Evaluation of CZ-resin vials for packaging protein-based parenteral formulations.

Due to the fact that some proteins have a tendency to bind to glass surfaces, plastic CZ-resin vials were evaluated as an alternative material to glass vials for packaging protein-based parenteral formulations. Physico-chemical tests including protein binding, extractable evaluation, oxygen permeation, light transmission and moisture loss were performed. Data show that two proteins (A and B) were found to bind to USP type I glass but not to CZ-resin. The CZ-resin vials passed all USP test specifications for extractables (organic extractable, non-volatile residue and residue on ignition). The oxygen permeation rate (79.06 cm(3)mm/m(2)24 h atm) was consistent with that reported by the vendor (67 cm(3)mm/m(2)24 h atm). The value obtained for light transmission, which was also found to be consistent with that reported by the vendor, shows that these vials offer no protection from light. The average moisture loss from 2 cm(3) vials filled with water was gravimetrically determined to be 0.04 mg/day/vial when the vials were stored at 40 degrees C/75% relative humidity (RH). Assuming a 1cm(3) product fill, this corresponds to approximately a 3% loss over a 2-year period. However, moisture loss was found to be negligible at the typical storage condition of 5 degrees C for protein formulations. The physico-chemical tests indicate that CZ-resin vial is a suitable candidate for packaging parenteral formulations since it shows low moisture loss at typical storage condition of 5 degrees C, and does not leach out extractables. However, it should not be used for light-sensitive and oxygen-sensitive parenteral formulations. For proteins A and B, the CZ-resin vial is a viable alternate to the use of glass vials since it offered significantly less protein binding. Protein binding in general, should be evaluated on a case by case basis, since it may vary for different proteins and under different formulation conditions.

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.

Effect of buffer constituents on the determination of therapeutic proteins by capillary electrophoresis.

Capillary electrophoresis has proved to be a versatile method for the determination of proteins, peptides and amino acids in pharmaceutical formulations. For quantification of the capillary electrophoresis data, however, significant errors may result if the analysis is performed using improper separation conditions. The peak area response for protein analytes, which is generally low in conventional UV detection, may also vary dramatically depending on the nature of the buffer used in the separation. This paper describes the effects of various buffer constituents and analytical conditions on the capillary electrophoretic separation and quantification of a humanized monoclonal antibody in bulk form and in a typical therapeutic formulation. For optimum peak area response and reproducibility, protein derivatization with an appropriate chromophore (e.g., fluorescamine) and separation in the presence of a moderate ionic strength buffer containing lithium chloride, tetramethylammonium chloride or trimethylammonium propylsulfonate is recommended. General guidelines for the determination of proteins by capillary electrophoresis and a rationale for the use of internal standards to improve the quantification of data are also discussed.

Assay of protein drug substances present in solution mixtures by fluorescamine derivatization and capillary electrophoresis.

A method is described to enhance the resolution and detection sensitivity of proteins, peptides, and amino acids in capillary electrophoretic analysis of solution mixtures. The method consists of derivatizing the analytes with fluorescamine, which is normally used as a fluorogenic reagent for compounds containing a reactive primary amine functional group, and then using the derivative as an ultraviolet chromophore to enhance detection sensitivity (measured at 280 nm) in capillary electrophoresis. The results demonstrated a significant improvement in the separation and detection sensitivity of the derivatized analytes as compared to their underivatized counterparts. The use of chromophores, such as fluorescamine, in capillary electrophoresis facilitates the analysis of components of solution mixtures, such as pharmaceutical formulations, that could not be resolved and/or detected by conventional capillary electrophoresis procedures.

Conformation and activity of recombinant human fibroblast interferon-beta.

