact silicone oil-water interfaces for their entire shelf lives. Protein
adsorption to silicone oil-water interfaces may induce aggregation and particle
formation which potentially may in turn result in reduced product efficacy, or
worse, unwanted immune responses in patients. We hypothesize that the formation
of aggregated recombinant human interleukin-1 receptor antagonist (rhIL-1ra) in
PFS stems primarily from the formation of viscoelastic gel-like protein layers
at the silicone oil-water interface and that conditions fostering stronger
interfacial gels lead to increased protein aggregation.

1 mg mL^-1
rhIL-1ra was incubated in siliconized and unsiliconized PFS at room temperature
both quiescently and with end-over-end rotation (3.5RPM). Formulation buffer was
varied from 60, 150mM, 500mM, to 800mM NaCl in 10mM MES, at pH 6.5. The extent
of protein monomer loss at those ionic strengths was monitored using size
exclusion chromatography. We employed a static light scattering instrument to
measure bulk protein-protein interactions through measurements of the osmotic
second virial coefficient B₂₂. A magnetic rod interfacial
shear rheometer was used to determine interfacial gelation times and interfacial
gel strengths by measuring the elastic and viscous moduli. To probe for
interfacial aggregation, front-face fluorescence of Thioflavin T was used as a
marker of intermolecular beta-sheet formation in gelled layers of rhIL-1ra
adsorbed at silicone oil-water interfaces.

Up to 15%
monomer loss was observed in silicone oil-coated PFS that underwent rotation, whereas
less than 4% monomer loss was detected in quiescently incubated siliconized PFS
and in unsiliconized PFS that were either quiescently incubated or that were
rotated. Protein monomer loss was most pronounced in low ionic strength buffer
and diminished with increasing ionic strength. These results correlated well
with static light scattering B₂₂measurements where net
attractive forces were measured at 60mM and at 150mM and net repulsive forces
were measured at 500mM and 800mM NaCl. To determine the cause of aggregation
inside PFS, rotation was stopped in siliconized PFS after 14 days of incubation
and no further monomer loss was observed over a 7 day period indicating that
protein aggregation occurred at the silicone oil-water interface. Shear
rheometer measurements showed that rhIL-1ra formed strong gels at the silicone
oil-water interface under low ionic strength conditions while increasing NaCl
concentration led to five times weaker gels. From front face fluorescence
measurements, it was possible to determine that those strong gel layers were
formed by interfacial intermolecular beta sheets as fluorescence intensity of
Thioflavin T increased by up to 600% in rhIL-1ra samples incubated with
silicone oil in 60mM buffer.  

We showed
that agitation-induced exposure to silicone oil-water interfaces caused
rhIL-1ra aggregation in siliconized PFS. This aggregation was greatly increased
at low ionic strengths where strong gels formed and interfacial intermolecular
beta sheet structures predominated for protein adsorbed at the silicone
oil-water interface.