Purification of recombinant brain derived neurotrophic factor using reversed phase displacement chromatography.

This work investigates the utility of RPLC displacement chromatography for the purification of recombinant brain derived neurotrophic factor (rHu-BDNF) from its variants and E. coli. protein (ECP) impurities. The closely associated variants (six in total) differ by one amino acid from the native BDNF and thus pose a challenging separation problem. Several operational parameters were investigated to study their effects on the yield of the displacement process. The results indicated that the concentration of trifluoroacetic acid (TFA) in the buffer was a key factor in achieving the desired purification. Displacement chromatography on an analytical scale column resulted in extremely high purity and yield in a single chromatographic step. The process was successfully scaled-up with respect to particle and column diameter. The production rate of a pilot scale RPLC displacement process was shown to be 23 times higher than the combined production rates of the current preparative ion exchange and hydrophobic interaction gradient elution steps that are used to remove variant and ECP impurities, respectively.

Hydrophobic displacement chromatography of proteins.

Displacement chromatography of proteins was successfully carried out in both hydrophobic interaction and reversed-phase chromatographic systems using low-molecular weight displacers. The displacers employed for hydrophobic displacement chromatography were water soluble, charged molecules containing several short alkyl and/or aryl groups. Spectroscopy was employed to verify the absence of structural changes to the proteins displaced on these hydrophobic supports. Displacement chromatography on a reversed-phase material was employed to purify a growth factor protein from its closely related variants, demonstrating the high resolutions that can be achieved by hydrophobic displacement chromatography. This process combines the high-resolution/high-throughput characteristics of displacement chromatography with the unique selectivity of these hydrophobic supports and offers the chromatographic engineer a powerful tool for the preparative purification of proteins.

Characterization of recombinant human brain-derived neurotrophic factor variants.

The purpose of this work was the chemical characterization of variants of the recombinant human brain derived neurotrophic factor (rHu-BDNF), expressed in Escherichia coli. This paper also addresses the question of the in vitro activity of these variants. Chemical characterization of the variants employed peptide mapping using Glu-C protease and cyanogen bromide digestion on reduced and alkylated variants followed by the analysis of the digested peptides using mass spectrometry and Edman sequencing. The BDNF variants in this work have been designated by the order of their elution as observed from the high temperature RPLC assay. It was determined that Peaks 1 and 2, which eluted just before the predominant BDNF peak, had methionine sulfoxide instead of methionine at positions 31 and 61, respectively. Peak 4, which is chromatographically a single peak, contained three variants. Two of these variants had norleucine instead of methionine, at positions 61 and 92, respectively, while the third had methionine sulfoxide instead of methionine at position 92. Peak 5 had norleucine at position 31 instead of methionine. All of these variants showed in vitro biological activity consistent with the BDNF standard, suggesting the preservation of the trkB receptor-ligand binding domain of the variants.

Ciliary neurotrophic factor is an endogenous pyrogen.

Fever is initiated by the action of polypeptide cytokines called endogenous pyrogens, which are produced by the host during inflammation, trauma, or infection and which elevate the thermoregulatory set point in the hypothalamus. Ciliary neurotrophic factor (CNTF) supports the differentiation and survival of central and peripheral neurons. We describe the activity of CNTF as intrinsically pyrogenic in the rabbit. CNTF induced a monophasic fever which rose rapidly (within the first 12 min) following intravenous injection; CNTF fever was blocked by pretreatment with indomethacin. The fever induced by CNTF was not due to contaminating endotoxins. Increasing doses of CNTF resulted in prolongation of the fever, suggesting the subsequent induction of additional endogenous pyrogenic activity. After passive transfer of plasma obtained during CNTF-induced fever, endogenous pyrogen activity was not present in the circulation; CNTF also did not induce the endogenous pyrogens interleukin 1, tumor necrosis factor, or interleukin 6 in vitro. Nevertheless, a second endogenous pyrogen may originate within the central nervous system following the systemic injection of CNTF. Of the four endogenous pyrogens described to date (interleukin 1, tumor necrosis factor, interferon, and interleukin 6), CNTF, like interleukin 6, utilizes the cell-surface gp 130 signal-transduction apparatus.

Xenotransplantation of canine, bovine, and porcine islets in diabetic rats without immunosuppression.

Permselective acrylic membranes were employed to prevent immune rejection of discordant islet xenografts isolated from various large animals. Canine, porcine, and bovine islets were seeded into tubular diffusion chambers and transplanted into the peritoneum of 27 nonimmunosuppressed streptozotocin-induced diabetic Lewis rats. Six recipients received islet grafts from bovine calves, 7 received grafts from pigs, and 14 received grafts from dogs. Four of the latter were removed at 1 month. In the control group of 10 diabetic rats, 4 received nonencapsulated canine islets, 3 received nonencapsulated bovine islets, and 3 received nonencapsulated porcine islets. Recipients of encapsulated islets promptly dropped from a pretransplantation plasma glucose level of 487 +/- 36 (mean +/- SEM) to 84 +/- 2 (canine), 81 +/- 4 (bovine), and 81 +/- 3 mg/dl (porcine) during the first week. All of the animals sustained these levels for at least 1 month. One rat spontaneously reverted to diabetes at 54 days posttransplantation; 4 other rats became hyperglycemic (glucose, greater than 600 mg/dl) after membrane removal on day 30. The remaining 22 rats maintained fasting euglycemia for greater than 10 weeks. In contrast, rats that received nonencapsulated islets became hyperglycemic in less than 7 days. Intravenous glucose tolerance test K values (decline in glucose levels, %/min) at 1 month for the canine and bovine encapsulated islet transplant group were 3.5 +/- 0.3 and 3.3 +/- 0.1 compared with 3.3 +/- 0.1 (P = 0.63) and 0.91 +/- 0.1 (P less than 0.0001) for normal (n = 4) and diabetic (n = 4) control groups. Morphologic studies of long-term functioning grafts (30-130 days) revealed well-preserved alpha, beta, and delta cells, with varying degrees of granulation. These results demonstrate that immune isolation of islet tissue using permselective artificial membranes can protect discordant islet xenografts from immune rejection in the absence of any immunosuppressive drugs.

