Liquid-liquid phase separation causes high turbidity and pressure during low pH elution process in Protein A chromatography.
Turbid elution pools and high column back pressure are common during elution of monoclonal antibodies (mAbs) by acidic pH in Protein A chromatography. This phenomenon has been historically attributed to acid-induced precipitation of incorrectly folded or pH-sensitive mAbs and host cell proteins (HCPs). In this work, we propose a new mechanism that may account for some observations of elution turbidity in Protein A chromatography. We report several examples of turbidity and high column back pressure occurring transiently under a short course of neutral conditions during Protein A elution. A systematic study of three mAbs displaying this behavior revealed phase separation characterized by liquid drops under certain conditions including neutral pH, low ionic strength, and high protein concentration. These liquid droplets caused solution turbidity and exhibited extremely high viscosity, resulting in high column back pressure. We found out that the droplets were formed through liquid-liquid phase separation (LLPS) as a result of protein self-association. We also found multiple factors, including pH, temperature, ionic strength, and protein concentration can affect LLPS behaviors. Careful selection of process parameters during protein A elution, including temperature, flow rate, buffer, and salt can inhibit formation of a dense liquid phase, reducing both turbidity (by 90%) and column back pressure (below 20 pounds per square inch). These findings provide both mechanistic insight and practical mitigation strategies for Protein A chromatography induced LLPS.
Purification Process Sciences, Medimmune LLC, One Medimmune Way, Gaithersburg, MD 20878, USA. Electronic address: Luoh@medimmune.com.
Haibin
H
Luo
Bioprocess Engineering, Medimmune LLC, One Medimmune Way, Gaithersburg, MD 20878, USA.
Nacole
N
Lee
Analytical Biochemistry, Medimmune LLC, One Medimmune Way, Gaithersburg, MD 20878, USA.
Xiangyang
X
Wang
Purification Process Sciences, Medimmune LLC, One Medimmune Way, Gaithersburg, MD 20878, USA.
Yuling
Y
Li
Cell Culture and Fermentation Sciences, Medimmune LLC, One Medimmune Way, Gaithersburg, MD 20878, USA.
Albert
A
Schmelzer
Purification Process Sciences, Medimmune LLC, One Medimmune Way, Gaithersburg, MD 20878, USA.
Alan K
AK
Hunter
Purification Process Sciences, Medimmune LLC, One Medimmune Way, Gaithersburg, MD 20878, USA.
Timothy
T
Pabst
Purification Process Sciences, Medimmune LLC, One Medimmune Way, Gaithersburg, MD 20878, USA.
William K
WK
Wang
D000911:Antibodies, Monoclonal / Q000737:chemistry; D001120:Arginine / Q000737:chemistry; D002021:Buffers; D002853:Chromatography, Liquid / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D059625:Liquid-Liquid Extraction / Q000379:methods*; D009391:Nephelometry and Turbidimetry; D011312:Pressure; D012965:Sodium Chloride / Q000737:chemistry; D012996:Solutions; D013205:Staphylococcal Protein A / Q000302:isolation & purification*; D013696:Temperature
D000911:Antibodies, Monoclonal; D002021:Buffers; D012996:Solutions; D013205:Staphylococcal Protein A; D012965:Sodium Chloride; D001120:Arginine
Column back pressure; Dense phase; Liquid-liquid phase separation (LLPS); Monoclonal antibody (mAb); Protein A chromatography; Reversible self-association; Transient turbidity; Viscosity