Label-free quantification of host cell protein impurity in recombinant hemoglobin materials

Area of Science:

• Biochemistry and Proteomics
• Analytical Chemistry
• Biotechnology

Background:

• Accurate quantitative analysis requires pure primary calibrators with known impurity mass fractions.
• Host cell proteins (HCPs) are critical impurities in bioengineered proteins used as calibration standards.
• Label-free quantification (LFQ) is explored as a method for HCP impurity determination.

Purpose of the Study:

• To evaluate label-free quantification (LFQ) for determining host cell protein (HCP) mass fractions in bioengineered protein standards.
• To assess the reliability and accessibility of LFQ for protein calibration.
• To demonstrate the universal applicability of LFQ across different proteomes.

Main Methods:

• Purified hemoglobin-A2 (HbA2) from E. coli, including natural and U15N-labeled forms, was used.
• LFQ quantified HCP impurities by integrating MS1 intensities of E. coli proteins relative to HbA2.
• Calibration was performed using E. coli lysate spiked with known mass ratios of pure HbA2; yeast and human K562 proteomes were used for validation.

Main Results:

• LFQ determined HbA2 mass fractions of 923 and 928 mg/g with expanded uncertainties of 2.8% and 1.3%, respectively.
• LFQ contributed approximately 3% to the overall uncertainty in HbA2 quantification.
• A minimum of five proteins in a calibrator mix was sufficient to maintain sampling error below 15%.

Conclusions:

• LFQ is a viable and accessible method for assigning mass fractions of HCP impurities in protein calibrators.
• The method demonstrates universal applicability across different proteomes and reduced sample complexity.
• Using LFQ with the original materials is practical when an overall uncertainty of 5% is acceptable, avoiding extensive purification.