Impact of Stress on the Immunogenic Potential of Adalimumab.

Monoclonal antibodies against tumor necrosis factor-alpha (TNFα) are widely used for treatment of inflammatory diseases. However, despite the inhibitory effect this class of drugs has on the immune system, anti-drug antibodies are often formed with continuous use. Particles formed during stress conditions, which can be used to simulate storage and handling conditions of commercial antibodies, have previously been associated with the formation of anti-drug antibodies. This study investigates the relationship between particles, oligomerization, folding and chemical degradation on the in vitro cytokine response toward the TNFα inhibitor adalimumab. Adalimumab aggregates generated using stir and heat stress were fractionated into distinct sub-populations, and their structure and immunogenic potential were evaluated. A chemically degraded sample of adalimumab was included to compare particle composition with the milder accelerated heat and stir stressed conditions. Particles from stressed adalimumab samples induced elevated cytokine levels and CD4+ T cell proliferation in vitro compared to non-stressed samples. Samples enriched with both submicron and subvisible particles of adalimumab induced the strongest cytokine release and the strongest CD4+ T cell proliferation despite maintaining some TNFα inhibitory functionality. Samples that were stressed and subsequently purified of subvisible and submicron particles did not elicit a significantly higher cytokine response or show increased CD4+ T cell proliferation compared to a non-stressed sample. Oxidation-induced chemical modifications in adalimumab, mainly in Met, His, Trp, and Tyr, were not found to be sufficient in absence of particle formation to induce increased CD4+ T cell proliferation or cytokine release despite less decreased TNFα inhibitory activity of adalimumab. These observations provide further evidence that particles do indeed potentiate the immunogenic potential of adalimumab.

Spectroscopic Evidence of Tertiary Structural Differences Between Insulin Molecules in Fibrils.

Protein fibrils are of great interest due to their involvement in several pathologies and their roles in the degradation of many therapeutic protein products. Fibrils share highly similar secondary structural motifs across different proteins and applied stress conditions. However, fibril morphology differs according to the surrounding conditions, with aromatic and hydrophobic amino acids playing important roles in mature fibril formation. In this study, we use Raman microscopy, by means of the aromatic amino acids in insulin molecules as markers, to probe for tertiary structure differences within fibrils. We compared 2 different fibril types, linear fibril bundles and spherulites. Generation of linear fibril bundles was undertaken in an acetic acid-containing formulation, whereas spherulites were generated in a hydrochloric acid-containing formulation. The Raman intensities of tyrosine and phenylalanine side chains suggest that there are significant differences between the fibril bundles. The findings suggest that the insulin components of the fibril strands are not arranged identically in the 2 fibril types and that this gives rise to differences in their tertiary structures. Overall, the work indicates that the physicochemical properties of fibril structures can be altered by changing the formulation and that these alterations can be monitored by Raman spectroscopy.

Optimization of Infrared Microscopy to Assess Secondary Structure of Insulin Molecules Within Individual Subvisible Particles in Aqueous Formulations.

The analysis of subvisible particles is currently challenging but pivotal to the understanding and control of the quality of protein therapeutics. While a range of characterization methods is available for subvisible particles, information on the protein conformation in a particle-considered a possible parameter in eliciting unwanted immunogenicity of protein therapeutics-is especially challenging in the lower micrometer range using existing analytical technologies. Using 6 different protein particle populations, we show that transmission Fourier transform infrared (FTIR) microscopy can determine protein secondary structure in single particles down to 10 µm. The analytical setup presented here is able to immobilize protein particles and obtain transmission FTIR spectra on individual protein particles in their intact aqueous environment. Spectra of dried particles, on the other hand, were found to occasionally differ from spectra of particles in aqueous environment. In summary, using the analytical setup described in this study, transmission FTIR microscopy uniquely provides information on single protein particles in particle populations in their aqueous environment without interference from the background protein solution.

A Platform for Preparing Homogeneous Proteinaceous Subvisible Particles With Distinct Morphologies.

Regulatory authorities and scientific communities are increasingly attentive to the known and universal presence of small particulates in biological drug products. The underlying concern is that these particulates may cause unwanted formation of antidrug antibodies in patients. Pharmacological studies, however, have to date not succeeded in unambiguously identifying risk-prone particle properties. This lack of success may be partly due to a lack of available, well-defined, homogenous particle material. Protein particles arising from stress of protein drug products are by nature often highly heterogeneous in size, morphology, and structure of the constituent protein in the particles. Here, we present simple and pharmaceutically relevant stress conditions to produce 8 different highly homogenous micrometer-sized protein particles from human insulin, representing very different morphologies and conformation of the constituent protein molecules in the particles generated. Insulin's self-association patterns were varied by formulation approaches to create diverse starting materials. The resulting collection of homogenous particles underlines that the particle formation is not necessarily a random process but a consequence of formulation and specific stress condition. Owing to the inherent homogenicity of these populations, the particle materials can act as a standard platform for further studies on insulin subvisible particles in drug products.