In vitro and in vivo immunogenicity assessment of protein aggregate characteristics.

The immunogenicity risk of therapeutic protein aggregates has been extensively investigated over the past decades. While it is established that not all aggregates are equally immunogenic, the specific aggregate characteristics, which are most likely to induce an immune response, remain ambiguous. The aim of this study was to perform comprehensive in vitro and in vivo immunogenicity assessment of human insulin aggregates varying in size, structure and chemical modifications, while keeping other morphological characteristics constant. We found that flexible aggregates with highly altered secondary structure were most immunogenic in all setups, while compact aggregates with native-like structure were found to be immunogenic primarily in vivo. Moreover, sub-visible (1-100 µm) aggregates were found to be more immunogenic than sub-micron (0.1-1 µm) aggregates, while chemical modifications (deamidation, ethylation and covalent dimers) were not found to have any measurable impact on immunogenicity. The findings highlight the importance of utilizing aggregates varying in few characteristics for assessment of immunogenicity risk of specific morphological features and may provide a workflow for reliable particle analysis in biotherapeutics.

Characterization of Insulin Dimers by Top-Down Mass Spectrometry.

High-molecular weight products (HMWP) are an important critical quality attribute in research and development of insulin biopharmaceuticals. We here demonstrate on two case studies of covalent insulin dimers, induced by Fe2+ incubation or ultraviolet (UV) light stress, that de novo characterization in top-down mass spectrometry (MS) workflows can identify cross-link types and sites. On the MS2 level, electron-transfer/higher-energy collision dissociation (EThcD) efficiently cleaved the interchain disulfide bonds in the dimers to reveal cross-link connectivities between chains. The combined utilization of EThcD and 213 nm ultraviolet photodissociation (UVPD) facilitated identification of the chemical composition of the cross-links. Identification of cross-link sites between chains at residue level was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds. UVPD provided identification of cross-link sites in the Fe2+-induced dimer without MS3, while cross-link site identification with MS2 was not possible for the UV light-induced dimer. Thus, using varied multistage approaches, it was discovered that in the UV light-induced dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain. In the Fe2+-induced dimer, Phe1 from both B-chains were cross-linked through a -CH2-. The UV chromophoric side chain of Phe1 was indicated in the cross-link, explaining why UVPD-MS2 was effective in fragmenting the cross-link and nearby backbone bonds. Our results demonstrated that higher-energy collisional dissociation (HCD), EThcD, and UVPD combined with MS3 were powerful tools for direct de novo characterization of cross-linked insulin dimers.

Direct Ultraviolet Laser-Induced Reduction of Disulfide Bonds in Insulin and Vasopressin.

Ultraviolet (UV) light has been shown to induce reduction of disulfide bonds in proteins in solution. The photoreduction is proposed to be a result of electron donation from excited Tyr or Trp residues. In this work, a powerful UV femtosecond laser was used to generate photoreduced products, while the hypothesis of Tyr/Trp mediation was studied with spectroscopy and mass spectrometry. With limited irradiation times of 3 min or less at 280 nm, the laser-induced reduction in arginine vasopressin and human insulin led to significant yields of ∼3% stable reduced product. The photogenerated thiols required acidic pH for stabilization, while neutral pH primarily caused scrambling and trisulfide formation. Interestingly, there was no direct evidence that Tyr/Trp mediation was a required criterion for the photoreduction of disulfide bonds. Intermolecular electron transfer remained a possibility for insulin but was ruled out for vasopressin. We propose that an additional mechanism should be increasingly considered in UV light-induced reduction of disulfide bonds in solution, in which a single UV photon is directly absorbed by the disulfide bond.

Characterization of Ultraviolet Photoreactions in Therapeutic Peptides by Femtosecond Laser Catalysis and Mass Spectrometry.

Peptides and proteins have diverse ultraviolet (UV) photoreaction pathways that can be activated by the energy of the UV photons absorbed. Simple light sources such as lamps are conventionally used to study these photoreactions in solution. This work provides a proof of concept that femtosecond laser technology can function as a highly potent UV source in rapidly conducting UV photostability studies of peptides. Correspondingly, sufficient quantities of photoproducts were generated in 1 min or less, allowing for identification of known and new photomodifications in the therapeutic peptides somatostatin-14 and arginine vasopressin. Identical photoproducts were also generated with a conventional continuous source. The major modifications included N-formylkynurenine, a cross-link between Trp and Phe, a Tyr product with an NH3 loss, and disruption of an unstable disulfide bond into a complex mixture of a trisulfide bond and multiple scrambled dimeric products. In conclusion, femtosecond lasers are extremely useful to drive fast UV-induced reactions for high throughput screening of photostability and modifications in amino acid polymers.