No abstract

Biologics such as monoclonal antibodies are much more complex than small-molecule drugs, which raises challenging questions for the development and regulatory evaluation of follow-on versions of such biopharmaceutical products (also known as biosimilars) and their clinical use once patent protection for the pioneering biologic has expired. With the recent introduction of regulatory pathways for follow-on versions of complex biologics, the role of analytical technologies in comparing biosimilars with the corresponding reference product is attracting substantial interest in establishing the development requirements for biosimilars. Here, we discuss the current state of the art in analytical technologies to assess three characteristics of protein biopharmaceuticals that regulatory authorities have identified as being important in development strategies for biosimilars: post-translational modifications, three-dimensional structures and protein aggregation.

Since the wide adoption of liquid chromatography/tandem mass spectrometry (LC/MS/MS), the ion suppression/enhancement phenomenon is the latest barrier to high-throughput analysis. This consequence of a nonoptimized analytical method can lead to adverse effects during quantitation (i.e. poor accuracy and precision). Previous papers have reported that ion suppression is a direct result of endogenous material present in biological samples. However, in the case of a solid-phase liquid chromatography/tandem mass spectrometry (SPE/LC/MS/MS) system, the measured result is the combination of several operating conditions and parameters. Little has been done to effectively monitor and/or choose optimized conditions for the complete sequence of extraction, clean up, separation and analysis. This paper describes a simple setup for quantification of ion suppression/enhancement. Several mobile phase additives, ion-pairing agents and SPE extracts were measured and compared against a standard reference. The results demonstrated that a clean up of plasma extracts based on ion exchange leads to minimal ion suppression/enhancement for the compounds that were investigated.

HPLCremains the workhorse technique in liquidphase separations. The method has two fundamental roots. One is gel-permeation chromatography for the characterization of polymers (1); the other is GC. HPLC began to emerge when GC researchers turned their attention to liquid-phase separations (2, 3). The original LC columns were typically 50-100 cm long, with a 1-2 mm i.d., and packed with 50-200-µm-diam particles. Shorter diffusion distances were known to provide better performance. However, it was not possible to prepare high-performance columns with particles with diameters <30 µm by using the dry-packing techniques that had been applied successfully in GC. One improvement was the development of superficially porous particles that resulted in better column performance at low retention factors (4,5).A breakthrough was achieved in the early 1970s, when slurry packing techniques were developed for fully porous particles <30 µm in diameter (6-8). Researchers rapidly learned to pack efficient columns with 10-, 5-, and 3-µm particles (9-11). The first commercial columns packed with 10-µm particles became available in 1973 (12). During these early investigations, it became clear that the pressures required to operate long, small-particle columns at suitable velocities (above the minimum of the plate-height-velocity curve) heat up the mobile phase because of friction (13,14). This heat would ultimately limit the benefits of using smaller particle sizes unless measures were taken to compensate for this effect.Today, two driving forces continue to test the limits of HPLC. One is the need for faster separations, such as analyses of either simple samples or a few constituents in a complex sample (15-17 ). The second is the desire to achieve greater separation power to quantify or identify all the constituents of a complex sample or to compare the contents of complex samples with each other (18)(19)(20)(21)(22) A new separati on techni que takes advantage of sub-2-µm porous parti cl es.

Monoclonal antibodies are typically glycosylated at asparagine residues in the Fc domain, and glycosylation heterogeneity at the Fc sites is well known. This paper presents a method for rapid analysis of glycosylation profile of the therapeutic monoclonal antibody trastuzumab from different production batches using electrospray quadrupole ion-mobility time-of-flight mass spectrometry (ESI-Q-IM-TOF). The global glycosylation profile for each production batch was obtained by a fast LC-MS analysis, and comparisons of the glycoprofiles of trastuzumab from different lots were made based on the deconvoluted intact mass spectra. Furthermore, the heterogeneity at each glycosylation site was characterized at the reduced antibody level and at the isolated glycopeptide level. The glycosylation site and glycan structures were confirmed by performing a time-aligned-parallel fragmentation approach using the unique dual-collision cell design of the instrument and the incorporated ion-mobility separation function. Four different production batches of trastuzumab were analyzed and compared in terms of global glycosylation profiles as well as the heterogeneity at each glycosylation site. The results show that each batch of trastuzumab shares the same types of glycoforms but relative abundance of each glycoforms is varied. (J Am Soc Mass

This study shows that state-of-the-art liquid chromatography (LC) and mass spectrometry (MS) can be used for rapid verification of identity and characterization of sequence variants and posttranslational modifications (PTMs) for antibody products. A candidate biosimilar IgG1 monoclonal antibody (mAb) was compared in detail to a commercially available innovator product. Intact protein mass, primary sequence, PTMs, and the micro-differences between the two mAbs were identified and quantified simultaneously. Although very similar in terms of sequences and modifications, a mass difference observed by LC-MS intact mass measurements indicated that they were not identical. Peptide mapping, performed with data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode (LC-MS(E)), located the mass difference between the biosimilar and the innovator to a two amino acid residue variance in the heavy chain sequences. The peptide mapping technique was also used to comprehensively catalogue and compare the differences in PTMs of the biosimilar and innovator mAbs. Comprehensive glycosylation profiling confirmed that the proportion of individual glycans was different between the biosimilar and the innovator, although the number and identity of glycans were the same. These results demonstrate that the combination of accurate intact mass measurement, released glycan profiling, and LC-MS(E) peptide mapping provides a set of routine tools that can be used to comprehensively compare a candidate biosimilar and an innovator mAb.

No abstract

In order to achieve fast chromatographic separations of unknown entities, very rapid gradients need to be performed. We have studied the influence of temperature on such rapid gradient separations carried out under reversed phase conditions. The tool by which the quality of a gradient separation is measured is the peak capacity. Elevated temperature has two major influences. The first one is a reduction in viscosity, which reduces the backpressure for a given set of operating conditions. The second effect is an improvement in the diffusivity of the analytes, which speeds up mass transfer. We have assessed the influence of flow rate, gradient duration, and temperature on peak capacity using columns with different particle sizes and column lengths. The beneficial effect of temperature on both the viscosity and the diffusivity of the sample results not only in an improvement of the speed of the separations but also in a reduction of the back pressure under optimal operating conditions. At reasonable pressures, good separation power can be achieved in as little as one minute or less.