Myelin water imaging in relapsing multiple sclerosis treated with ocrelizumab and interferon beta-1a.

BACKGROUND: Myelin water imaging is a magnetic resonance imaging (MRI) technique that quantifies myelin damage and repair in multiple sclerosis (MS) via the myelin water fraction (MWF). OBJECTIVE: In this substudy of a phase 3 therapeutic trial, OPERA II, MWF was assessed in relapsing MS participants assigned to interferon beta-1a (IFNb-1a) or ocrelizumab (OCR) during a two-year double-blind period (DBP) followed by a two-year open label extension (OLE) with ocrelizumab treatment. METHODS: MWF in normal appearing white matter (NAWM), including both whole brain NAWM and 5 white matter structures, and chronic lesions, was assessed in 29 OCR and 26 IFNb-1a treated participants at weeks 0, 24, 48 and 96 (DBP), and weeks 144 and 192 (OLE), and in white matter for 23 healthy control participants at weeks 0, 48 and 96. RESULTS: Linear mixed-effects models of data from baseline to week 96 showed a difference in the change in MWF over time favouring ocrelizumab in all NAWM regions. At week 192, lesion MWF was lower for participants originally randomised to IFNb-1a compared to those originally randomised to OCR. Controls showed no change in MWF over 96 weeks in any region. CONCLUSION: Ocrelizumab appears to protect against demyelination in MS NAWM and chronic lesions and may allow for a more permissive micro environment for remyelination to occur in focal and diffusely damaged tissue.

High-resolution glycoform profiling of intact therapeutic proteins by hydrophilic interaction chromatography-mass spectrometry.

Glycosylation is considered a critical quality attribute of therapeutic proteins. Protein heterogeneity introduced by glycosylation includes differences in the nature, number and position of the glycans. Whereas analysis of released glycans and glycopeptides provides information about the composition and/or position of the glycan, intact glycoprotein analysis allows assignment of individual proteoforms and co-occurring modifications. Yet, resolving protein glycoforms at the intact level is challenging. We have explored the capacity of hydrophilic liquid chromatography-mass spectrometry (HILIC-MS) for assessing glycosylation patterns of intact pharmaceutical proteins by analyzing the complex glycoproteins interferon-beta-1a (rhIFN-ß - 1a) and recombinant human erythropoietin (rhEPO). Efficient glycoform separation was achieved using a superficially-porous amide HILIC stationary phase and trifluoroacetic acid (TFA) as eluent additive. In-source collision-induced dissociation proved to be very useful to minimize protein-signal suppression effects by TFA. Direct injection of therapeutic proteins in aqueous formulation was possible without causing extra band dispersion, provided that the sample injection volume was not larger than 2 µL. HILIC-MS of rhIFN-ß - 1a and rhEPO allowed the assignment of, respectively, 15 and 51 glycoform compositions, next to a variety of posttranslational modifications, such as succinimide, oxidation and N-terminal methionine-loss products. MS-based assignments showed that neutral glycan units significantly contributed to glycoform separation, whereas terminal sialic acids only had a marginal effect on HILIC retention. Comparisons of HILIC-MS with the selectivity provided by capillary electrophoresis-MS for the same glycoproteins, revealed a remarkable complementarity of the techniques. Finally it was demonstrated that by replacing TFA for difluoroacetic acid, peak resolution somewhat decreased, but rhEPO glycoforms with relative abundances below 1% could be detected by HILIC-MS, increasing the overall rhEPO glycoform coverage to 72.

Rapid Conformational Analysis of Protein Drugs in Formulation by Hydrogen/Deuterium Exchange Mass Spectrometry.

Hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) has become an established method for analysis of protein higher order structure. Here, we use HDX-MS methodology based on manual solid-phase extraction (SPE) to allow fast and simplified conformational analysis of proteins under pharmaceutically relevant formulation conditions. Of significant practical utility, the methodology allows global HDX-MS analyses to be performed without refrigeration or external cooling of the setup. In mode 1, we used dimethyl sulphoxide-containing solvents for SPE, allowing the HDX-MS analysis to be performed at acceptable back-exchange levels (<30%) without the need for cooling any components of the setup. In mode 2, SPE and chromatography were performed using fast isocratic elution at 0°C resulting in a back-exchange of 10%-30%. Real-world applicability was demonstrated by HDX-MS analyses of interferon-ß-1a in formulation, using an internal HDX reference peptide (P7I) to control for any sample-to-sample variations in back-exchange. Advantages of the methodology include low sample use, optimized excipient removal using multiple solvents, and fast data acquisition. Our results indicate that HDX-MS can provide a reliable approach for fast conformation analysis of proteins in their intended formulations, which could facilitate an increased use of the technique in pharmaceutical development research.

Capillary Zone Electrophoresis-Mass Spectrometry of Intact Proteins.

Capillary electrophoresis (CE) coupled with mass spectrometry (MS) has proven to be a powerful analytical tool for the characterization of intact proteins. It combines the high separation efficiency, short analysis time, and versatility of CE with the mass selectivity and sensitivity offered by MS detection. This chapter focuses on important practical considerations when applying CE-MS for the analysis of intact proteins. Technological aspects with respect to the use of CE-MS interfaces and application of noncovalent capillary coatings preventing protein adsorption are treated. Critical factors for successful protein analysis are discussed and four typical CE-MS systems are described demonstrating the characterization of different types of intact proteins by CE-MS. These methodologies comprise the use of sheath-liquid and sheathless CE-MS interfaces, and various types of noncovalent capillary coatings allowing efficient and reproducible protein separations. The discussion includes the analysis of lysozyme-drug conjugates and the therapeutic proteins human growth hormone, human interferon-ß-1a, and human erythropoietin.