Recombinant granulocyte colony stimulating factor (filgrastim) of prolonged action: Optimization of conditions for extraction and purification from inclusion bodies

A unified technology platform for the production of two medical products with nonprolonged and prolonged action on the basis of the recombinant human granulocyte colony-stimulating factor (filgrastim) was developed. The production cost for the previously developed domestic medical product was reduced by the unified technology. Moreover, the introduction of an additional step for the PEGylation of the recombinant granulocyte colony stimulating factor to the technological line allowed the start of the production of a new generation of the medical product and standardization of the documenting procedure for the technological process in accordance with GMP requirements.     

Levels of human serum granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor under pathological conditions

Levels of serum granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) in patients with various leukocyte disorders were estimated by enzyme linked immunosorbent assay (ELISA). Some cases of acute myelogenous leukemia and aplastic anemia showed elevated serum levels of G-CSF and/or GM-CSF, whereas almost all of 23 healthy controls showed G-CSF and GM-CSF levels lower than 100 pg/ml. High levels of both types of CSF were noted in patients with granulocytosis due to infection. These levels became lower after resolution of the infection. Daily changes in serum CSF levels were also examined in a patient with autoimmune neutropenia, and it was found that the peripheral neutrophilic granulocyte count changed almost in parallel with the serum G-CSF level but not with GM-CSF, following the pattern with a delay of about 4–5 h, suggesting the possibility that G-CSF mainly regulates peripheral neutrophil circulation.     

Chemical modification of recombinant human granulocyte colony-stimulating factor by polyethylene glycol increases its biological activity in vivo.

Recombinant human granulocyte colony-stimulating factor (rHuG-CSF) produced in Escherichia coli was chemically modified by polyethylene glycol (PEG) of molecular weights 4,500 or 10,000. The neutrophils observed at 32 hours after intravenous injection of the rHuG-CSF modified with PEG (4,500) or PEG (10,000) to mice were, respectively, 2.5 times and 5 times more than that observed after the injection of the unmodified rHuG-CSF. These results show that the covalent attachment of PEG to rHuG-CSF enhanced its pharmacological activity in vivo and that the modification with the larger PEG molecule is more effective to enhance the in vivo activity of rHuG-CSF.     

Recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) induces human macrophage production of transforming growth factor-alpha

Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is known as an inducer of proliferation and functional activation of myeloid cells. This study was carried out to characterize the effect of purified recombinant human GM-CSF (rhGM-CSF) on induction of TGF-alpha in macrophages. Using Northern blot analysis and immunoassays, we show here that rhGM-CSF markedly stimulates production of TGF-alpha messenger RNA and protein in normal tonsil macrophages. The findings are consistent with macrophages being a normal inducible source of TGF-alpha which may be an important mediator of various activities of GM-CSF both in hematopoietic and non-hematopoietic cells.     

Recombinant granulocyte colony-stimulating factor induces production of human neutrophil peptides in lung cancer patients with neutropenia

Human neutrophil peptides (HNPs) 1, 2 and 3 are antimicrobial peptides localized in the azurophil granules of neutrophils. We investigated the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the biosynthesis of HNPs 1-3 using a sensitive radioimmunoassay and Northern blot analysis. Seven patients with lung cancer were first treated with various anticancer agents for 3 days (days 1-3) followed by treatment with rhG-CSF (2 microgram/kg weight/day) for 7 days (days 8-14). Chemotherapy caused neutropenia but the neutrophil count increased biphasically between days 8 and 14. Chemotherapy did not change the baseline plasma concentration of HNPs 1-3 (74.1+/-2.1 pmol/ml) but the concentration increased from day 12, 5 days after commencement of rhG-CSF therapy, to reach a peak value of 430.8+/-57.0 pmol/ml on day 15, 1 day after the last administration of rhG-CSF. Baseline HNPs 1-3 content per neutrophil was 0.59+/-0.02 fmol, decreased to 0.30+/-0.07 fmol on day 9, then increased to 0.78+/-0.07 fmol on day 15. Analyses of peripheral blood neutrophils by Northern blot and reverse-phase high-performance liquid chromatography showed that the amounts of HNPs 1-3 mRNA and precursors of HNPs 1-3 markedly increased in response to rhG-CSF. Our results indicate that recombinant hG-CSF does not only increase neutrophil count but stimulates HNPs 1-3 biosynthesis in neutrophils, thus enhancing the host defense system of compromised hosts with neutropenia.     

