Monitoring of glycoprotein products in cell culture lysates using lectin affinity chromatography and capillary HPLC coupled to electrospray linear ion trap-Fourier transform mass spectrometry (LTQ/FTMS).
In this paper, we describe the combination of lectin chromatography with capillary LC coupled to a linear ion trap-Fourier transform mass spectrometer (LTQ/FTMS) to enrich and characterize overexpressed glycoproteins from a cell culture lysate. A well-characterized glycoprotein, recombinant tissue plasminogen activator (rt-PA), was used as a standard, and we demonstrated that the three N-linked glycopeptides (including glycan structures) present in a tryptic digest of the rt-PA standard could be characterized in the new hybrid MS platform. A feature of this approach is that a significant amount of information can be obtained about the carbohydrate structures by direct analysis of the tryptic digest without the need for additional time-consuming sample preparation protocols. A combination of lectins was then studied for improved recovery of captured glycopeptides and was related to the selectivity of different lectins for specific glycosylation motifs. This approach was then extended to the lysate of a cell line routinely used in biotechnology manufacture (Chinese hamster ovary, CHO). This study showed that the combinations of lectins could enrich glycoproteins significantly from a CHO cell lysate. We also demonstrated that with this level of enrichment and with the new hybrid mass spectrometer, we could study the structures of N-linked glycopeptides of rt-PA present in a crude CHO cell lysate, at a ratio of 1:200 (rtPA:total cell lysate protein, w/w) by accurate mass measurement in the FTMS and tandem MSn in the linear ion trap. The generic and high throughput nature of the lectin approach combined with the ability to directly analyze the glycan structures in the tryptic digest suggest that this platform has the potential to routinely monitor glycoprotein products at early stage manufacturing in the biotech industry.
Wang
Yonghui
Y
Barnett Institute, Northeastern University, 360 Huntington Ave., Boston, Massachusetts 02115, USA.
Wu
Shiaw-Lin
SL
Hancock
William S
WS
D000818:Animals; D016466:CHO Cells; D002457:Cell Extracts / Q000737:chemistry*; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006224:Cricetinae; D006023:Glycoproteins / Q000032:analysis* / Q000096:biosynthesis* / Q000737:chemistry; D006801:Humans; D037102:Lectins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000592:standards; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000592:standards
D002457:Cell Extracts; D006023:Glycoproteins; D037102:Lectins; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator