Characterization by LC-MS/MS of oxidized products identified in synthetic peptide somatostatin and cetrorelix submitted to forced oxidative stress by hydrogen peroxide: Two case studies.

In a broader scenario, the forced degradation studies provided by the ICH guidelines for Q1A, Q1B, and Q2B degradation studies allow to know the CQA of the molecule used as a drug product, to determine the appropriate analytical methods, excipients, and storage conditions ensuring the quality of the drug, its efficacy, and patient safety. In this study, we focused our attention on understanding how oxidative stress is performed by H2 O2 -impacted small synthetic peptides that do not contain residues susceptible to oxidation such as methionine. Among the amino acids susceptible to oxidation, methionine is the most reactive and depending on the structure of the protein where it is exposed, it tends to oxidize by converting into methionine sulfone or methionine sulfoxide by oxidation of its sulfur atom. Scouting experiments obtained by forced oxidative stress conditions are presented on two small synthetic peptides that do not contain any methionine residues spiked with different amounts of H2 O2 , and they are analyzed by LC-MS/MS. Less frequent oxidation products than those commonly observed on proteins/peptides-containing methionine have been characterized on both peptides. The study demonstrated that somatostatin, by means of one residue of tryptophan on the molecule, can generate traces of several oxidized products detected by UPLC-MS. Furthermore, even at a negligible level, oxidation on tyrosine and proline in cetrorelix that does not contain methionine nor tryptophan has been detected by UHPLC-MS/MS. Identification and quantification of oxidized species were achieved by high-resolution MS and MS/MS experiments. Thus, FDSs undoubtedly aid the evaluation of the CQAs as an important component of the characterization package as recommended by HAs and ICH, facilitating the understanding of unforeseen features of the studied molecule used as drugs.

Comprehensive investigation of a structural variant in a bi-specific, N-and C-terminal Fc-fusion molecule, and its monitoring with LC-MS based method.

There is an increasing interest in the generation of Fc-fusion molecules to exploit the effector functions of Fc and the fusion partner, towards improving the therapeutic potential. The Fc-fusion molecules have unique structural and functional attributes that impart various advantages. However, the manufacturing of Fc-fusion molecules possesses certain challenges in the biopharmaceutical development. The fusion of unnaturally occurring two or more domains in a construct can pose problems for proper folding and are prone to aggregation and degradation. Reshuffling of disulfide bridges represents a posttranslational event that affects folding. This can play a critical role in the correct structure of a molecule and leads to structural heterogeneity in biotherapeutics; it may also impact the in vivo biological activities, safety, and efficacy of the biopharmaceutical. Our work presents an investigation case of a doublet band, as observed only in nonreducing sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) for a bi-specific, N- and C-terminal Fc-fusion molecule. Other characterization and orthogonal methods from the analytical panel did not indicate the presence of two distinct species, including the orthogonal CE-SDS (Caliper Lab Chip GXII). Therefore, it was necessary to determine if the phenomenon was an analytical artifact or a real variant of our Fc-fusion molecule. With the comprehensive mass spectrometry-based characterization, we were able to determine that the doublet band was related to the reshuffling of one disulfide bridge in one of the fused domains. Our work illustrates the application of nonreducing peptide mapping by mass spectrometry to characterize and identify disulfide variants in a complex N- and C-terminal Fc-fusion molecule, and further adoption to monitor the disulfide structural variants in the intermediate process samples to drive the manufacturing of a consistent product with the desired quality attributes.

Characterization of disulfide bridges pattern of recombinant human interleukin 12 fusion protein p40 subunit and identification and quantification of cysteinylated free cysteine 252.

