Pascal Bailon

PEG-modified biopharmaceuticals.

PEGylation is a process in which one or more units of chemically activated polyethylene glycol reacts with a biomolecule, usually a protein, peptide, small molecule or oligonucleotide, creating a putative new molecular entity possessing physicochemical and physiological characteristics that are distinct from its predecessor molecules. In recent years, PEGylation has been used not only as a drug delivery technology but used also as a drug modification technology to transform existing biopharmaceuticals clinically more efficacious than before their PEGylation. PEGylation bestows several useful properties upon the native molecule, resulting in improved pharmacokinetic and pharmacodynamic properties, which in turn enable the native molecule to achieve maximum clinical potency. In addition, PEGylation results in sustained clinical response with minimal dose and less frequency of dosing, leading to improved quality of life via increased patient compliance and reduced cost. During the course of development of various pegylated protein therapeutics, several new insights have been gained. This review article focuses on the approaches, strategies and the utilization of modern PEGylation concepts in the design and development of well-characterized pegylated protein therapeutics.

Polyethylene glycol-conjugated pharmaceutical proteins

Abstract Polyethylene glycol (PEG)-conjugated proteins belong to a new class of biomolecules that are neither proteins nor polymers, but which are hybrids of the two. PEG conjugation of biopharmaceuticals is now common practice in efforts to achieve sustained clinical response. However, very little is known about the strategies and criteria used to produce a well-characterized pegylated biomolecule. In this review, the issues of pegylation reagent selection, reaction conditions, process considerations and purification will be addressed, as well as biochemical and biological characterizations. The isolation of positional isomers and the determination of pegylation sites will also be included.

Affinity purification methods. Improved procedures for cyanogen bromide reaction on agarose.

Abstract The high solubility of cyanogen bromide in N -methyl-2-pyrrolidone has afforded an efficient means of controlling the amount of the reagent used to activate an agarose preparation. By controlling the activation step, one can determine the final concentration of the leash moiety. The upper limits of e-aminocaproic acid incorporation seem to be about 5, 20, and 60 μequiv/ml gel, respectively, for 2, 4, and 6% agarose gels. It is important to conduct the cyanogen bromide reaction at 4–10°C, but the coupling step may be done at room temperature.

Membrane-based receptor affinity chromatography.

Membrane-based receptor affinity chromatography (MRAC), which utilizes the molecular recognition between an immobilized receptor and its soluble protein ligand, has been developed for the purification of human interleukin-2 and related biomolecules. The multi-purpose affinity membrane used in this study consisted of a soluble form of interleukin-2 receptor (IL-2R) chemically bonded to hollow-fiber membranes in an oriented fashion. A model system involving anti-Tac-H (a humanized monoclonal antibody to IL-2R) was used to study the important factors influencing the performance of MRAC, including support morphology, mass transfer rate and adsorption kinetics. All three are shown to be highly efficient. MRAC has been successfully applied to the purification of anti-Tac-H, recombinant human interleukin-2 (rIL-2) and interleukin 2-Pseudomonas exotoxin fusion protein (IL2-PE40). Overall, MRAC was found to be a viable, scalable and extremely productive affinity purification method.

Affinity purification methods. Nonspecific adsorption of proteins due to ionic groups in cyanogen bromide treated agarose.

Abstract The coupling of aliphatic amines to agarose by the cyanogen bromide reaction yields isourea linkages which are positively charged at pH 7. The presence of these cationic sites in affinity gels causes significant non-specific adsorption of proteins. Serum albumin was found to bind to a number of derivatized gels which possessed these charged groups. The use of adipic dihydrazide as the leash moiety yielded affinity gels which were noncharged at pH 7. Serum albumin failed to adsorb to these gels. Beta-galactosidase from Escherichia coli was found to be sensitive to both ionic and hydrophobic groups in an affinity gel. A sample of active-site inhibited enzyme was found to bind to an affinity gel which contained both the cationic isourea and a phenyl structure in the leash. Thus it was concluded that the affinity purification of this enzyme has yet to be demonstrated. These studies dictate against the use of salt and pH gradients to desorb enzymes from affinity sorbents.

Lyotropic salt effects in hydrophobic chromatography.

Abstract Pure hydrophobic chromatography can be observed with agarose gels containing caprylyl hydrazide. These nonionic gels show increased avidity in protein adsorption with higher content of caprylyl groups. Lyotropic salt effects can be used to control chromatographic behavior of proteins. Salting-out agents enhance binding of protein to sorbent, while salting-in agents diminish this binding. Based on these findings, the hydrophobic separation of β-lactoglobulin, ovalbumin, and bovine serum albumin is demonstrated.

Brain administration of OB protein (leptin) inhibits neuropeptide-Y-induced feeding in ob/ob mice

OB protein (or leptin) administration causes a long-lasting reduction in food intake and body weight in obese ob/ob mice. Neuropeptide Y, a stimulator of feeding, has been proposed to be a major mediator of the biological actions of OB protein. To test this hypothesis, the interaction of brain administration of exogenous OB protein and NPY on the feeding behavior of ob/ob mice was examined. Human OB protein, in a dose-dependent manner, partially or completely blocked feeding induced by exogenous NPY. These results demonstrate that OB protein can functionally antagonize and dominate the actions of exogenous NPY on feeding.

Identification of peptides that bind to the constant region of a humanized IgG1 monoclonal antibody using phage display

The pFc′ fragments of a humanized IgG1 monoclonal antibody were generated by digestion with immobilized pepsin. These pFc′ fragments were separated from F(ab′)2 fragments by affinity chromatography. The pFc′ fragments corresponding to the constant region of the humanized IgG1 monoclonal antibody were used as targets for phage display using variable‐length peptide libraries. Interacting phage‐displayed peptides were selected by repetitious cycles of target screening and phage amplification. Peptide sequences, deduced by sequencing DNA from isolated phage, were aligned and analyzed for amino acid motifs against each other and protein A. These results indicated that an amino acid motif has been identified using phage display technology that is sufficient for pFc′ binding. Furthermore, the peptides derived from this study may prove useful in the development of peptidomimetic alternatives to protein A for use in affinity chromatography. Copyright

Medium-scale ligand-affinity purification of two soluble forms of human interleukin-2 receptor.

Recombinant technology has facilitated the production of two soluble forms of human interleukin-2 receptor (IL-2R) in Chinese hamster ovary cells. We have developed a ligand-affinity method for the medium-scale purification of these two IL-2Rs, based on the biochemical interactions between the matrix-bound ligand (interleukin-2) and its soluble receptor. The affinity-purified IL-2R is further purified by anion-exchange chromatography followed by gel filtration. This method has provided enough highly pure IL-2R for structure and function studies and for use in practical applications such as high-flux drug screening assays and the receptor-affinity purification of human recombinant interleukin-2.

Structural analysis of recombinant soluble human interleukin-2 receptor primary structure, assignment of disulfide bonds and core IL-2 binding structure

A purified soluble and functional form of recombinant human interleukin-2 receptor, engineered and expressed in Chinese hamster ovary cells, was structurally characterized. The primary sequence of this 224 amino acid recombinant protein which lacks most of the carboxy-terminal transmembrane and cytoplasmic portions of the intact protein was established by sequence analyses. The disulfide bonds were assigned by comparative peptide mapping of the reduced and non-reduced peptide digests. As in the case of natural interleukin-2 receptor they occur between cysteines 3-147, 46-104, 131-163, and 28/30-59/61. Based on assignment of the disulfide bonds, a structural model of the interleukin-2 receptor for interleukin-2 binding is proposed.