Host cell protein quantification workflow using optimized standards combined with data-independent acquisition mass spectrometry

Hessmann, Steve; Chéry, Cyrille C.; Sikora, Anne-Sophie; Gervais, Annick; Carapito, Christine

doi:10.1016/j.jpha.2023.03.009

Cited by 4 publications

(2 citation statements)

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“…Although discussed here in the context of value-assignment to materials to be used as primary calibrators with protein quantification, LFQ is increasingly also being used in areas, such as process optimization and quality control of pharmaceutical products [ 15 , 27 – 31 ]. Typically in most of these applications, it is about quantification of individual proteins, rather than aiming at a mass fraction as a whole for HCPs.…”

Section: Discussionmentioning

confidence: 99%

“…Central to the functioning of LFQ is that an amount of any peptide produces a specific amount of MS1 intensity per mass of protein regardless of what the individual peptides (proteins) are. Unlike in most applications of LFQ, [ 5 , 6 , 14 , 15 ] in our context, peptide intensities are not collected separately per protein, but are rather integrated over all proteins identified from the host cell proteome. We demonstrate that this quantity can be calibrated against known amounts of the host cell proteome.…”

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confidence: 99%

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

Henrion,

Arsene,

Liebl

et al. 2023

Anal Bioanal Chem

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Quantitative analysis relies on pure-substance primary calibrators with known mass fractions of impurity. Here, label-free quantification (LFQ) is being evaluated as a readily available, reliable method for determining the mass fraction of host cell proteins (HCPs) in bioengineered proteins which are intended for use as protein calibration standards. In this study a purified hemoglobin-A2 (HbA2) protein, obtained through its overexpression in E. coli, was used. Two different materials were produced: natural and U15N-labeled HbA2. For the quantification of impurities, precursor ion (MS1-) intensities were integrated over all E. coli proteins identified and divided by the intensities obtained for HbA2. This ratio was calibrated against the corresponding results for an E. coli cell lysate, which had been spiked at known mass ratios to pure HbA2. To demonstrate the universal applicability of LFQ, further proteomes (yeast and human K562) were then alternatively used for calibration and found to produce comparable results. Valid results were also obtained when the complexity of the calibrator was reduced to a mix of just nine proteins, and a minimum of five proteins was estimated to be sufficient to keep the sampling error below 15%. For the studied materials, HbA2 mass fractions (or purities) of 923 and 928 mg(HbA2)/g(total protein) were found with expanded uncertainties (U) of 2.8 and 1.3%, resp. Value assignment by LFQ thus contributes up to about 3% of the overall uncertainty of HbA2 quantification when these materials are used as calibrators. Further purification of the natural HbA2 yielded a mass fraction of 999.1 mg/g, with a negligible uncertainty (U = 0.02%), though at a significant loss of material. If an overall uncertainty of 5% is acceptable for protein quantification, working with the original materials would therefore definitely be viable, circumventing the need of further purification.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%