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Screening mAbs with Recombinant Lectins and Ion Mobility Spectrometry

· Ion Mobility,mAbs,Lectins,Electrospray

Introduction:

Accelerated drug development requires a new way to screen glycoproteins. IonDX Inc. has developed a bench-top analytical system to address this need. Our patented atmospheric pressure ion mobility spectrometer, IMgenius™, along with recombinant prokaryotic lectin (RPL) reagents, quantifies specific glycans and binding efficiency.• Platform technology – 3 min assay• Improved specificity over plant-based lectins• Mobility shifts reveal subpopulations • Minimum sample preparation• Detection of surface glycans

IMgenius™ system and methodology:

IMgenius determines the mobility (K) of a population of molecular ions as they travel through a gas under the influence of an electric field. Small ions have higher mobility than larger ions because they experience less drag as they travel through a gas. Inverse mobility (1/K) plots are easily interpreted because 1/K scales with molecular size. The width of a peak in an ion mobility spectrum correlates with molecular heterogeneity. Wider peaks indicate greater heterogenity. Peak position (modal 1/K value) indicates molecular size. 1/K values are directly convertible to collision cross section (CCS).

Deglycosylation:

Ion mobility spectra of Tocil and Cetux before and after deglycosylation with PNGaseF. Deglycosylation reduces the size of the mAbs and causes the spectra to shift to the left, indicating a decrease in molecular size.

RPL Binding:

After native Tocil binds to αGal, the complex shifts from 43 to 47 and the peak for the complex becomes 22% wider. Examination of the peak for the Tocil-aGal complex shows the peak expanded on the right side, which indicates that the larger conformers bind more efficiently than smaller conformers. Deglycosylated Tocil shifts minimally after binding with αGal. 

Sample preparation and ion generation:

Reactions between glycoproteins and RPLs are performed in suitable buffers and then buffered exchanged into 25 mM NH4Ac. Desalted samples are electrosprayed at 0.3 µL/min followed by passing the electrospray droplets through a charge-reducing chamber where droplet charge is reduced to a single charge. Charge reduction is accomplished by exposing the droplets to bipolar air ions produced when alpha particles emitted by a safely disposable sealed Polonium source. In this way protein ions never pass through high charge states and remain in their native conformation, allowing only surface glycans to be detected when native deglycosylation is used.

Application take-away:

IonDX’s platform ion mobility technology, along with highly-specific recombinant prokaryotic lectins (RPLs), provides a rapid new glycoprofiliing methodology for assaying glycoproteins such as biotherapeutic antibodies.

Additional Applications of IMgenius:

IonDX’s industry-wide collaborations have allowed us to develop analytical solutions across many additional classes of biotherapeutics, including the characterization of:

  • Non-covalent protein complexes
  • High molecular weight aggregates
  • mRNA
  • Glycoproteins
  • CRISPR-Cas
  • Virus particles
  • Lipid nanoparticles

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