RATIONALE: We show evidence of cysteinylation on Cys252 of recombinant human p40 subunit of interleukin 12 (IL-12). This was reported in 1996. However, no paper detailing this concept has been published yet. Our paper reports the quantification of Cys252 cysteinylation as well as the full disulfide bridges assignment by nonreducing peptide mapping using mass spectrometry (MS) detection. METHODS: Nonreducing peptide mapping was applied for disulfide bridges assignment. This study presents an ad hoc method in which applying a neutral pH in the presence of an alkylating agent allowed to mitigate the formation of artifacts such as reshuffled disulfide bridges and permitted the detection of free cysteine. Ultra-high-performance liquid chromatography-MS analysis was performed on a Waters quadrupole time-of-flight Xevo G2-XS mass spectrometer acquiring data in MSE mode. MS data were processed using Expressionist MS Refiner 13.5 (Genedata). RESULTS: Scouting experiments were performed using two batches of drug substance. An in-depth study of the LC tandem mass spectrometry profiles revealed the presence of additional species related to "free" Cys252; this cysteine residue was also detected in its S-cysteinylated and S-homocysteinylated forms. This result is consistent with that reported in literature so far. The relative abundance of overall "cysteinylated" species resulted in the range between 46% and 36%, which has also been confirmed using orthogonal techniques such as Ellman's assay. CONCLUSIONS: Our data clearly demonstrate that the free cysteine (Cys252) on the p40 subunit of recombinant IL-12 is also present in its cysteinylated and homocysteinylated forms at a considerable rate. Our observations, although based on results obtained on an IL-12-derived fusion protein, are consistent with the current literature.

In vitro biological characterization of IFN-ß-1a major glycoforms.

Recombinant human interferon ß-1a (IFN-ß-1a) is extensively used as the first-line treatment of relapsing forms of multiple sclerosis. Its glycosylation is recognized as having a complex impact on a wide range of molecule characteristics and functions. The present study reports the enrichment of IFN-ß-1a glycoforms and their physicochemical and biological characterization by means of electrospray ionization-mass spectrometry, sialic acid content, thermal denaturation and various in vitro bioassays (antiproliferative, antiviral, immunomodulatory and reporter gene assay). The glycoforms were fractionated by means of cation-exchange chromatography using recombinant IFN-ß-1a derived from Chinese Hamster Ovary cell culture as starting material. The obtained fractions contained bi- and higher-antennarity glycans as described in the European Pharmacopoeia monograph (Nr. 1639E, Interferon beta 1a concentrated solution). The in vitro bioassay responses revealed a correlation mainly with the glycan antennarity. It is therefore suggested that all glycoforms have biological activity and play a role in modulating the overall IFN-ß biological activity with higher-antennarity glycoforms being able to better sustain IFN-ß-1a bioactivity over time. These data indicate the role of IFN-ß-1a glycosylation in vivo and shed new light on the role of the glycosylation heterogeneity, in particular with regard to antennarity, on biological properties of glycoproteins.

Heterogeneity of commercial recombinant human growth hormone (r-hGH) preparations containing a thioether variant.

The objective of the present study was to assess (I) the potential presence of a recently discovered thioether variant in commercially available recombinant human growth hormone (r-hGH) preparations, and (II) the impact of the thioether modification on the in-vivo bioactivity and the receptor binding kinetics. Samples were tested employing European (EP) and US Pharmacopeia (USP) Somatropin monograph and mass spectrometry methods. None of the international standards contained this variant. All products conformed to EP specifications but six out of eight lots contained the variant. An artificially enriched thioether sample exhibited a significantly reduced in vivo biopotency and altered receptor-binding properties compared with a control. The absence of the variant in the pituitary hGH standard, and the possibility to generate it artificially suggests that it is not naturally occurring and that it may arise from an uncontrolled manufacturing process. Controlled studies may be required to assess its clinical efficacy and safety. EP and USP methods may need to be adapted to reliably detect the presence of the variant.

Characterisation of a novel growth hormone variant comprising a thioether link between Cys182 and Cys189.

A novel variant of recombinant human growth hormone (r-hGH), isolated from biopharmaceutical preparations produced in E. coli, was identified and characterised. This variant contains a nonreducible thioether bridge near the C terminus between Cys182 and Cys189 and was characterised using various analytical techniques. As previous work by Cunningham and Wells (1993) highlighted the involvement of several residues in this part of the sequence in the binding and affinity of the molecule to its receptor, the presence of this modified intramolecular link may have important implications with regard to the biological behaviour of the molecule. Furthermore, as the conversion of a disulfide into a thioether was previously reported for a therapeutic monoclonal antibody (Tous et al., 2005), this may imply that disulfide bridges located in this part of the molecule have a generic susceptibility to thioether formation. This in turn is relevant to the biopharmaceutical industry for monitoring the integrity of disulfide bridges near the protein C terminus. The present study exhibits a state of the art physicochemical investigation for the unequivocal elucidation of a novel structure involving peptide mapping with mass spectrometry and de novo peptide sequencing. Changes in the higher order structure of the molecule were highlighted by near UV circular dichroism and molecular modelling.