title: A Micro-Polyethylene Glycol Precipitation Assay as a Relative Solubility Screening Tool for Monoclonal Antibody Design and Formulation Development.
abstract: Adequate protein solubility is an important prerequisite for development, manufacture, and administration of biotherapeutic drug candidates, especially for high-concentration protein formulations. A previously established method for determining the relative apparent solubility (thermodynamic activity) of proteins using polyethylene glycol (PEG) precipitation is adapted for screening and comparing monoclonal antibody (mAb) candidates where only limited quantities (≤1 mg) are available. This micro-PEG assay is used to evaluate various broadly neutralizing mAb candidates to HIV-1 viral spike (gp120 and gp41 glycoproteins). Using ∼1 mg of VRC01-WT mAb per assay, the precision of the micro-PEG assay was established. A series of 7 different broadly neutralizing mAbs to the HIV-1 viral spike proteins were compared by curve shape (%PEG vs. protein concentration), %PEGmidpoint determinations, and extrapolated apparent solubility values. Numerous formulation conditions were then evaluated for their relative effects on the VRC01-WT mAb. The PEGmidpt and apparent solubility values of VRC01-WT mAb decreased as the solution pH increased and increased as NaCl and arginine were added. A final optimization of the micro-PEG assay established that amounts as low as 0.1-0.2 mg can be used. Thus, the micro-PEG assay has significant potential as a relative solubility screening tool during candidate selection and early formulation development.
authors.affiliation: Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, 2030 Becker Drive, Lawrence, Kansas 66047.
authors.forename: Vishal M
authors.initials: VM
authors.lastname: Toprani
authors.affiliation: Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, 2030 Becker Drive, Lawrence, Kansas 66047.
authors.forename: Sangeeta B
authors.initials: SB
authors.lastname: Joshi
authors.affiliation: Vaccine Production Program, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Kueltzo
authors.affiliation: Vaccine Production Program, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Schwartz
authors.affiliation: Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, 2030 Becker Drive, Lawrence, Kansas 66047.
authors.forename: C Russell
authors.initials: CR
authors.lastname: Middaugh
authors.affiliation: Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, 2030 Becker Drive, Lawrence, Kansas 66047. Electronic address: volkin@ku.edu.
authors.forename: David B
authors.initials: DB
authors.lastname: Volkin
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000080908:Broadly Neutralizing Antibodies; D011232:Chemical Precipitation; D004339:Drug Compounding / Q000379:methods*; D015195:Drug Design; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120 / Q000276:immunology; D015700:HIV Envelope Protein gp41 / Q000276:immunology; D057166:High-Throughput Screening Assays; D011092:Polyethylene Glycols / Q000737:chemistry*; D012995:Solubility
chemical_list: D000911:Antibodies, Monoclonal; D000080908:Broadly Neutralizing Antibodies; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D015700:HIV Envelope Protein gp41; C578595:VRC01 monoclonal antibody; C514793:gp120 protein, Human immunodeficiency virus 1; C514787:gp41 protein, Human immunodeficiency virus 1; D011092:Polyethylene Glycols
keywords: HIV; VRC01; candidate selection; comparability; formulation; monoclonal antibody; polyethylene glycol; solubility; stability