Four‐step approach to efficiently develop capillary gel electrophoresis methods for viral vaccine protein analysis

Figures

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  Figure 1. Effects of gel buffer concentration and capillary temperature on (A) the viscosity and (B) the conductivity.

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  Figure 2. The effects of gel buffer dilution on injection, sample stacking and CGE separation. Dilution of the gel buffer decreases the viscosity as well as decreases the ionic strength of the buffer. A lower viscosity results in an increased conductivity, whereas a lower ionic strength results in a decreased conductivity. In the case of diluting the Sciex CE‐SDS gel buffer with water, the total effect was that the conductivity was decreased (see text). The decreases in viscosity and in conductivity ultimately affect the sample injection, band broadening and sample stacking.

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  Figure 3. (A) The list of critical method parameters and actions required to develop and validate a CE‐SDS method for vaccine protein analysis. (B) Set of four steps for fast CE‐SDS viral vaccine protein method development and prospective validation.

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  Figure 4. Feasibility and optimization of CE‐SDS for G1 mini‐HA protein purity analysis, with the MW, sizing standard (MW‐standard), mini‐HA formulation buffer (Blank), non‐reduced mini‐HA from the feasibility experiment (Non‐reduced), reduced mini‐HA from the feasibility experiment (Reduced), deglycosylated and reduced mini‐HA at the selected conditions for sample preparation and separation (Optimal). The Internal standard (IS) is 10 KD standard. Separation conditions were 100% gel buffer, 20°C capillary temperature, hydrodynamic injection at 100 mbar for 100 s, and a capillary effective length of 24.5 cm for MW‐standard, Blank, Non‐reduced and Reduced. For the Optimal condition the same conditions applied with a capillary effective length of 8.5 cm instead of 24.5 cm. At the selected conditions, a system peak was observed at 5.0–5.6 min that was separated from the IS at 5.7 min, and the deglycosylated‐monomeric‐mini‐HA peak was observed at 7.2 min.

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  Figure 5. IPV protein (VP1‐4) analysis CE‐SDS method development. (A) Electropherograms of separation optimization results of default conditions with electrokinetic injection (Default), hydrodynamically injected IPV (Hydrodynamic) and the optimal conditions (Optimal), with hydrodynamic injection, 80% gel buffer, and an applied voltage of 20 KV. (B) Electropherograms of three different IPV strains.