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

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 m² V⁻¹ s⁻¹ in uncoated to 4.52x10⁻⁸ m² 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.