Circular permutation of granulocyte colony-stimulating factor

The sequence of granulocyte colony-stimulating factor (G-CSF) has been circularly permuted by introducing new chain termini into interhelical loops and by constraining the N- and C-terminal helices, either by direct linkage of the termini (L0) or by substitution of the amino-terminal 10-residue segment with a seven-residue linker composed of glycines and serines (L1). All the circularly permuted G-CSFs (cpG-CSFs) were able to fold into biologically active structures that could recognize the G-CSF receptor. CD and NMR spectroscopy demonstrated that all of the cpG-CSFs adopted a fold similar to that of the native molecule, except for one [cpG-CSF(L1)[142/141]] which has the new termini at the end of loop 34 with the shorter L1 linker. All of the cpG-CSFs underwent cooperative unfolding by urea, and a systematically lower free energy change (DeltaGurea) was observed for molecules with the shorter L1 linker than for those molecules in which the original termini were directly linked (the L0 linker). The thermodynamic stability of the cpG-CSFs toward urea was found to correlate with their relative ability to stimulate proliferation of G-CSF responsive cells. Taken together, these results indicate that the G-CSF sequence is robust in its ability to undergo linear rearrangement and adopt a biologically active conformation. The choice of linker, with its effect on stability, seems to be important for realizing the full biological activity of the three-dimensional structure. The breakpoint and linker together are the ultimate determinants of the structural and biological profiles of these circularly permuted cytokines. In the following paper [McWherter, C. A., et al. (1999) Biochemistry 38, 4564-4571], McWherter and co-workers have used circularly permuted G-CSF sequences to engineer chimeric dual IL-3 and G-CSF receptor agonists in which the relative spatial orientation of the receptor agonist domains is varied. Interpreting the differences in activity for the chimeric molecules in terms of the connectivity between domains depends critically on the results reported here for the isolated cpG-CSF domains.     

Structural identification and biological activity of positional isomers of long-acting and mono-PEGylated recombinant human granulocyte colony-stimulating factor with trimeric-structured methoxy polyethylene glycol N-hydroxysuccinimidyl functional group

The individual positional isomers from the mono-PEGylated recombinant human granulocyte colony-stimulating factor (rhG-CSF) were successfully isolated with additional strong cation exchange chromatography using Source 15S. The three isolated individual positional isomers were found to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), analytical size exclusion high-performance liquid chromatography (SE-HPLC), and analytical cation exchange HPLC (CIE-HPLC) and were also characterized with respect to site of PEGylation by enzymatic digestion with endoproteinase Lys-C and N-terminal sequencing. In addition, in vitro biological activity was determined by cell proliferation assay. It was determined that the three isolated individual positional isomers were PEGylated at Lys35, Met(N-terminal), and Lys17 of the rhG-CSF molecule with a 23-kDa trimer-structured methoxy polyethylene glycol N-hydroxysuccinimidyl functional group (mPEG-NHS). All individual positional isomers (Lys35-PEGylated rhG-CSF, Met(N-terminal)-PEGylated rhG-CSF, and Lys17-PEGylated rhG-CSF) retained in vitro biological activity and were found to be 18.5%, 37.6%, and 7.1%, respectively, compared with the rhG-CSF molecule. The significantly different in vitro biological activities observed in the individual positional isomers could be presumably due to interference of receptor binding or active sites on the rhG-CSF molecule. In conclusion, the individual positional isomers isolated from the mono-PEGylated rhG-CSF were well characterized with respect to the site of PEGylation involving Lys35, Met(N-terminal), and Lys17. This characterization of the individual positional isomers would be critical to provide a basis for establishing consistency in the manufacturing process.     

