Profiling preparations of recombinant birch pollen allergen Bet v 1a with capillary zone electrophoresis in pentamine modified fused-silica capillaries

Punzet, M.; Ferreira, Fátima; Briza, Peter; Ree, Ronald van; Malissa, H.; Stutz, Hanno

doi:10.1016/j.jchromb.2006.01.031

Cited by 21 publications

(37 citation statements)

References 70 publications

(101 reference statements)

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“…Even without the addition of a wall-coating reagent, a minor fraction is partially resolved from the major peak of each cluster (Fig. 1A, peaks 1b, 2b, and 3b) which was not visible in the phosphate buffer [39]. In total, nine different fractions could be distinguished with MES without addition of TEPA.…”

Section: Czementioning

confidence: 97%

“…The preparation procedure for rBet v 1a is outlined elsewhere [4]. Due to the employment of urea in course of the applied modified manufacturing procedure, Bet v 1a is known to be partially carbamylated in this batch [39]. The lyophilized material was reconstituted either in 500 or 1000 mL ultrapure water to give stock solutions of 2.0 and 1.0 mg/mL, respectively.…”

Section: Allergens and Proteinsmentioning

confidence: 99%

“…Particularly, so-called "good buffers" provide favorable results due to their low conductivity and reduced Joule heating. Based on an optimized separation system for Bet v 1a published recently [39], MES was employed instead of phosphate, however, maintaining a concentration of 100 mmol/L and the pH at 6.50. Three peak clusters can be distinguished which represent Bet v 1a and its single and double carbamylated variants.…”

Section: Czementioning

confidence: 99%

“…However, applications of CZE [32][33][34][35], CIEF [31,36], CITP [37], and MEKC [32,33,38] in allergen specification are limited and mainly related to natural crude extracts [33,38]. Recently, our group has optimized and validated a CZE separation for a profiling of different preparations of recombinant Bet v 1a [39] and revealed a progressive chemical modification (carbamylation) of the target allergen in one preparation due to the implementation of urea in the manufacturing procedure. According to the relevant ICH guidelines [18,19], the objective of the following approach was to develop and optimize both CZE and CIEF separation for the aforementioned heterogeneous preparation of recombinant Bet v 1a and to compare the respective allergen profiles as well as the relative purity of the target allergen, in order to assess the compliance of CE with regulations for biotechnological products [18,19,21].…”

Section: Introductionmentioning

confidence: 99%

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Profiling recombinant major birch pollen allergen Bet v 1a and carbamylated variants with CZE and CIEF

2007

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A preparation of recombinant birch pollen allergen of Betula verrucosa isoform 1a (Bet v 1a) containing chemically modified (carbamylated) variants has been analyzed by CZE and CIEF. In CZE, employing a 100 mmol/L MES buffer at pH 6.50, with 0.4 mmol/L tetraethylenepentamine (TEPA) added, allowed for the resolution of 17 protein fractions. The CIEF profiling of the allergen preparation required a combination of a wide-pH-range carrier ampholyte (CA) of pH 3-10 with two narrow-range CAs of pH 5-6 and 5-7. For CIEF, 91 mmol/L of glycine at pH 2.12 and 20 mmol/L of CHES at pH 10.00 were applied as anolyte and catholyte, respectively. The generated pH gradient was nonlinear with a flat slope for pH 4-6, thus providing an improved resolution. In CIEF, up to 18 protein fractions were distinguished as well. The pI of the target allergen Bet v 1a was 4.9 as determined by means of two pI marker compounds flanking the allergen. Relative purity of the target allergen within the preparation containing carbamylated variants was in accordance for both separation systems and varied between 40.7 and 42.8%.

