Quantifying protein residues in APIs: Bradford assay mechanism and limitations, outperformed by HILIC-MS/MS

Yangrui Zhang; Yizhen Liu; Yangyang Chen; Chen Guo; Fengting Ou; Junhuan Lin; Hanmeng Guo; Tao Ke; Lushan Yu

doi:10.1016/j.jpha.2026.101552

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Yangrui Zhang, Yizhen Liu, Yangyang Chen, Chen Guo, Fengting Ou, Junhuan Lin, Hanmeng Guo, Tao Ke, Lushan Yu. Quantifying protein residues in APIs: Bradford assay mechanism and limitations, outperformed by HILIC-MS/MS[J]. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2026.101552

Citation:

Yangrui Zhang, Yizhen Liu, Yangyang Chen, Chen Guo, Fengting Ou, Junhuan Lin, Hanmeng Guo, Tao Ke, Lushan Yu. Quantifying protein residues in APIs: Bradford assay mechanism and limitations, outperformed by HILIC-MS/MS[J]. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2026.101552

Citation:

Yangrui Zhang, Yizhen Liu, Yangyang Chen, Chen Guo, Fengting Ou, Junhuan Lin, Hanmeng Guo, Tao Ke, Lushan Yu. Quantifying protein residues in APIs: Bradford assay mechanism and limitations, outperformed by HILIC-MS/MS[J]. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2026.101552

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Quantifying protein residues in APIs: Bradford assay mechanism and limitations, outperformed by HILIC-MS/MS

doi: 10.1016/j.jpha.2026.101552

Yangrui Zhang^1,2,
Yizhen Liu^1,2,
Yangyang Chen^1,2,
Chen Guo³,
Fengting Ou⁴,
Junhuan Lin^1,2,
Hanmeng Guo^1,2,
Tao Ke^1,2,
Lushan Yu^{1,2,4
,
,}

1. Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China;
2. National Key Laboratory of Advanced Drug Delivery and Release Systems, Zhejiang University, Hangzhou, 310058, China;
3. Shenzhen Audencia Financial Technology Institute, Shenzhen University, Shenzhen, Guangdong, 518000, China;
4. Jinhua Institute of Zhejiang University, Jinhua, Zhejiang, 321036, China

Funds:

This work was supported by the Jinhua Science and Technology Plan Project (2023-1-119), the National Natural Science Foundation of China (82274003) and the Ten-Thousand Talents Program of Zhejiang Province (2021R52013).

Received Date: Aug. 06, 2025
Accepted Date: Jan. 13, 2026
Rev Recd Date: Jan. 09, 2026
Available Online: Jan. 15, 2026

Abstract

Abstract

The molecular complexity of active pharmaceutical ingredients (APIs) has increased over recent decades, with production largely relying on microbial processes such as fermentation. However, these processes introduce non-therapeutic protein impurities. Accurate quantification of protein impurities in APIs is critical, as underestimation risks safety and efficacy, while overestimation leads to costly over-purification. Here, we evaluated five conventional protein assays with diverse standards and found their accuracy compromised by protein structural diversity. To deconstruct the mechanism of this limitation, we examined the Bradford assay as a case study. Machine learning models trained on data from over 140 standards revealed that, beyond the known roles of basic and aromatic residues, other amino acids exert positive or negative effects on the Bradford assay response—as predicted by Lasso regression and validated with five designed peptides. Together with molecular docking and interaction analysis, which identified electrostatic interactions as the primary driving force, these findings collectively account for the inherent bias of such structure-dependent assays. The systematic evaluation and mechanistic insight prompted us to develop a hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS) method for amino acid analysis, enabling accurate protein quantification at ppm levels in matrices. Applied to eight commercial APIs, the method revealed substantial variation in protein residue levels (9.53–5570 ppm). By providing precise measurement of protein impurities in small-molecule APIs, this study helps ensure drug safety and efficacy at no excessive purification costs, while also offering insights into the molecular mechanisms underlying the Bradford assay.
- Active Pharmaceutical Ingredient (API),
- Protein Residues,
- Bradford Assay,
- HILIC-MS/MS

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References(51)

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