Carrier ampholytes for IEF, on their fortieth anniversary (1967–2007), brought to trial in court: The verdict

Abstract

The present review summarizes the data accumulated in 1-year work, by exploring, via a 3-D methodology (Rotofor fractionation followed by CE-MS), all the narrow (2 pH unit wide) carrier ampholyte (CA) compounds for IEF, produced by three companies (Pharmacia with Pharmalyte and Ampholine, BioRad with Bio-Lyte and Serva with Servalyt). All species have been assessed by measuring the types of pH gradient produced, the total number of individual chemicals (with M(r) values) and isoforms and their focusing behavior ('good' or 'poor' ampholytes). Servalyt contains a grand total of 686 chemical entities and no less than 3899 isoforms; Pharmalyte 643 and 2211; Bio-Lyte 255 and 1192; Ampholine 294 and 1182, respectively. In terms of M(r) distribution, although all 2-pH-unit ranges start with the same low M(r) values (ca. 200) their upper limits are quite different. Thus, Pharmalyte reaches an upper M(r) value of 1179 (in the pH 4-6 range), versus 907 for Servalyt, 835 for Bio-Lyte and 893 for Ampholine. In general, in going towards the more alkaline pH intervals (e.g. pH 8-10) the molecular mass of carrier ampholytes (CAs) is reduced to as low as 491 (Bio-Lyte), indicating that the alkaline species are probably made with shorter oligoamines and are, in general, less substituted. All acidic pH intervals (up to pH 6-8) appear to be constituted by a very large proportion of well focusing species, indicating small values of DeltapK across their pI. Above pH 8, all brands of CAs worsen, the vast majority being unable to focus properly and sustain adequately the pH gradient. General guidelines are given for the synthesis of new alkaline species for improving the basic pH ranges.

Online CIEF‐ESI‐MS in glycerol–water media with a view to hydrophobic protein applications

“…One explanation for this could be that the basic frontier was going past the detector first, whereas the acidic frontier could be more perturbed due to the longer influence of pressure application, as it went past the detector last. The shift for the acidic frontier was observed for all the analytes tested in this study, which could also indicate a possible distortion of the pH gradient zone on the acidic side, may be due to the presence of more ampholytes in this pH region [25]. Finally, a 6-min focusing time was considered optimum as it provided satisfactory results in less total analysis time.…”

“…the presence of ampholytes and an anticonvective gel to establish and stabilize the pH gradient. Although the m/z values of ampholytes are mostly beyond the m/z range of most proteins [25], their presence in the sprayed solution causes a reduction in the MS signal intensity due to ion suppression [16,[26][27][28] and background signal, and decreases the mass resolution. Approaches aiming at preventing the ampholytes from entering the ion source have been developed.…”

IEF in microfluidic devices

“…Svennson (who changed his name to Rilbe in 1968) envisioned the concept of carrier ampholytes (CAs) -a series of amphoteric compounds with closely spaced pIs that migrate within an applied electric field to invoke a corresponding pH gradient and boast sufficient buffering capacity to provide continuous current across the separation zone. The tireless development of these compounds [60] led to the emergence of CA-IEF as both a preparatory and analytical tool.…”

Evaluation of capillary zone electrophoresis for charge heterogeneity testing of monoclonal antibodies

“…IEC which allows MS characterization of different charge species after desalting of single fractions by dialysis and/or RP-HPLC separation [15][16][17] is currently the most important method which is preferred for biopharmaceuticals during late stage and market supply. Due to very small amounts and MS incompatible compounds like ampholytes or running buffer components identification of CE fractions is more difficult [18] and requires indirect approaches like Rotofor [19], free flow electrophoresis (FFE) [20] offgel-fractionation [21] or complex online coupling technology [18]. In case of charge differences that are buried within the three dimensional structure IEF and CZE are expected to be advantageous whereas in case of changes of the surface charge pattern or changes without influence on the net charge IEC may be preferred [7].…”