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Section: Czementioning

confidence: 97%

Section: Allergens and Proteinsmentioning

confidence: 99%

Section: Czementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Profiling recombinant major birch pollen allergen Bet v 1a and carbamylated variants with CZE and CIEF

2007

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“…CE has the capability to cover major physico-chemical aspects of Ag products as demonstrated recently [36][37][38]. Moreover, CE has been employed in the quality control and characterization of mAbs [39][40][41][42][43].…”

Section: Introductionmentioning

confidence: 99%

Confirmation of immuno‐reactivity of the recombinant major birch pollen allergen Bet v 1a by affinity‐CIEF

et al. 2009

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Affinity-CIEF has been applied to characterize a recombinant product of the major birch pollen allergen Betula verrucosa isoform 1a (Bet v 1a) immuno-chemically. For this purpose mAbs of the IgG-type have been produced in-lab from two murine hybridoma lines, specified as clones 2 and 5.1. Both IgG clones were characterized by SDS-PAGE, MALDI-TOF-MS and CIEF. The purified IgG solutions had to be dialysed against 10 mmol/L phosphate (pH 7.4) to prevent IgG precipitation and to ensure appropriate CIEF separation. Both tested monoclonal IgGs (mIgGs) comprised four constituents covering pI ranges of 6.98-7.09 and 6.78-7.03 for clones 2 and 5.1 with major peaks at pI 7.09 and 7.03, respectively. When increasing amounts of Bet v 1a (pI 4.95) were incubated with 2.0 mumol/L mIgG, novel peaks were progressively induced in a pI range slightly more acidic than the focusing region of mIgGs. These peaks grew on the expense of original mIgG peaks. All pI values were calculated using two pI marker compounds with a repeatability of better than 0.03 units. New peaks represent complexes between Bet v 1a and mIgG either of 1:1 or of 2:1 binding stoichiometry. At a molar ratio of 2:1, saturation of both IgG paratopes with allergen (Ag) molecules was achieved as indicated by unbound Bet v 1a. The current CIEF approach addresses the proof of single epitope integrity in the course of immuno-chemical characterization of Bet v 1a. Contrary to traditional immunoassays, affinity CIEF allows for a distinction and relative quantification of mAbs, Ag-antibody complexes and Ag variants coexisting in one sample.

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Glucosylceramide Mimics: Highly Potent GCase Inhibitors and Selective Pharmacological Chaperones for Mutations Associated with Types 1 and 2 Gaucher Disease

et al. 2013

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A series of iminoxylitol derivatives carrying a C-linked di-O-acyl or di-O-alkyl glyceryl substituent were prepared and characterized. All of these compounds, which were designed as glucosylceramide (GlcCer) mimics, were nanomolar inhibitors of lysosomal β-glucosidase (glucocerebrosidase, GCase). Two of these pseudoglycolipids were further evaluated for their ability to enhance the activity of mutant GCase in human Gaucher cells. Although the di-O-hexyl ether was surprisingly devoid of chaperoning activity on both N370S and L444P GCases, the di-O-decanoyl ester was a potent chaperone of the L444P hydrolase, capable of increasing the residual activity of the enzyme by a factor of two at a very low concentration (50 nM); such a significant effect on the L444P mutation in human fibroblasts has not yet been observed. In heat-stress studies, the diether was found to be much more effective in stabilizing the wild-type enzyme than the diester. Four representative pseudoglycolipids were also assayed as inhibitors of GlcCer synthase, because such compounds could find use in the substrate reduction therapy approach to treat lysosomal storage diseases, but these compounds revealed only moderate activity. As efficient pharmacological chaperones, new structures such as the di-C10 -ester constitute leads for the development of therapeutic agents for types 2 and 3 Gaucher disease, the most severe neuronopathic forms of this lysosomal disease.

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Profiling preparations of recombinant birch pollen allergen Bet v 1a with capillary zone electrophoresis in pentamine modified fused-silica capillaries

Cited by 21 publications

References 70 publications

Profiling recombinant major birch pollen allergen Bet v 1a and carbamylated variants with CZE and CIEF

Profiling recombinant major birch pollen allergen Bet v 1a and carbamylated variants with CZE and CIEF

Confirmation of immuno‐reactivity of the recombinant major birch pollen allergen Bet v 1a by affinity‐CIEF

Glucosylceramide Mimics: Highly Potent GCase Inhibitors and Selective Pharmacological Chaperones for Mutations Associated with Types 1 and 2 Gaucher Disease

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