التفاصيل البيبلوغرافية
| العنوان: |
Minimal purification method enables developability assessment of recombinant proteins |
| المؤلفون: |
Rodriguez‐Aponte, Sergio A., Naranjo, Christopher A., Johnston, Ryan S., Dalvie, Neil C., Crowell, Laura E., Bajoria, Sakshi, Kumru, Ozan S., Joshi, Sangeeta B., Volkin, David B., Love, J. Christopher |
| المصدر: |
Biotechnology and Bioengineering ; ISSN 0006-3592 1097-0290 |
| بيانات النشر: |
Wiley |
| سنة النشر: |
2023 |
| المجموعة: |
Wiley Online Library (Open Access Articles via Crossref) |
| الوصف: |
Analytical characterization of proteins is a critical task for developing therapeutics and subunit vaccine candidates. Assessing candidates with a battery of biophysical assays can inform the selection of one that exhibits properties consistent with a given target product profile (TPP). Such assessments, however, require several milligrams of purified protein, and ideal assessments of the physicochemical attributes of the proteins should not include unnatural modifications like peptide tags for purification. Here, we describe a fast two‐stage minimal purification process for recombinant proteins secreted by the yeast host Komagataella phaffii from a 20 mL culture supernatant. This method comprises a buffer exchange and filtration with a Q‐membrane filter and we demonstrate sufficient removal of key supernatant impurities including host‐cell proteins (HCPs) and DNA with yields of 1–2 mg and >60% purity. This degree of purity enables characterizing the resulting proteins using affinity binding, mass spectrometry, and differential scanning calorimetry. We first evaluated this method to purify an engineered SARS‐CoV‐2 subunit protein antigen and compared the purified protein to a conventional two‐step chromatographic process. We then applied this method to compare several SARS‐CoV‐2 RBD sequences. Finally, we show this simple process can be applied to a range of other proteins, including a single‐domain antibody, a rotavirus protein subunit, and a human growth hormone. This simple and fast developability methodology obviates the need for genetic tagging or full chromatographic development when assessing and comparing early‐stage protein therapeutics and vaccine candidates produced in K. phaffii . |
| نوع الوثيقة: |
article in journal/newspaper |
| اللغة: |
English |
| DOI: |
10.1002/bit.28385 |
| الإتاحة: |
https://doi.org/10.1002/bit.28385Test |
| حقوق: |
http://creativecommons.org/licenses/by/4.0Test/ |
| رقم الانضمام: |
edsbas.728837F0 |
| قاعدة البيانات: |
BASE |
