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PO-CON1852E Affinity purification of IdeZ digest for Glycosylation profile of Immunoglobulins using a linear benchtop MALDI-TOFMS. ASMS 2019 WP-053 Y. Yamazaki 1 , S. Nakaya 1 1 Shimadzu Corporation, Kyoto, Japan
2 Affinity purification of IdeZ digest for Glycosylation profile of Immunoglobulins using a linear benchtop MALDI-TOFMS. Introduction Glycosylation on protein plays wide-range vital roles in biological processes from stabilization of protein conformation to expression of binding specificity. In this view, a characterization of the N-/O-linked glycan is quite significant, especially, in development of biopharmaceuticals. To date, whereas intensive efforts were conducted to characterize glycans precisely with high-end mass spectrometers, conventional instruments without time consuming preparation has been anticipated for batch analysis in screening or QA/QC. A newly developed bench-top MALDI-TOFMS is expected to be the conventional instrument in terms of sufficient specification, through-put, and cost effectiveness. We attempted to characterize glycosylation of IgG without a release of glycan using the bench-top MALDI-TOFMS. To do this, we examined a preparation using affinity beads and enzymatic cleavage. Methods • IgGs and their preparations NIST mAb, IgG from mouse serum, and myeloma IgG were subject to MS analysis. IdeZ and Protein-A magnetic beads (Mag Sepharose) were purchased from Promega, and GE Healthcare respectively. The IgGs dissolved in tris-NaCl buffer were incubated with IdeZ at 37°C for two hours. Resultant solutions including Fc and F(ab’)2 were applied to the beads equilibrated with the tris-buffer, then washed with the same buffer several times. Fc associated with Protein-A was eluted with acidic solution, then desalted with ZipTip C18. • MALDI-TOFMS All MS analysis in positive ion mode was performed with a bench-top linear MALDI-TOFMS (MALDI-8020, Shimadzu Corp., Japan). Sinapinic acid (SA) and Ferulic acid (FA) were applied to MS analysis as matrix at the same condition as follows; 20 mg/mL in 50% acetonitrile/milliQ water with 0.1% TFA. • Statistical data analysis eMSTAT Solution (Shimadzu Corp., Japan) was applied to perform classification/differentiation of glycosylated-Fc regions. Overview • A batch analysis of glycosylation of IgG using a bench-top linear MALDI-TOFMS was intended. • A magnet beads linked with protein A was applied to affinity purification, following a digestion of IgG by IdeZ. • Three different glycosylated Fc were clearly observed to classify them with a statistical software.
3 Affinity purification of IdeZ digest for Glycosylation profile of Immunoglobulins using a linear benchtop MALDI-TOFMS. MALDI-8020 Fig.1 MS of whole IgGs Features: • Compact design • > 5,000 MS resolution • High through-put with solid state laser and fast stage motion • Rapid sample introduction • UV laser-based source cleaning • Quiet operation Results NIST mAb Mouse serum Mouse myeloma
4 Affinity purification of IdeZ digest for Glycosylation profile of Immunoglobulins using a linear benchtop MALDI-TOFMS. Fig.2 Work-flow of Fc affinity separation/detection IgG (150kDa) (Fab’)2 (100kDa) Fc x 2 (25kDa) IdeZ, 2hrs, 37ºC Protein A beads, 15 min. Fix the beads & Wash Magnet Elution of Fc under Acidic condition MALDI-TOFMS
5 Affinity purification of IdeZ digest for Glycosylation profile of Immunoglobulins using a linear benchtop MALDI-TOFMS. Fig.4 A comparison of Fc measured with SA and FA under the identical MS condition Fig.3 MS of NIST mAb treated with IdeZ (Fab’)2 Fc Fc, +2 SA FA
6 Affinity purification of IdeZ digest for Glycosylation profile of Immunoglobulins using a linear benchtop MALDI-TOFMS. Fig.5 A comparison of three Fc separated with the work-flow NIST mAb Mouse serum Mouse myeloma
Affinity purification of IdeZ digest for Glycosylation profile of Immunoglobulins using a linear benchtop MALDI-TOFMS. 7 Fig.6 Classification of three Fc using eMSTAT Solution Generation of Score Plot in a minute NIST mAb Mouse serum Mouse myeloma Data sets for various Fc Save peak lists in each folder Open “IgG” folder in eMSTAT Solution eMSTAT Solution IgG >
First Edition: May, 2019 © Shimadzu Corporation, 2019 Affinity purification of IdeZ digest for Glycosylation profile of Immunoglobulins using a linear benchtop MALDI-TOFMS. Conclusions • Purification of enzymatically fragmented IgGs by affinity-beads enables batch analysis for the glycosylation using a bench-top MALDI-TOFMS. • MS resolution of the bench-top MALDI-TOFMS is sufficient to recognize three Fc that differ in glycosylation mainly. • A statistical analysis by eMSTAT Solution enables a classification of three glycosylated Fc smoothly and quickly, which could be applicable to QA/QC. For Research Use Only. Not for use in diagnostic procedures. This presentation may contain references to products that are not available in your country. All rights reserved. Information subject to change without notice.