Characterization of peripheral blood stem cell grafts mobilized by granulocyte colony-stimulating factor and plerixafor compared with granulocyte colony-stimulating factor alone

Background aimsThis study aimed to characterize the immune effectors contained in apheresis samples obtained from patients with grafts mobilized with plerixafor and granulocyte colony-stimulating factor (G-CSF) (P+G) compared with grafts mobilized with G-CSF alone (G).MethodsAliquots of apheresis samples were obtained from 36 patients with malignant diseases after mobilization with G (n = 18) or P+G (n = 18). The phenotype and cytokine secretion profile of T cell and dendritic cell subsets were characterized by multicolor cytometry including intracellular cytokine staining.ResultsIn grafts collected after mobilization with P+G, there was a significantly higher percentage of CD3⁺ T cells compared with samples collected after mobilization with G alone. On a functional level, a significant increase of interferon-γ and tumor necrosis factor-α secreting CD8⁺ T cells was observed in the P+G group compared with the G group. CD4⁺Foxp3⁺ regulatory T cells were similar in both groups but exhibited a lower expression of inducible costimulatory molecule and a significantly higher expression of CD127 in the P+G group. Myeloid dendritic cells (MDCs) and BDCA3⁺ dendritic cells were similar in both groups. In contrast, plasmacytoid dendritic cells (PDCs) (CD123⁺BDCA2⁺HLA-DR⁺) were significantly increased in the P+G grafts, leading to a higher PDC-to-MDC ratio. PDCs mobilized by P+G displayed different functional markers—a higher percentage of ILT7⁺ PDCs and decreased expression of CD86—suggesting a potential regulatory capacity of PDCs mobilized by P+G.ConclusionsGrafts mobilized with P+G exhibited major different functional features compared with grafts mobilized with G alone, suggesting that such grafts may have an impact on patient outcome after autologous stem cell transplantation.     

Protein tailoring of human granulocyte colony-stimulating factor

Summary Recombinant human granulocyte-colony stimulating factor (rhG-CSF) was modified by site-directed mutagenesis and chemical modification in order to improve its pharmacological activity and its thermostability. The mutant rhG CSF which 17th cysteine was substituted with alanine was chemically modified by activated polyethylene glycol. The chemically modified mutant rhG-CSF greatly increased both its biological activityin vivo and its thermostability. This is a successful example of protein tailoring in which site-directed mutagenesis and chemical modification were used at the same time.     

Antifungal activities of recombinant antifungal protein by conjugation with polyethylene glycol.

Tenecin 3, an antifungal protein isolated from coleopteran insect Tenebrio molitor larvae, inhibited growth of the fungus Candida albicans. We have previously reported that tenecin 3 has a propensity of random structure with very loose turn-like elements by circular dichroism (CD) analysis and 2D nuclear overhauser effect spectroscopy [Lee et al. (1999)]. However, the antifungal mechanism of tenecin-3 has not yet been studied due to its very low availability from natural sources. As an initial step to study the antifungal mechanism of tenecin 3, recombinant tenecin 3 (RT-3) obtained from an expression system in Escherichia coli showed antifungal activity against C. albicans as did natural tenecin 3. To elucidate the antifungal mechanism of RT-3 and to explore the possibility of preparing polyethylene glycol (PEG) conjugated derivative, we synthesized PEG conjugated RT-3 (RT-3-PEG) and examined its antifungal activity against C. albicans in vitro. RT-3-PEG showed greater antifungal activity against C. albicans than RT-3 alone at the same dose. When C. albicans was treated with RT-3-PEG in vitro, K+ in the C. albicans cell was leaked out rapidly compared to the C. albicans treated with RT-3 alone. When the morphological change of RT-3-PEG treated C. albicans was examined by scanning electron microscopy, string-like substances, which may have been derived from the fungus, were stacked around the cell whose wall was damaged. Also, no appreciable hemolysis of mouse erythrocytes was detected under conditions in which 1% melittin caused 100% hemolysis. These results suggested that the RT-3-PEG derivative probably does not interact with mammalian cell appreciably, although it has antifungal activity.     