Large-scale animal cell culture: a biological perspective.

It is generally recognized that no one cell culture system can be universally applied to all cell types commonly used for biopharmaceutical manufacture. The analogous concept that no single cell type may be useful for the expression of all biopharmaceutical products may also gain credence in the biotechnology community. It may be that like specialized bioreactors, there will come to exist a variety of cell types that will be used for the production of different types of biopharmaceutical products. In addition, it may not be enough in the future just to demonstrate the stability of expression of the amino acid backbone of the protein only; the carbohydrate portion of the molecule may become the subject of real scrutiny. Questions such as how the carbohydrate side chain affects the performance of the molecule in vivo are being asked of more DNA constructs. The next question becomes, how can we control the expression of carbohydrate moieties on the molecule? Such questions are in the future of the biotech manufacturing field. Aside from those examples mentioned above dealing with the insertion of receptors, other more subtle attempts at modifying cellular metabolism are taking place. It was reported at a recent meeting that the sialyltransferase gene was inserted into a CHO line which did not normally express this enzyme (116). The transfected line was capable of expressing the transferase and, more importantly, the enzyme functioned correctly in sialylating glycoproteins. Other very complex relationships exist between the substratum and the cell that could have very direct consequences on culture maintenance. For example, researchers recently published results indicating that collagenase synthesis and secretion is stimulated in rabbit fibroblasts by autocrine factors. They determined that these autocrine proteins had sequence homology to serum amyloid-A and beta-2-microglobulin. It may be that using serum supplements in the medium in those systems that couple fibroblast and collagen substratum may not be prudent, especially for long-term culture. The traditional selection of a cell type for expressing heterologous proteins has generally been limited to the more "common" cell types such as CHO cells, C127 cells, and myeloma cells. In many cases these cell types were selected because there was a great deal of preexisting literature on the cell type (i.e., "cookbook" methods of transfection for the cell) or the cell was simply being carried in the lab at the time the effort was made to express a biopharmaceutical product.(ABSTRACT TRUNCATED AT 400 WORDS)

Manufacture of pharmaceutical proteins from hybridomas and other cell substrates.

Murine monoclonal antibodies are now routinely produced from highly expressing hybridoma cell lines for human clinical applications. Epstein Barr Virus transformed human lymphocytes are used to produce human monoclonal antibodies but these cells have generally exhibited low expression and poor stability in culture when compared with typical murine hybridomas. Alternative cells are therefore finding wider usage. Backfusions and tribrids are being used to increase and/or prolong the expression of monoclonal antibodies. In addition, genetically engineered cells producing antibodies with human constant and murine variable regions may reduce antigenicity of the chimeric protein used for injection. Pharmaceutical acceptability of the final product is also strongly dependent on culture and purification methods that are used in the manufacturing process. Large scale perfusion technology has allowed high density, efficient monoclonal production in the absence of antibiotics in low serum or serum free media. These methods reduce the cell protein/DNA challenge to the purification process by dead and dying cells and also prevent product degradation by reducing product residence time within the bioreactor. Gentle purification procedures that avoid extremes of pH and salt concentration can preserve structural integrity and biological activity of the protein and can provide high yields and purity in excess of 99%. Such purification procedures alone or in combination with specific viral inactivation steps can be validated to have the capability overall to reduce virus concentration by a factor of 10(8) to 10(12). The combination of carefully developed cell substrates, with advanced perfusion culture and purification technology can consistently produce acceptable protein-based pharmaceutical products in compliance with cGMP's.

Phosphorylation of ribosome-associated proteins during the mammalian cell cycle. Unique phosphorylation of a specific protein during mitosis.

Chinese hamster ovary cells in monolayer culture were incubated with [32P] phosphate. Ribosome-associated proteins, including both structural proteins and those tightly bound to washed, centrifuged ribosomes, were isolated and separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. The electrophoretic pattern showed five major regions or peaks of 32P radioactivity which represented phosphorylated ribosome-associated proteins with molecular weights of 17 500, 23 000, 30 000, 38 000, and 57 000. When asynchronous cells were pulse-labeled with [32P] phosphate, the predominant peak of 32P radioactivity was associated with the protein of 38 000 daltons. Similar results were obtained with cells synchronized in the G1, S, or G2 phase of the mammalian cell cycle. Conversely, proteins isolated from the ribosomes of mitotic cells, collected and labeled with [32P] phosphate in the presence of colcemid, showed a new and predominant peak of 32P radioactivity migrating with a protein of 45 000 daltons. When cells labeled in mitosis were allowed to progress into G1 phase, this peak of 32P radioactivity rapidly disappeared from the electrophoretic pattern. These results suggest that a specific protein associated with the ribosomes was phosphorylated uniquely during the mitotic phase of the cell cycle.