title: Reproducible production of a PEGylated dual-acting peptide for diabetes.
abstract: A PEGylated glucagon-like peptide-1 (GLP-1) agonist and glucagon antagonist hybrid peptide was engineered as a potential treatment for type 2 diabetes. To support preclinical development of this PEGylated dual-acting peptide for diabetes (DAPD), we developed a reproducible method for PEGylation, purification, and analysis. Optimal conditions for site-specific PEGylation with 22 and 43 kDa maleimide-polyethylene glycol (maleimide-PEG) polymers were identified by evaluating pH, reaction time, and reactant molar ratio parameters. A 3-step purification process was developed and successfully implemented to purify PEGylated DAPD and remove excess uncoupled PEG and free peptide. Five lots of 43 kDa PEGylated DAPD with starting peptide amounts of 100 mg were produced with overall yields of 53% to 71%. Analytical characterization by N-terminal sequencing, amino acid analysis, matrix-assisted laser desorption/ionization mass spectrometry, and GLP-1 receptor activation assay confirmed site-specific attachment of PEG at the engineered cysteine residue, expected molecular weight, correct amino acid sequence and composition, and consistent functional activity. Purity and safety analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), analytical ion-exchange chromatography, reversed-phase high-performance liquid chromatography, and limulus amebocyte lysate test showed that the final products contained <1% free peptide, <5% uncoupled PEG, and <0.2 endotoxin units per milligram of peptide. These results demonstrate that the PEGylation and purification process we developed was consistent and effective in producing PEGylated DAPD preclinical materials at the 100 mg (peptide weight basis) or 1.2 g (drug substance weight basis) scale.
authors: Bayer HealthCare Pharmaceuticals, Biotechnology, Berkeley, CA 94701, USA.
authors: Irene
authors: I
authors: Tom
authors: Vivian
authors: V
authors: Lee
authors: Michael
authors: M
authors: Dumas
authors: Melanie
authors: M
authors: Madanat
authors: Jun
authors: J
authors: Ouyang
authors: Joanne
authors: J
authors: Severs
authors: John
authors: J
authors: Andersen
authors: Joanne M
authors: JM
authors: Buxton
authors: James P
authors: JP
authors: Whelan
authors: Clark Q
authors: CQ
authors: Pan
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003924:Diabetes Mellitus, Type 2 / Q000188:drug therapy; D015195:Drug Design; D052216:Glucagon-Like Peptide 1 / Q000819:agonists*; D007004:Hypoglycemic Agents / Q000138:chemical synthesis* / Q000494:pharmacology / Q000627:therapeutic use; D010455:Peptides / Q000138:chemical synthesis* / Q000494:pharmacology / Q000627:therapeutic use; D011092:Polyethylene Glycols / Q000138:chemical synthesis* / Q000494:pharmacology / Q000627:therapeutic use; D051381:Rats
chemical_list: D007004:Hypoglycemic Agents; C518718:PEG-DAPD peptide; D010455:Peptides; D011092:Polyethylene Glycols; D052216:Glucagon-Like Peptide 1