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Protein Variant Analysis Protein Variant Analysis

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Selective Separations for Quality Control and R&D

Advances with our PolyAspartic Acid (PolyCAT A®) columns make it possible to separate and quantitate many protein variants that differ by a single residue. In the past, such detection usually required protein digestion and peptide mapping. Variants form as purified recombinant proteins degrade in storage; the rate and pathway is influenced by the solution pH. At high pH, the main pathway is deamidation of susceptible Asn- residues to yield a succinimide ring.; this hydrolyzes to a racemic mixture of Asp- and isoAsp- residues. At neutral pH, the fastest pathway is dehydration of Asp- to yield first a succinimidyl- intermediate, then Asp- and isoAsp-. The effects on biological activity range from trivial to serious.

Human Growth Hormone (HGH) has an Asp- at position 130. In the example below, this form is resolved from the variants with isoAsp- and succinimide at position 130.

Recombinant Protein Dehydration Variant Analysis

-Human Growth Hormone after 6 Days at 37C-

Formulation at pH 6.8 leads to increased dehydration (Asp converts to isoAsp)

Protein Variant Dehydration Graphic

Column: PolyCAT A (1000Å); p/n: P1850-1-204 (or 204CT0510)

Gradient: 130-145 mM NH4-acetate, pH 4.0, with 40% MeCN; 30C

Resolution from deamidation variants is even better. HGH also undergoes nonenzymatic loss of the first two residues. This desPhe-Pro- form is well separated from HGH, as are its corresponding variants.

Recombinant Protein Deamidation Variant Analysis

-Human Growth Hormone after 6 Days at 37C-

Formulation at pH 8.3 leads to increased deamidation (Asn converts to Asp + isoAsp)

Protein Variant Deamidation Graphic

Column: PolyCAT A (1000Å); p/n: P1850-1-204 (or 204CT0510)

Gradient: 130-145 mM NH4-acetate, pH 4.0, with 40% MeCN; 30C

Data courtesy of Benny Welinder, Novo Nordisk A/S)

Ordering Information

Polyaspartic Acid WCX column (5; 1000Å PolyCAT A)

Dimensions         Part Number    (PolyLC p/n)       
200 x 4.6mm           P204CT0510      204CT0510         
200 x 2.1mm           P202CT0510      202CT0510        
100 x 4.6mm           P104CT0510      104CT0510        
100 x 2.1mm           P102CT0510      102CT0510        

For other part numbers and prices, please see the PolyLC Price List .


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Last Updated: 12/02/16