ABSTRACT
As an increasing number of recombinant therapeutic glycoproteins are manufactured and investigated, the importance of their attached glycans is becoming more widely reported and understood. Regulatory agencies expect detailed "extended characterization" of the glycoprotein as well as routine, well-controlled "release assays" with specifications to be employed for quality control of each manufactured lot. In this chapter we will briefly discuss relevant glycan issues in the area of therapeutic recombinant glycoprotein manufacture and describe in detail two assays that are employed in the development of, for example, recombinant Factor VIII for the treatment of hemophilia.
Subject(s)
Glycoproteins/chemistry , Glycoproteins/therapeutic use , Recombinant Proteins/chemistry , Recombinant Proteins/therapeutic use , Chromatography, High Pressure Liquid , Factor VIII/metabolism , Factor VIII/therapeutic use , Glycoproteins/metabolism , Glycosylation , Hemophilia A/drug therapy , Humans , Mass Spectrometry , Oligosaccharides/metabolism , Proteolysis , Recombinant Proteins/metabolism , Thrombin/metabolismABSTRACT
A long-acting factor VIII (FVIII) as a replacement therapy for hemophilia A would significantly improve treatment options for patients with hemophilia A. To develop a FVIII with an extended circulating half-life, but without a reduction in activity, we have engineered 23 FVIII variants with introduced surface-exposed cysteines to which a polyethylene glycol (PEG) polymer was specifically conjugated. Screening of variant expression level, PEGylation yield, and functional assay identified several conjugates retaining full in vitro coagulation activity and von Willebrand factor (VWF) binding.PEGylated FVIII variants exhibited improved pharmacokinetics in hemophilic mice and rabbits. In addition, pharmacokinetic studies in VWF knockout mice indicated that larger molecular weight PEG may substitute for VWF in protecting PEGylated FVIII from clearance in vivo. In bleeding models of hemophilic mice, PEGylated FVIII not only exhibited prolonged efficacy that is consistent with the improved pharmacokinetics but also showed efficacy in stopping acute bleeds comparable with that of unmodified rFVIII. In summary site-specifically PEGylated FVIII has the potential to be a long-acting prophylactic treatment while being fully efficacious for on-demand treatment for patients with hemophilia A.
Subject(s)
Coagulants/pharmacokinetics , Factor VIII/pharmacokinetics , Hemophilia A/drug therapy , Polyethylene Glycols/pharmacokinetics , Animals , Blotting, Western , Coagulants/administration & dosage , Coagulants/chemistry , Delayed-Action Preparations , Electrophoresis, Polyacrylamide Gel , Factor VIII/administration & dosage , Factor VIII/chemistry , Half-Life , Mice , Mice, Knockout , Mutagenesis, Site-Directed , Polyethylene Glycols/administration & dosage , Polyethylene Glycols/chemistry , Rabbits , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
To optimize the stability of a peptide development candidate for the treatment of type II diabetes, formulation studies were initiated in organic solvents and compared to results obtained in aqueous solutions. Stability was assessed by reversed phase liquid chromatography (RPLC) and electrospray ionization mass spectrometry (ESI-MS). Previous studies had shown deamidation and hydrolysis to be the primary mechanisms of degradation in aqueous formulations. Surprisingly, the use of an organic solvent did not decrease the rate of degradation and, as presented here, produced degradation products including dimers. We propose here that deamidation can readily occur in polar anhydrous organic solvents such as DMSO and that the dimer forms through intermolecular nucleophilic attack of an amino acid side chain on a stabilized cyclic imide intermediate.
Subject(s)
Asparagine/chemistry , Aspartic Acid/chemistry , Dimethyl Sulfoxide/chemistry , Pituitary Adenylate Cyclase-Activating Polypeptide/chemistry , Amino Acid Sequence , Chromatography, Gel , Chromatography, High Pressure Liquid , Dimerization , Molecular Sequence Data , Spectrometry, Mass, Electrospray IonizationABSTRACT
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.
