Host cell protein quantification of an optimized purification method by mass spectrometry

Area of Science:

• Biotechnology
• Protein Purification
• Vaccine Development

Background:

• Recombinant ExoProtein A (EPA), a detoxified Pseudomonas aeruginosa Exotoxin A, serves as a crucial protein carrier in vaccine formulation.
• Scaled manufacturing of EPA using Escherichia coli led to elevated levels of E. coli host cell proteins (HCPs), nearing or exceeding acceptable limits per human dose.

Purpose of the Study:

• To redevelop the EPA purification process to effectively reduce HCP content in the final product.
• To evaluate the efficacy of the revised purification strategy using orthogonal analytical methods.
• To establish a high-throughput mass spectrometry (MS) method for HCP analysis.

Main Methods:

• Orthogonal analytical techniques including a platform-specific immunoassay and 2D/LC-MS^E were employed to quantify HCP levels.
• A revised purification process was implemented to minimize HCP contamination.
• 1D/LC-MS/MS was developed and utilized as a high-throughput method for HCP profiling.

Main Results:

• The original purification method resulted in HCP levels of 1,830 ppm (0.18% w/w) by immunoassay.
• The revised purification process reduced HCP levels below the immunoassay's detection limit.
• 2D/LC-MS^E identified 57 unique HCPs totaling 37,811 ppm (3.78% w/w) in the reference sample; revised process lots showed significantly lower levels (730 and 598 ppm).
• 1D/LC-MS/MS analysis demonstrated good correlation with 2D/LC-MS^E data, validating its utility.

Conclusions:

• The revised purification process effectively minimizes HCP contamination in Recombinant ExoProtein A.
• 1D/LC-MS/MS is a suitable high-throughput method for HCP analysis in EPA manufacturing.
• These advancements support the reliable production of EPA as a protein carrier for vaccine applications.