Monoclonal Antibodies Follow Distinct Aggregation Pathways During Production-Relevant Acidic Incubation and Neutralization
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
Standard
Monoclonal Antibodies Follow Distinct Aggregation Pathways During Production-Relevant Acidic Incubation and Neutralization. / Pedersen, Thomas Skamris; Tian, Xinsheng; Thorolfsson, Matthias; Karkov, Hanne Sophie; Rasmussen, Hanne B; Langkilde, Annette E; Vestergaard, Bente.
I: Pharmaceutical Research, Bind 33, Nr. 3, 03.2016, s. 716-28.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
Harvard
APA
Vancouver
Author
Bibtex
}
RIS
TY - JOUR
T1 - Monoclonal Antibodies Follow Distinct Aggregation Pathways During Production-Relevant Acidic Incubation and Neutralization
AU - Pedersen, Thomas Skamris
AU - Tian, Xinsheng
AU - Thorolfsson, Matthias
AU - Karkov, Hanne Sophie
AU - Rasmussen, Hanne B
AU - Langkilde, Annette E
AU - Vestergaard, Bente
PY - 2016/3
Y1 - 2016/3
N2 - PURPOSE: Aggregation aspects of therapeutic monoclonal antibodies (mAbs) are of common concern to the pharmaceutical industry. Low pH treatment is applied during affinity purification and to inactivate endogenous retroviruses, directing interest to the mechanisms of acid-induced antibody aggregation.METHODS: We characterized the oligomerization kinetics at pH 3.3, as well as the reversibility upon neutralization, of three model mAbs with identical variable regions, representative of IgG1, IgG2 and IgG4 respectively. We applied size-exclusion high performance liquid chromatography and orthogonal analytical methods, including small-angle X-ray scattering and dynamic light scattering and supplemented the experimental data with crystal structure-based spatial aggregation propensity (SAP) calculations.RESULTS: We revealed distinct solution behaviors between the three mAb models: At acidic pH IgG1 retained monomeric, whereas IgG2 and IgG4 exhibited two-phase oligomerization processes. After neutralization, IgG2 oligomers partially reverted to the monomeric state, while on the contrary, IgG4 oligomers tended to aggregate. Subclass-specific aggregation-prone motifs on the Fc fragments were identified, which may lead to two distinct pathways of reversible and irreversible aggregation, respectively.CONCLUSIONS: We conclude that subtle variations in mAb sequence greatly affect responses towards low-pH incubation and subsequent neutralization, and demonstrate how orthogonal biophysical methods distinguish between reversible and irreversible mAb aggregation pathways at early stages of acidic treatment.
AB - PURPOSE: Aggregation aspects of therapeutic monoclonal antibodies (mAbs) are of common concern to the pharmaceutical industry. Low pH treatment is applied during affinity purification and to inactivate endogenous retroviruses, directing interest to the mechanisms of acid-induced antibody aggregation.METHODS: We characterized the oligomerization kinetics at pH 3.3, as well as the reversibility upon neutralization, of three model mAbs with identical variable regions, representative of IgG1, IgG2 and IgG4 respectively. We applied size-exclusion high performance liquid chromatography and orthogonal analytical methods, including small-angle X-ray scattering and dynamic light scattering and supplemented the experimental data with crystal structure-based spatial aggregation propensity (SAP) calculations.RESULTS: We revealed distinct solution behaviors between the three mAb models: At acidic pH IgG1 retained monomeric, whereas IgG2 and IgG4 exhibited two-phase oligomerization processes. After neutralization, IgG2 oligomers partially reverted to the monomeric state, while on the contrary, IgG4 oligomers tended to aggregate. Subclass-specific aggregation-prone motifs on the Fc fragments were identified, which may lead to two distinct pathways of reversible and irreversible aggregation, respectively.CONCLUSIONS: We conclude that subtle variations in mAb sequence greatly affect responses towards low-pH incubation and subsequent neutralization, and demonstrate how orthogonal biophysical methods distinguish between reversible and irreversible mAb aggregation pathways at early stages of acidic treatment.
KW - Acids
KW - Antibodies, Monoclonal
KW - Chromatography, High Pressure Liquid
KW - Humans
KW - Hydrogen-Ion Concentration
KW - Immunoglobulin G
KW - Kinetics
KW - Protein Multimerization
KW - Solutions
KW - Journal Article
KW - Research Support, Non-U.S. Gov't
U2 - 10.1007/s11095-015-1821-0
DO - 10.1007/s11095-015-1821-0
M3 - Journal article
C2 - 26563206
VL - 33
SP - 716
EP - 728
JO - Pharmaceutical Research
JF - Pharmaceutical Research
SN - 0724-8741
IS - 3
ER -
ID: 169741335