title: Detection and characterization of subvisible aggregates of monoclonal IgG in serum.
abstract: OBJECTIVE
  To detect and characterize the aggregation of therapeutic monoclonal antibodies in undiluted biological fluids.
  
  
  METHODS
  Fluorescently labeled subvisible IgG aggregates formed by applying either heat stress or by pH-shift were investigated immediately after addition to human serum, and after 24 h. Unstressed and stressed IgG formulations were analyzed by fluorescence single particle tracking, confocal laser scanning microscopy and flow cytometry.
  
  
  RESULTS
  Unstressed formulations remained free from subvisible aggregates in serum, whereas heat-stressed and pH-shift stressed formulations showed dissimilar aggregation behaviors. The aggregation profile of the heat-stressed formulation diluted in serum remained practically the same as the one diluted in buffer, even after the 24 h incubation period. The pH-shift stressed formulation had strikingly smaller and more numerous subvisible aggregates immediately after dilution in serum compared to buffer. These aggregates became noticeably larger in both diluents after 24 h, but in serum they appeared to be formed by other types of constituents than the labeled protein itself.
  
  
  CONCLUSIONS
  These results show that subvisible therapeutic protein aggregates may undergo changes in number, type and size distribution upon contact with human serum. This emphasizes the importance of analytical strategies for monitoring aggregation in undiluted biological fluids.
authors.affiliation: Division of Drug Delivery Technology Leiden/Amsterdam Center for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands.
authors.forename: Vasco
authors.initials: V
authors.lastname: Filipe
authors.forename: Robert
authors.initials: R
authors.lastname: Poole
authors.forename: Olubukayo
authors.initials: O
authors.lastname: Oladunjoye
authors.forename: Kevin
authors.initials: K
authors.lastname: Braeckmans
authors.forename: Wim
authors.initials: W
authors.lastname: Jiskoot
mesh_terms: D000911:Antibodies, Monoclonal / Q000097:blood* / Q000737:chemistry*; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000097:blood* / Q000737:chemistry*; D018613:Microscopy, Confocal; D010316:Particle Size; D011487:Protein Conformation; D013388:Succinimides / Q000737:chemistry
chemical_list: C492694:Alexa Fluor 488 carboxylic acid succinimidyl ester; D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D013388:Succinimides