Consideration of conformational transitions and racemization during process development of recombinant glucagon-like peptide-1.

Physicochemical characterization of dry, excipient-free recombinant glucagon-like peptide-1 (rGLP-1) indicates the conformation and purity of the bulk peptide is dependent on the purification scheme and the in-process storage and handling. The recombinant peptide preparations were highly pure and consistent with the expected primary structure and bioactivity. However, variations in solubility were observed for preparations processed by different methods. The differences in solubility were shown to be due to conformational differences induced during purification. A processing scheme was identified to produce rGLP-1 in its native, soluble form, which exhibits FT-IR spectra, consistent with glucagon-like peptide-1 synthesized by solid-state peptide synthesis. rGLP-1 was also found to undergo base-catalyzed amino acid racemization. Racemization can impact the yield and impurity profile of bulk rGLP-1, since the peptide is exposed to alkali during its purification. A combination of enzymatic digestion using leucine aminopeptidase (which cleaves N-terminal L-amino acids >> D-amino acids) and matrix-assisted laser desorption ionization mass spectrometry was used to identify racemization as a degradation pathway. The racemization rate increased with increasing temperature and base concentration, but decreased with increasing peptide concentration. The racemized peptides were shown to be less bioactive than rGLP-1.

Aqueous stability of recombinant human thrombopoietin as a function of processing schemes.

Preformulation studies conducted with recombinant human thrombopoietin (rhTPO), a 332 amino acid glycoprotein which stimulates platelet production, show distinctions in degradation profiles as a function of processing schemes. The stability-limiting degradation pathways change as a function of purification stage and method and are dependent upon the presence of contaminating protease. The stability-limiting degradation pathway of affinity-purified and in-process rhTPO preparations is primarily attributed to proteolysis initiated by a protease present as a fermentation contaminant. The proteolysis increases with increasing pH as a function of temperature. The degradation profiles for these preparations show that bioactivity initially increases and then decreases with increasing pH as a function of temperature. This is consistent with proteolysis to active forms which ultimately undergo degradation to less active forms. Similar studies conducted with rhTPO preparations purified by a combination of more conventional chromatographic steps show different stability-limiting degradation pathways and a different pH-stability profile when compared to affinity purified or in-process preparations. In this case, degradation is accompanied by decreases in activity under all conditions, consistent with the conversion to less active forms. These results illustrate the importance of preformulation and stability characterization of protein pharmaceuticals in support of both process and formulation development. Issues related to storage and handling of inprocess preparations differ from those with formulated product since the stability-limiting degradation pathways change as a function of purification stage.

Aqueous stability of human epidermal growth factor 1-48.

Human epidermal growth factor 1-48 (hEGF 1-48, Des(49-53)hEGF) is a single chain polypeptide (48 amino acids; 3 disulfide bonds; 5445 Da) possessing a broad spectrum of biologic activity including the stimulation of cell proliferation and tissue growth. In this study, three primary aqueous degradation products of hEGF 1-48 were isolated using isocratic, reverse phase/ion-pair HPLC. The degradation products were characterized using amino acid sequencing, electrospray ionization mass spectrometry, isoelectric focusing, and degradation kinetics. Results indicate that hEGF 1-48 degrades via oxidation (Met21), deamidation (Asn1), and succinimide formation (Asp11). The relative contribution of each degradation pathway to the overall stability of hEGF 1-48 changes as a function of solution pH and storage condition. Succinimide formation at Asp11 is favored at pH < 6 in which aspartic acid is present mostly in its protonated form. Deamidation of Asn1 is favored at pH > 6. The relative contribution of Met21 oxidation is increased with decreasing temperature, storage as a frozen solution (-20 degrees C), and exposure to fluorescent light.

Fibrin based drug delivery systems.

Fibrinogen is the natural substrate for thrombin, the final proteolytic enzyme of the blood coagulation cascade. Following reaction with thrombin, fibrinogen is converted into fibrin monomers which then spontaneously polymerize into fibrin threads forming the fibrin matrix. This biochemical reaction between fibrinogen and thrombin has been used to produce natural, biodegradable, biocompatible systems for sustained drug delivery. Three potential delivery systems have been explored: 1) fibrin microparticles with drug entrapped are produced by an emulsion process, 2) reaction between fibrinogen adsorbed on suspended drug and thrombin in solution results in the formation of fibrin-coated drug particulates, and 3) fibrin sheets suitable as implants have been cast with drug entrapped. The drug release behavior of both small and large molecular weight substances from these systems was evaluated.

Evaluation of antiproliferative agents using a cell-culture model.

Chinese hamster ovarian (CHO) cells in culture were used to evaluate the relative antiproliferative potential of drugs. These agents have been used to improve the clinical response after glaucoma filtering surgery. The following drugs were evaluated: 5-fluorouracil (5-FU) as the benchmark, 5-fluorouridine (FUR), 5-fluorodeoxyuridine (FUDR), 5'-deoxy-5-fluorouridine (DFUR), bleomycin, and cytarabine (ARA-C). In addition to cell counting, a colorimetric assay based on the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to follow growth response. The MTT assay was found to be extremely convenient and an indirect measure of cell activity, offering an alternate or addition to a measure of cell number. All agents tested were shown to inhibit cellular proliferation. Dose-response curves for each agent indicate the following absolute potency: FUDR greater than FUR greater than ARA-C greater than 5-FU = bleomycin greater than DFUR. Besides absolute potency, an evaluation of the effects of equivalent inhibitory concentrations of each drug on growth rate was assessed. Several agents affected the proliferation rate patterns differently. Based on these studies, it is suggested that the in vitro model can identify potential agents through an assessment of their overall activity profile in CHO cells, which includes not only their potency based on dose response, but their onset of activity, duration of effect, and potential for toxicity.