Atypical Asparagine Deamidation of NW Motif Significantly Attenuates the Biological Activities of an Antibody Drug Conjugate
Mingyan Cao,
G. Patrick Hussmann,
Yeqing Tao,
Ellen O’Connor,
Conner Parthemore,
Diana Zhang-Hulsey,
Dengfeng Liu,
Yang Jiao,
Niluka de Mel,
Meagan Prophet,
Samuel Korman,
Jaytee Sonawane,
Christina Grigoriadou,
Yue Huang,
Scott Umlauf,
Xiaoyu Chen
Affiliations
Mingyan Cao
Department of Process and Analytical Sciences, Biopharmaceutical Development, BioPharmaceuticals R&D, AstraZeneca, One Medimmune Way, Gaithersburg, MD 20878, USA
G. Patrick Hussmann
Department of Process and Analytical Sciences, Biopharmaceutical Development, BioPharmaceuticals R&D, AstraZeneca, One Medimmune Way, Gaithersburg, MD 20878, USA
Yeqing Tao
Department of Process and Analytical Sciences, Biopharmaceutical Development, BioPharmaceuticals R&D, AstraZeneca, One Medimmune Way, Gaithersburg, MD 20878, USA
Ellen O’Connor
Department of Process and Analytical Sciences, Biopharmaceutical Development, BioPharmaceuticals R&D, AstraZeneca, One Medimmune Way, Gaithersburg, MD 20878, USA
Conner Parthemore
Department of Process and Analytical Sciences, Biopharmaceutical Development, BioPharmaceuticals R&D, AstraZeneca, One Medimmune Way, Gaithersburg, MD 20878, USA
Diana Zhang-Hulsey
Department of Process and Analytical Sciences, Biopharmaceutical Development, BioPharmaceuticals R&D, AstraZeneca, One Medimmune Way, Gaithersburg, MD 20878, USA
Dengfeng Liu
Department of Process and Analytical Sciences, Biopharmaceutical Development, BioPharmaceuticals R&D, AstraZeneca, One Medimmune Way, Gaithersburg, MD 20878, USA
Yang Jiao
Department of Process and Analytical Sciences, Biopharmaceutical Development, BioPharmaceuticals R&D, AstraZeneca, One Medimmune Way, Gaithersburg, MD 20878, USA
Niluka de Mel
Department of Process and Analytical Sciences, Biopharmaceutical Development, BioPharmaceuticals R&D, AstraZeneca, One Medimmune Way, Gaithersburg, MD 20878, USA
Meagan Prophet
Department of Process and Analytical Sciences, Biopharmaceutical Development, BioPharmaceuticals R&D, AstraZeneca, One Medimmune Way, Gaithersburg, MD 20878, USA
Samuel Korman
Department of Process and Analytical Sciences, Biopharmaceutical Development, BioPharmaceuticals R&D, AstraZeneca, One Medimmune Way, Gaithersburg, MD 20878, USA
Jaytee Sonawane
Department of Process and Analytical Sciences, Biopharmaceutical Development, BioPharmaceuticals R&D, AstraZeneca, One Medimmune Way, Gaithersburg, MD 20878, USA
Christina Grigoriadou
Department of Process and Analytical Sciences, Biopharmaceutical Development, BioPharmaceuticals R&D, AstraZeneca, One Medimmune Way, Gaithersburg, MD 20878, USA
Yue Huang
Department of Integrated Bioanalysis, Clinical Pharmacology & Safety Sciences, R&D, AstraZeneca, 121 Oyster Point Boulevard, South San Francisco, CA 94080, USA
Scott Umlauf
Department of Process and Analytical Sciences, Biopharmaceutical Development, BioPharmaceuticals R&D, AstraZeneca, One Medimmune Way, Gaithersburg, MD 20878, USA
Xiaoyu Chen
Department of Process and Analytical Sciences, Biopharmaceutical Development, BioPharmaceuticals R&D, AstraZeneca, One Medimmune Way, Gaithersburg, MD 20878, USA
Asparagine deamidation is a post-translational modification (PTM) that converts asparagine residues into iso-aspartate and/or aspartate. Non-enzymatic asparagine deamidation is observed frequently during the manufacturing, processing, and/or storage of biotherapeutic proteins. Depending on the site of deamidation, this PTM can significantly impact the therapeutic’s potency, stability, and/or immunogenicity. Thus, deamidation is routinely monitored as a potential critical quality attribute. The initial evaluation of an asparagine’s potential to deamidate begins with identifying sequence liabilities, in which the n + 1 amino acid is of particular interest. NW is one motif that occurs frequently within the complementarity-determining region (CDR) of therapeutic antibodies, but according to the published literature, has a very low risk of deamidating. Here we report an unusual case of this NW motif readily deamidating within the CDR of an antibody drug conjugate (ADC), which greatly impacts the ADC’s biological activities. Furthermore, this NW motif solely deamidates into iso-aspartate, rather than the typical mixture of iso-aspartate and aspartate. Interestingly, biological activities are more severely impacted by the conversion of asparagine into iso-aspartate via deamidation than by conversion into aspartate via mutagenesis. Here, we detail the discovery of this unusual NW deamidation occurrence, characterize its impact on biological activities, and utilize structural data and modeling to explain why conversion to iso-aspartate is favored and impacts biological activities more severely.
