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A High-Throughput Bioluminescent Assay to Monitor the Deamidation of Asparagine and Isomerization of Aspartate Residues in Therapeutic Proteins and Antibodies.

Abstract Since the introduction of Herceptin and Rituximab in 1986 therapeutic antibodies have gained tremendous momentum in treatment of broad range of several diseases such as cancer and inflammation. Selection of the clinical candidate mAb usually starts with large-scale in vitro screening and profiling of multiple mAbs to identify candidates that show high in vitro or in vivo activity, and thus it is necessarily to identify and eliminate potentially unstable mAbs during the lead selection process. Antibodies undergo a variety of degradation reactions which may result in compromised bioactivity and safety profile. The non-enzymatic post-translational modification of both deamidation of asparagine (Asn) and isomerization of aspartate (Asp) residues is one of the major chemical reactions occurring in proteins during production and storage resulting in formation of protein variants that may affect the quality, safety, and functionality of the therapeutic proteins. Current Methods (HPLC and LC/MS) for monitoring isoaspartate (isoAsp) formation are time consuming, require specialized equipment and trained personnel and are not amenable to high throughput scale (HTS). We have developed a robust, homogenous, high throughput formatted, and sensitive assay to accurately monitor the formation of isoAsp under several conditions such as new formulations, storage periods, and temperature.
PMID
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Authors

Mayor MeshTerms
Keywords
Journal Title journal of pharmaceutical sciences
Publication Year Start




PMID- 28263844
OWN - NLM
STAT- Publisher
DA  - 20170306
LR  - 20170306
IS  - 1520-6017 (Electronic)
IS  - 0022-3549 (Linking)
DP  - 2017 Mar 02
TI  - A High-Throughput Bioluminescent Assay to Monitor the Deamidation of Asparagine
      and Isomerization of Aspartate Residues in Therapeutic Proteins and Antibodies.
LID - S0022-3549(17)30131-4 [pii]
LID - 10.1016/j.xphs.2017.02.022 [doi]
AB  - Since the introduction of Herceptin and Rituximab in 1986 therapeutic antibodies 
      have gained tremendous momentum in treatment of broad range of several diseases
      such as cancer and inflammation. Selection of the clinical candidate mAb usually 
      starts with large-scale in vitro screening and profiling of multiple mAbs to
      identify candidates that show high in vitro or in vivo activity, and thus it is
      necessarily to identify and eliminate potentially unstable mAbs during the lead
      selection process. Antibodies undergo a variety of degradation reactions which
      may result in compromised bioactivity and safety profile. The non-enzymatic
      post-translational modification of both deamidation of asparagine (Asn) and
      isomerization of aspartate (Asp) residues is one of the major chemical reactions 
      occurring in proteins during production and storage resulting in formation of
      protein variants that may affect the quality, safety, and functionality of the
      therapeutic proteins. Current Methods (HPLC and LC/MS) for monitoring
      isoaspartate (isoAsp) formation are time consuming, require specialized equipment
      and trained personnel and are not amenable to high throughput scale (HTS). We
      have developed a robust, homogenous, high throughput formatted, and sensitive
      assay to accurately monitor the formation of isoAsp under several conditions such
      as new formulations, storage periods, and temperature.
CI  - Copyright (c) 2017. Published by Elsevier Inc.
FAU - Hsiao, Kevin
AU  - Hsiao K
AD  - Research and Development, Promega Corp., Madison WI 53711.
FAU - Alves, Juliano
AU  - Alves J
AD  - Research and Development, Promega Corp., Madison WI 53711.
FAU - Patel, Rushikesh
AU  - Patel R
AD  - Research and, Development, Drug Product Science and Technology, Bristol-Myers
      Squibb Pharmaceutical, New Brunswick, NJ, 08903.
FAU - Adams, Monica
AU  - Adams M
AD  - Research and, Development, Drug Product Science and Technology, Bristol-Myers
      Squibb Pharmaceutical, New Brunswick, NJ, 08903.
FAU - Nashine, Vishal
AU  - Nashine V
AD  - Research and, Development, Drug Product Science and Technology, Bristol-Myers
      Squibb Pharmaceutical, New Brunswick, NJ, 08903.
FAU - Goueli, Said
AU  - Goueli S
AD  - Research and Development, Promega Corp., Madison WI 53711; Dept. of Pathology and
      Laboratory Medicine, University of Wisconsin Madison School of Medicine and
      Public Health, Madison, WI, 53705; USA.
LA  - eng
PT  - Journal Article
DEP - 20170302
PL  - United States
TA  - J Pharm Sci
JT  - Journal of pharmaceutical sciences
JID - 2985195R
EDAT- 2017/03/07 06:00
MHDA- 2017/03/07 06:00
CRDT- 2017/03/07 06:00
PHST- 2017/01/12 [received]
PHST- 2017/02/04 [revised]
PHST- 2017/02/13 [accepted]
AID - S0022-3549(17)30131-4 [pii]
AID - 10.1016/j.xphs.2017.02.022 [doi]
PST - aheadofprint
SO  - J Pharm Sci. 2017 Mar 2. pii: S0022-3549(17)30131-4. doi:
      10.1016/j.xphs.2017.02.022.

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