Conformation of highly purified recombinant human fibroblast interferon-beta (rHuIFN-beta) was correlated with its biological activity. The extent of ordered secondary structure was determined by circular dichroic (CD) spectroscopy in various buffer conditions to establish conditions of protein stability and its potential for helix formation. The highest "helicity" (about 50 +/- 5% of alpha-helices) and the highest antiviral activities (4-10 x 10(7) units/mg) were found in 50% ethylene glycol, 1 M NaCl and 0.05 M Na3PO4, pH 7.2 (Buffer I); 80 mM citric acid, 20 mM Na2HPO4, pH 2.9 (Buffer II); and 25 mM NH4OAc, 125 mM NaCl, pH 5.1 (Buffer III). Both helicity and antiviral activity of the IFN-beta decrease in parallel with denaturation by urea, heat, and/or by repeated cycles of freezing and thawing. Low pH (pH 2.9 Buffer II) exhibits a distinct stabilizing effect on the structure and antiviral activity of IFN-beta against heat denaturation.

Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector.

A cDNA coding for human interleukin 2 (IL-2) was inserted into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the polyhedrin promoter. Cells infected with recombinant virus produced high levels of Mr 15,500 IL-2 polypeptide, the majority of which was secreted into the culture medium during infection. The recombinant IL-2 was able to stimulate the growth of an IL-2-dependent cell line. The N-terminal amino acid sequence of the insect-derived IL-2 was identical to that of natural IL-2. Thus, a mammalian signal peptide was recognized and properly removed in insect cells.

Effects of recombinant and hybrid recombinant human leukocyte interferons on cytotoxic activity of natural killer cells.

Two recombinant human leukocyte interferons (A and D), five hybrid interferons containing varying portions of A and D, and one fibroblast recombinant interferon were tested over a wide range of concentrations for their ability to modulate cytolytic activity of human natural killer (NK) cells. All of the interferons tested were purified to homogeneity. Although all the interferons were active, there were significant quantitative differences in their ability to augment cytolysis and the rank order of potency was reproducible among donors. The various recombinant interferons were also tested for their ability to augment mouse NK activity and the parental D and the A/D hybrids exhibited significant augmentation of cytolysis, which was consistent with their interspecies reactivity in viral neutralization assays. There generally was a direct correlation between antiviral activity and the ability of interferon to augment mouse NK activity; however, this correlation was not evident when tested on human cells. The study of these hybrids led to the identification of two molecules (A/D Bgl and A/D Pvu) which are very active in augmenting mouse NK activity. In addition, considerable insight has been obtained regarding the structure-function relationship of these leukocyte interferons and their ability to boost murine NK. This biological activity was associated with the COOH-terminal portion of the D interferon.

Molecular weight of the functional unit of human leukocyte, fibroblast, and immune interferons.

There is good agreement between the target molecular weight and the known molecular weight of human leukocyte interferons (about 20,000). The target molecular weight of fibroblast interferon, 31,000 to 42,000, is significantly larger than the monomer molecular weight of 21,000 to 24,000, suggesting that the dimer may be the predominant active functional unit in solution. A range from 63,000 to 73,000 for the target molecular weight of several different fractions of immune interferon (including natural crude as well as the recombinant form) indicates that the functional form of the immune interferon may be a trimer or tetramer. Thus, these studies indicate that the functional unit of leukocyte interferon is the monomer, that of fibroblast interferon is a dimer, and that of immune interferon is probably a tetramer (or trimer).

Amino acid sequence of a human leukocyte interferon.

The primary structures of three major species of human leukocyte interferon differ from the structure predicted from the DNA sequence of recombinants containing leukocyte interferon-coding regions. Compared to the recombinant interferon produced in bacteria, three of the purified natural proteins isolated from leukocytes lack the 10 COOH-terminal amino acids suggested by the DNA sequence.

Current concepts of the structure and nature of mammalian salivary mucous glycoproteins.

The visco-elastic properties of salivary secretions are due to high molecular-weight glycoproteins, known as mucins. Mucins are composed of numerous oligosaccharide side-chains O-glycosidically linked through 2-acetamido-2-deoxy-alpha-D-galactose to the hydroxyl groups of seryl and threonyl residues of the protein core; on the average, every fourth amino acid residue is involved in such a bond. This work conveys their isolation and purification, compiles the compositional analysis of several mammalian submaxillary and sublingual mucins; defines the conditions of the alkaline beta-elimination reaction, its mechanism, and importance in structural studies of glycoprotens, and briefly discusses the influence of stimuli on mucous secretions, as well as biosynthesis, structural diversity, and physiological role of salivary mucous glycoproteins.