Minimal purification method enables developability assessment of recombinant proteins
Name
Biotech Bioengineering - 2023 - Rodriguez‐Aponte - Minimal purification method enables developability assessment of.pdf
Description
Published version
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1.91 MB
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Checksum (MD5)
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Author(s)
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
Date Issued
March 17, 2023
Journal
Biotechnology and Bioengineering
Publisher
Wiley
Citation
Analytical characterization of proteins is a critical task for developingtherapeutics and subunit vaccine candidates. Assessing candidates with abattery of biophysical assays can inform the selection of one that exhibitsproperties consistent with a given target product profile (TPP). Such assess-ments, however, require several milligrams of purified protein, and idealassessments of the physicochemical attributes of the proteins should notinclude unnatural modifications like peptide tags for purification. Here, wedescribe a fast two‐stage minimal purification process for recombinant proteinssecreted by the yeast host Komagataella phaffii from a 20 mL culturesupernatant. This method comprises a buffer exchange and filtration with aQ‐membrane filter and we demonstrate sufficient removal of key supernatantimpurities including host‐cell proteins (HCPs) and DNA with yields of 1–2 mgand >60% purity. This degree of purity enables characterizing the resultingproteins using affinity binding, mass spectrometry, and differential scanningcalorimetry. We first evaluated this method to purify an engineered SARS‐CoV‐2subunit protein antigen and compared the purified protein to a conventionaltwo‐step chromatographic process. We then applied this method to compareseveral SARS‐CoV‐2 RBD sequences. Finally, we show this simple process can beapplied to a range of other proteins, including a single‐domain antibody, arotavirus protein subunit, and a human growth hormone. This simple and fastdevelopability methodology obviates the need for genetic tagging or fullchromatographic development when assessing and comparing early‐stageprotein therapeutics and vaccine candidates produced in K. phaffii.
Version
Final published version
Abstract
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.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Koch Institute for Integrative Cancer Research at MIT
Massachusetts Institute of Technology. Department of Chemical Engineering
Ragon Institute of MGH, MIT and Harvard
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DOI of Published Version
https://doi.org/10.1002/bit.28385
