Successive multiple ionic polymer layer coated capillaries in the separation of proteins – Recombinant allergen variants as a case study

Abstract

A successive multiple ionic polymer layer (SMIL) coating consisting of two pairs of poly(diallyldimethylammonium chloride) and dextran sulfate (DS) layers was applied for the separation of recombinant products of the major birch pollen allergen Betula verrucosa (Bet v 1a). The combination with volatile ammonium bicarbonate buffer at pH 6.70 offers the possibility for future MS hyphenation. The negative net charge of allergens required DS as terminal SMIL layer. The EOF was accelerated from 3.17x10(-8) m(2) V(-1) s(-1) in uncoated to 4.52x10(-8) m(2) V(-1) s(-1) in SMIL capillaries. Fresh prepared SMIL capillaries showed slight EOF acceleration due to gradual re-organization of SMIL structure until stabilization was achieved. Dry storage of SMIL capillaries prevented fluctuations in EOF and migration times and improved coating durability. However, the gradual reconstitution of entangled SMIL layers affected efficiency, but was cured by a 10 mmol/L NaOH rinsing step. Durability of SMIL capillaries in MS-applicable dimension was confirmed for > 70 runs and in total 42 h of voltage application with average intra-day precision of 0.22 and 0.79% and inter-day-precision of 0.91 and 1.17% for migration times of EOF and Bet v 1a, respectively. Final SMIL coating allowed for the separation of Bet v 1a, a hypoallergenic isoform and carbamylated variants with 150,000-685,000 plates.

Capillary electrophoresis-mass spectrometry using noncovalently coated capillaries for the analysis of biopharmaceuticals

“…The former capillary coatings can be produced simply by flushing the capillary with solutions of highly adsorptive agents. Very stable and uniform adsorbed coatings can be produced by applying multiple layers of oppositely charged polymers, as indicated for the first time by Katayama et al [20, 21] and demonstrated by others subsequently [22–29]. …”

Separation and characterization of nitrated variants of the major birch pollen allergen by CZE‐ESI‐μTOF MS

“…ammonium acetate and ‐formate (data not shown). Based on the facile degassing at pH 6.5, the related pH drift and the screening results (data not shown), an initial pH of 8.0 was selected for subsequent optimization of NG and SL flow rates.…”

“…Remarkably, only one CZE-UV separation has been reported in this context to date [10], although CZE seems predestined for this task since it can equally address minute changes in the protein net charge and the conformation [24][25][26][27]. The applicability of various CE-UV modes, such as CZE, CIEF, and affinity CIEF, for the characterization of modified recombinant allergens has been described previously [28][29][30][31][32]. In comparison with UV, MS detection exhibits a higher sensitivity and allows for a further confirmation of identity by mass information of intact allergen variants.…”

Polyelectrolyte multilayer coatings for the separation of proteins by capillary electrophoresis: Influence of polyelectrolyte nature and multilayer crosslinking

“…Protein adsorption was observed on fused silica capillary and on PB/DS by the increase of layer thickness, while no increase could be measured for PB or PB/DS/PB coatings [17]. SMIL coatings based on two pairs of PDADMA/DS have been used for the separation of recombinant allergen variants in ammonium bicarbonate buffer at pH 6.7 with excellent repeatability/reproducibility [18]. PDADMA/PSS SMIL could be also deposited on poly(methyl methacrylate) microchip [19].…”