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Product Manual for ProPac
4.3.
Monitoring Forced Aspargine Deamidation of Glycoproteins by Cation Exchange
Chromatography
Deamidation of Asn residues or the isomerization of Asp residues occurs in a variety of protein-based pharmaceuticals
including human growth hormone [1], tissue plasminogen activator [2], hirudin [3], monoclonal antibodies [4], acidic
fibroblast growth factor [5], and interleukin 1 [6], with varying effects on the activity or stability of the therapeutic protein.
Hence, monitoring the deamidation of Asn residues in proteins is of interest to analytical and protein chemists in quality
control and process departments at biotechnology and pharmaceutical companies [7].
As described by A. D. Donato et al. [8], separation of the Asn67 deamidation products of ribonuclease A required cation
exchange on Mono S followed by hydrophobic interaction chromatography to resolve the two deamidation variants (Asp and
isoAsp at residue 67). In contrast, using only a ProPac WCX-10 Column, deamidation variant forms having Asp or isoAsp at
Asn67 were baseline-resolved from each other and from native ribonuclease A in a single chromatographic analysis (9, 10).
Figure 5 shows the separation of ribonuclease A and its deamidation products on ProPac SCX-20 at various incubation time
points during the course of the forced deamidation. The baseline separation made it possible to quantify the change in
amounts of each form within the mixture as a function of time. This forced deamidation of RNase A follows the method
described by Di Donato et al (8). For the deamidation experiment, combine 334 μL of 15 mg/mL RNase A in DI H
100 μL of 10% ammonium bicarbonate and 566 uL of DI H
A solution and incubated at 37 ºC. Aliquots were withdrawn (50 μL) intermittently and kept frozen until used for analysis.
When ready to analyze the samples, aliquots were thawed, diluted five-fold with the Eluent A (20 mM MES pH 5.6 + 60 mM
NaCl, See figure 5) in auto-sampler vials and used for analysis.
Document No. 065403-01
®
SCX-20
©2010 DIONEX CORPORATION
O in a 1.5 mL microcentrifuge tube to make a 5 mg/mL RNase
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O,
2
September 2010
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