Probing Native Protein Conformations with IMgenius™

· Ion Mobility,Electrospray,Native Structure


Based on the current industry drive for accelerated drug development, there is a heightened need for screening native protein structures. Atmospheric pressure Ion Mobility Spectrometry (IMS) is an emerging analytical technology that addresses this challenge. IonDX Inc. has developed a bench-top analytical system, IMgenius™, based on this technology. Our patented spectrometer simultaneously measures five important attributes of proteinsand biotherapeutics: 

• Size distribution

• Structural conformation

• Stability, including aggregation

• Comparability 

• Purity 

IMgenius SystemTM and Methodology:

Charge-reduced electrospray: Desalted samples are electrosprayed at 0.3 uL/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 are 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 native conformations to be studied.

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Atmospheric pressure ion mobility spectrometry: IMgenius determines the mobility (K) of a population of molecular ions as they travel through atmospheric pressure air and are detected as an ion current after they are deflected onto a pick-up electrode by voltage applied to a center rod. Small ions have higher mobility than larger ions because they experience less drag as they travel through a gas and therefore are deflected more easily.Inverse mobility (1/K) plots are easily interpreted as 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).

Native Conformation of Myoglobin:

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Ko values for myoglobulin (blue) ref obtained with IM-MS show variation across ion charge states, even for an individual charge state, which makes it difficult to identify the native conformation. Our ion source produces lowly�charged ions (red) and when extrapolated towards z = 0, actually passes through 0. The zero intercept confirms the preservation of native conformations. See below for discussion of the Mason-Schamp equation. 

Native Confirmation of NISTmAb:

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Ko values for NISTmAb (blue) ref obtained with IM-MS lead to a prediction of CCS = 68 nm2. This value is significantly less than 100 nm2 predicted from X-ray crystallography and attributable gas-phase collapse. conditions in our ion source and IMgenius minimize gas-phase collapse.

The Mason Schamp Equation provides an expression for determining CCS (sigma) from Ko. Plotting Ko vs z should be a straight line with a zero intercept. Plots with non-zero intercepts, as shown above, indicate the ions are stretched out and therefore not in their native conformation. Our data has a zero intercept and confirms our production of native conformations.

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