Agitation-induced aggregation and subvisible particulate formation in model proteins.
The kinetics of agitation-induced subvisible particle formation was investigated for a few model proteins - human serum albumin (HSA), hen egg white lysozyme (HEWL), and a monoclonal antibody (IgG2). Experiments were carried out for the first time under relatively low protein concentration and low agitation speed to investigate the details of subvisible particle formation at the initial phase of aggregation (<2%) process. Upon agitation, both soluble higher molecular mass species (HMMS) and subvisible particles (SbVPs) formed at different rates, and via different mechanisms. Agitation enhanced exposure of hydrophobic sites in HSA but did not cause detectable structural changes in HEWL and IgG2. SbVPs from HSA partially dissociates in a neutral pH buffer (SEC mobile phase) but does not upon dilution in the same formulation buffer. Opposite results were obtained for SbVPs from IgG2 and HEWL. Neither the relative hydrophobic surface area nor the Tm of the model proteins seems to be an indicator of tendency for agitation-mediated SbVP formation. Taken together, our data suggests that agitation-induced SbVP formation can occur through different mechanisms and can vary, depending on the protein and solution conditions.
Pharmaceutical Research and Development, Pfizer Inc, Chesterfield, MO, USA.
Murali
M
Jayaraman
Pharmaceutical Research and Development, Pfizer Inc, Chesterfield, MO, USA.
Patrick M
PM
Buck
Pharmaceutical Research and Development, Pfizer Inc, Chesterfield, MO, USA.
Arun Alphonse
AA
Ignatius
Pharmaceutical Research and Development, Pfizer Inc, Chesterfield, MO, USA.
Kevin R
KR
King
Pharmaceutical Research and Development, Pfizer Inc, Chesterfield, MO, USA. Electronic address: wei.2.wang@pfizer.com.
Wei
W
Wang
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002645:Chickens; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007074:Immunoglobulin G / Q000737:chemistry*; D007700:Kinetics; D008970:Molecular Weight; D009113:Muramidase / Q000737:chemistry*; D010316:Particle Size; D011487:Protein Conformation; D055550:Protein Stability; D012709:Serum Albumin / Q000737:chemistry*; D000075462:Serum Albumin, Human; D012995:Solubility; D013499:Surface Properties
C422455:ALB protein, human; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D012709:Serum Albumin; D009113:Muramidase; D000075462:Serum Albumin, Human
Aggregation; Agitation; Particle; Protein; Stability; Unfolding