title: The "New Polyethylene Glycol Dilemma": Polyethylene Glycol Impurities and Their Paradox Role in mAb Crystallization.
abstract: Polyethylene glycols (PEG) represent the most successful and frequently applied class of excipients used for protein crystallization. PEG auto-oxidation and formation of impurities such as peroxides and formaldehydes that foster protein drug degradation is known. However, their effect on mAb crystallization has not been studied in detail before. During the present study, a model IgG1 antibody (mAb1) was crystallized in PEG solutions. Aggregate formation was observed during crystallization and storage that was ascribed to PEG degradation products. Reduction of peroxide and formaldehyde levels prior to crystallization by vacuum and freeze-drying was investigated for its effect on protein degradation. Vacuum drying was superior in removal of peroxides but inferior in reducing formaldehyde residues. Consequently, double purification allowed extensive removal of both impurities. Applying of purified PEG led to 50% lower aggregate fractions. Surprisingly, PEG double purification or addition of methionine prior to crystallization prevented crystal formation. With increased PEG concentration or spiking with peroxides and formaldehydes, crystal formation could be recovered again. With these results, we demonstrate that minimum amounts of oxidizing impurities and thus in consequence chemically altered proteins are vital to initiate mAb1 crystallization. The present study calls PEG as good precipitant for therapeutic biopharmaceuticals into question.
authors: Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximillians-University Munich, Munich D-81377, Germany. Electronic address: Christian.Hildebrandt@cup.uni-muenchen.de.
authors: Christian
authors: C
authors: Hildebrandt
authors: Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximillians-University Munich, Munich D-81377, Germany.
authors: Lea
authors: L
authors: Joos
authors: NBE Formulation and Process Sciences, Drug Product Development, AbbVie GmbH and Company KG, Ludwigshafen D-67061, Germany.
authors: Rainer
authors: R
authors: Saedler
authors: Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximillians-University Munich, Munich D-81377, Germany.
authors: Gerhard
authors: G
authors: Winter
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D003460:Crystallization; D003890:Desiccation; D005079:Excipients / Q000737:chemistry*; D005557:Formaldehyde / Q000737:chemistry; D005612:Freeze Drying; D007074:Immunoglobulin G / Q000737:chemistry*; D010545:Peroxides / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry*; D066329:Protein Aggregates
chemical_list: D000911:Antibodies, Monoclonal; D005079:Excipients; D007074:Immunoglobulin G; D010545:Peroxides; D066329:Protein Aggregates; D005557:Formaldehyde; D011092:Polyethylene Glycols
keywords: crystallization; formaldehydes; freeze-drying; peroxides; polyethylene glycol (PEG); polymer chemical degradation; protein aggregation; proteins; vacuum drying