ABSTRACT
Polyethylene glycol (PEG) conjugation to proteins has emerged as an important technology to produce drug molecules with sustained duration in the body. However, the implications of PEG conjugation to protein aggregation have not been well understood. In this study, conducted under physiological pH and temperature, N-terminal attachment of a 20 kDa PEG moiety to GCSF had the ability to (1) prevent protein precipitation by rendering the aggregates soluble, and (2) slow the rate of aggregation relative to GCSF. Our data suggest that PEG-GCSF solubility was mediated by favorable solvation of water molecules around the PEG group. PEG-GCSF appeared to aggregate on the same pathway as that of GCSF, as evidenced by (a) almost identical secondary structural transitions accompanying aggregation, (b) almost identical covalent character in the aggregates, and (c) the ability of PEG-GCSF to rescue GCSF precipitation. To understand the role of PEG length, the aggregation properties of free GCSF were compared to 5kPEG-GCSF and 20kPEG-GCSF. It was observed that even 5kPEG-GCSF avoided precipitation by forming soluble aggregates, and the stability toward aggregation was vastly improved compared to GCSF, but only marginally less stable than the 20kPEG-GCSF. Biological activity measurements demonstrated that both 5kPEG-GCSF and 20kPEG-GCSF retained greater activity after incubation at physiological conditions than free GCSF, consistent with the stability measurements. The data is most compatible with a model where PEG conjugation preserves the mechanism underlying protein aggregation in GCSF, steric hindrance by PEG influences aggregation rate, while aqueous solubility is mediated by polar PEG groups on the aggregate surface.
Subject(s)
Granulocyte Colony-Stimulating Factor/chemistry , Granulocyte Colony-Stimulating Factor/metabolism , Polyethylene Glycols/metabolism , Proteins/chemistry , Chemical Precipitation , Copper/chemistry , Kinetics , Protein Structure, Secondary , Solubility , Structure-Activity RelationshipABSTRACT
A recombinant C-terminal truncated form of the human soluble tumor necrosis factor receptor type I (sTNF-RI) was produced in E. coli. This soluble receptor contains the first 2.6 of the 4 domains of the intact sTNF-RI molecule. A monoPEGylated form of this molecule was produced using a 30 kD methoxyPEG aldehyde with approximately 85% selectivity for the N-terminal amino group. This molecule was shown to be less immunogenic in primates than the full length (4.0 domain) molecule or other versions of sTNF-RI which were either PEGylated at different sites or with different molecular weight PEGs. The 30 kD PEG also has a longer serum half-life to the molecule than lower molecular weight PEGs. This molecule markedly blunts the inflammatory response in a number of rheumatoid arthritis animal models. In addition, phase I/II and early phase II data in humans indicate that PEG sTNF-RI is non-immunogenic and that weekly dosing with this drug can reduce the number of tender and swollen joints in rheumatoid arthritis patients. PEG sTNF-RI has comparable American College of Rheumatology (ACR) efficacy scores as other anti-TNF molecules currently used to treat rheumatoid arthritic patients.
Subject(s)
Antigens, CD , Arthritis, Rheumatoid/drug therapy , Receptors, Tumor Necrosis Factor , Animals , Antigens, CD/chemistry , Antigens, CD/genetics , Antigens, CD/metabolism , Antigens, CD/therapeutic use , Area Under Curve , Biological Availability , Disease Models, Animal , Female , Half-Life , Humans , Inflammation/drug therapy , Male , Metabolic Clearance Rate , Receptors, Tumor Necrosis Factor/chemistry , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor/metabolism , Receptors, Tumor Necrosis Factor/therapeutic use , Receptors, Tumor Necrosis Factor, Type I , Species Specificity , Structure-Activity RelationshipABSTRACT
A site-directed method of joining proteins to poly(ethylene glycol) is presented which allows for the preparation of essentially homogeneous PEG-protein derivatives with a single PEG chain conjugated to the amine terminus of the protein. This selectivity is achieved by conducting the reductive alkylation of proteins with PEG-aldehydes at lower pH. Working examples demonstrating the application of this method to improve the delivery characteristics and therapeutic value of several proteins are provided.
