LC-MS determination of Nicotiana benthamiana host plant proteins in the drug products of recombinant plant-produced pembrolizumab
Abstract Host proteins from Nicotiana benthamiana can remain in the recombinant biologic products after undergoing multiple steps of purification. These plant protein impurities may induce immunogenicity upon use. Therefore, controlling and monitoring host cell proteins is necessary throughout the p...
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Nature Portfolio
2025-07-01
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| Online Access: | https://doi.org/10.1038/s41598-025-11541-6 |
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| author | Ratama Daduang Pipob Suwanchaikasem Kaewta Rattanapisit Sarocha Vitayathikornnasak Theerakarn Srisangsung Christine Joy I. Bulaon Waranyoo Phoolcharoen |
| author_facet | Ratama Daduang Pipob Suwanchaikasem Kaewta Rattanapisit Sarocha Vitayathikornnasak Theerakarn Srisangsung Christine Joy I. Bulaon Waranyoo Phoolcharoen |
| author_sort | Ratama Daduang |
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| description | Abstract Host proteins from Nicotiana benthamiana can remain in the recombinant biologic products after undergoing multiple steps of purification. These plant protein impurities may induce immunogenicity upon use. Therefore, controlling and monitoring host cell proteins is necessary throughout the process of recombinant protein production. Liquid chromatography-mass spectrometry (LC-MS) has been successfully used to characterize protein species down to nanogram levels in various types of samples. In this study, LC-MS was applied to detect N. benthamiana plant proteins in plant-produced pembrolizumab anti-cancer antibody. Two types of purification techniques, gravity flow with manually packed column and automated system with prepacked column, were studied. After purification, the protein products were primarily assessed with SDS-PAGE and Western blot analyses and further examined with LC-MS to confirm protein identity and investigate plant protein contaminations. The pembrolizumab sequence was confirmed with more than 89% coverage. A higher number of host plant proteins were detected in the protein samples purified with gravity flow column. Luminal-binding protein 5 and ribulose bisphosphate carboxylase (RuBisCO) enzyme were predominant host plant proteins detected. Luminal-binding protein 5 was observed in the products purified with both purification methods. It was likely bound to pembrolizumab antibody and co-eluted into eluate fraction as its sequence is similar to binding immunoglobulin protein (BiP). RuBisCO enzyme was detected in the samples purified with gravity flow only. Its presence was likely due to incomplete wash by gravity flow chromatography. In summary, this study provided an important clue for plant proteins that could be contaminated in plant-produced products and suggested that second column chromatography is required to enhance purification efficiency. |
| format | Article |
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| language | English |
| publishDate | 2025-07-01 |
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| spelling | doaj-art-0ce48a2ca90e47d19a32a553ed8d1bfe2025-08-20T03:04:25ZengNature PortfolioScientific Reports2045-23222025-07-0115111210.1038/s41598-025-11541-6LC-MS determination of Nicotiana benthamiana host plant proteins in the drug products of recombinant plant-produced pembrolizumabRatama Daduang0Pipob Suwanchaikasem1Kaewta Rattanapisit2Sarocha Vitayathikornnasak3Theerakarn Srisangsung4Christine Joy I. Bulaon5Waranyoo Phoolcharoen6Baiya Phytopharm Co., LtdBaiya Phytopharm Co., LtdBaiya Phytopharm Co., LtdBaiya Phytopharm Co., LtdBaiya Phytopharm Co., LtdBaiya Phytopharm Co., LtdDepartment of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn UniversityAbstract Host proteins from Nicotiana benthamiana can remain in the recombinant biologic products after undergoing multiple steps of purification. These plant protein impurities may induce immunogenicity upon use. Therefore, controlling and monitoring host cell proteins is necessary throughout the process of recombinant protein production. Liquid chromatography-mass spectrometry (LC-MS) has been successfully used to characterize protein species down to nanogram levels in various types of samples. In this study, LC-MS was applied to detect N. benthamiana plant proteins in plant-produced pembrolizumab anti-cancer antibody. Two types of purification techniques, gravity flow with manually packed column and automated system with prepacked column, were studied. After purification, the protein products were primarily assessed with SDS-PAGE and Western blot analyses and further examined with LC-MS to confirm protein identity and investigate plant protein contaminations. The pembrolizumab sequence was confirmed with more than 89% coverage. A higher number of host plant proteins were detected in the protein samples purified with gravity flow column. Luminal-binding protein 5 and ribulose bisphosphate carboxylase (RuBisCO) enzyme were predominant host plant proteins detected. Luminal-binding protein 5 was observed in the products purified with both purification methods. It was likely bound to pembrolizumab antibody and co-eluted into eluate fraction as its sequence is similar to binding immunoglobulin protein (BiP). RuBisCO enzyme was detected in the samples purified with gravity flow only. Its presence was likely due to incomplete wash by gravity flow chromatography. In summary, this study provided an important clue for plant proteins that could be contaminated in plant-produced products and suggested that second column chromatography is required to enhance purification efficiency.https://doi.org/10.1038/s41598-025-11541-6Luminal-binding protein 5RuBisCOFast protein liquid chromatographyMonoclonal antibodyPlant molecular farmingProtein purification |
| spellingShingle | Ratama Daduang Pipob Suwanchaikasem Kaewta Rattanapisit Sarocha Vitayathikornnasak Theerakarn Srisangsung Christine Joy I. Bulaon Waranyoo Phoolcharoen LC-MS determination of Nicotiana benthamiana host plant proteins in the drug products of recombinant plant-produced pembrolizumab Scientific Reports Luminal-binding protein 5 RuBisCO Fast protein liquid chromatography Monoclonal antibody Plant molecular farming Protein purification |
| title | LC-MS determination of Nicotiana benthamiana host plant proteins in the drug products of recombinant plant-produced pembrolizumab |
| title_full | LC-MS determination of Nicotiana benthamiana host plant proteins in the drug products of recombinant plant-produced pembrolizumab |
| title_fullStr | LC-MS determination of Nicotiana benthamiana host plant proteins in the drug products of recombinant plant-produced pembrolizumab |
| title_full_unstemmed | LC-MS determination of Nicotiana benthamiana host plant proteins in the drug products of recombinant plant-produced pembrolizumab |
| title_short | LC-MS determination of Nicotiana benthamiana host plant proteins in the drug products of recombinant plant-produced pembrolizumab |
| title_sort | lc ms determination of nicotiana benthamiana host plant proteins in the drug products of recombinant plant produced pembrolizumab |
| topic | Luminal-binding protein 5 RuBisCO Fast protein liquid chromatography Monoclonal antibody Plant molecular farming Protein purification |
| url | https://doi.org/10.1038/s41598-025-11541-6 |
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