Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins.
Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5-6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5-6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development.
a Office of Biotechnology Products; Center for Drug Evaluation and Research; Food and Drug Administration ; Silver Spring ; MD 20993 , USA.
Shen
S
Luo
a Office of Biotechnology Products; Center for Drug Evaluation and Research; Food and Drug Administration ; Silver Spring ; MD 20993 , USA.
Baolin
B
Zhang
D000911:Antibodies, Monoclonal / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D001053:Apolipoproteins / Q000737:chemistry / Q000378:metabolism; D000068258:Bevacizumab / Q000097:blood / Q000737:chemistry / Q000378:metabolism; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid; D003176:Complement C3 / Q000737:chemistry / Q000378:metabolism; D003181:Complement C4 / Q000737:chemistry / Q000378:metabolism; D017242:Complement Factor H / Q000737:chemistry / Q000378:metabolism; D003165:Complement System Proteins / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism; D005947:Glucose / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D000069285:Infliximab / Q000097:blood / Q000737:chemistry / Q000378:metabolism; D007526:Isoelectric Point; D013058:Mass Spectrometry; D066263:Protein Aggregation, Pathological; D011485:Protein Binding; D000068878:Trastuzumab / Q000097:blood / Q000737:chemistry / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001053:Apolipoproteins; D001798:Blood Proteins; D003176:Complement C3; D003181:Complement C4; D005353:Fibronectins; D000068258:Bevacizumab; D017242:Complement Factor H; D003165:Complement System Proteins; D000069285:Infliximab; D005947:Glucose; D000068878:Trastuzumab
aggregation; compatibility; complement proteins; diluent; isoelectric point; monoclonal antibodies; plasma; product formulation; therapeutic proteins