Identification of distinct receptors for two hemopoietic growth factors (granulocyte colony-stimulating factor and multipotential colony-stimulating factor) by chemical cross-linking

Granulocyte colony-stimulating factor (G-CSF) and multipotential colony-stimulating factor (multi-CSF or interleukin 3) are two members of a family of hemopoietic growth and differentiation factors. Using biologically active radioiodinated derivatives and chemical cross-linking (predominantly with the homobifunctional reagent disuccinimidyl suberate) followed by gel electrophoresis and autoradiography, receptors for these two factors have been identified. The G-CSF receptor was identified as a single subunit protein of Mr approximately 150,000 while two molecular species able to specifically cross-link to 125I-multi-CSF were identified of Mr approximately 75,000 and 60,000. For both CSFs specificity of formation of cross-linked species was demonstrated by showing that the homologous unlabeled CSF (but not other CSFs) competed for formation of the complexes with the appropriate dose-response relation, by showing that saturation occurred over the appropriate range of 125I-CSF concentration and by showing that the cellular specificity of CSF binding paralleled that for cross-linked complex formation. The formation of cross-linked complexes was dependent on the concentration and type of chemical cross-linker, especially for cross-linking of 125I-multi-CSF. Based on a number of criteria it is suggested that the two species cross-linked to 125I-multi-CSF do not represent receptors of different affinity but, rather, two noncovalently associated subunits of a receptor complex.     

Soluble periplasmic production of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens

Cost-effective production of soluble recombinant protein in a bacterial system remains problematic with respect to expression levels and quality of the expressed target protein. These constraints have particular meaning today as "biosimilar" versions of innovator protein drugs are entering the clinic and the marketplace. A high throughput, parallel processing approach to expression strain engineering was used to evaluate soluble expression of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens. The human g-csf gene was optimized for expression in P. fluorescens and cloned into a set of periplasmic expression vectors. These plasmids were transformed into a variety of P. fluorescens host strains each having a unique phenotype, to evaluate soluble expression in a 96-well growth and protein expression format. To identify a strain producing high levels of intact, soluble Met-G-CSF product, more than 150 protease defective host strains from the Pfēnex Expression Technology? toolbox were screened in parallel using biolayer interferometry (BLI) to quantify active G-CSF binding to its receptor. A subset of these strains was screened by LC-MS analysis to assess the quality of the expressed G-CSF protein. A single strain with an antibiotic resistance marker insertion in the pfaI gene was identified that produced>99% Met-GCSF. A host with a complete deletion of the autotransporter-coding gene pfaI from the genome was constructed, and expression of soluble, active Met-GSCF in this strain was observed to be 350mg/L at the 1 liter fermentation scale.     

Purification and characterization of human granulocyte colony-stimulating factor (G-CSF).

A colony-stimulating factor (CSF) has been purified to homogeneity from the serum-free medium conditioned by one of the human CSF-producing tumor cell lines, CHU-2. The molecule was a hydrophobic glycoprotein (mol. wt 19,000, pI = 6.1 as asialo form) with possible O-linked glycosides. Amino acid sequence determination of the molecule gave a single NH2-terminal sequence which had no homology to the corresponding sequence of the other CSFs previously reported. The biological activity was apparently specific for a neutrophilic granulocyte-lineage of both human and mouse bone marrow cells with a specific activity of 2.7 X 10(8) colonies/10(5) non-adherent human bone marrow cells/mg protein. The purified CSF can be regarded as a G-CSF of human origin and will become a useful material for investigation of regulatory mechanisms of human granulopoiesis.     

Gene structure and function of granulocyte colony-stimulating factor

In the last few years, the molecular and genetic nature of the granulocyte colony-stimulating factor, which controls proliferation and differentiation of neutrophils, has been characterized. Recent clinical application of G-CSF proves that this hormone is effective in the treatment of patients suffering from neutropenia.     

Pretreatment with granulocyte colony-stimulating factor reduces myelopoiesis in irradiated mice

The purpose of this study was to investigate effects of the treatment prior to irradiation with granulocyte colony-stimulating factor (G-CSF) on hematopoiesis in B10CBAF1 mice exposed to a sublethal dose of 6.5 Gy of 60Co gamma radiation. G-CSF was administered in a 4-day regimen (3 microg/day); irradiation followed 3 h after the last injection of G-CSF. Such a treatment was found to stimulate granulopoiesis, as shown by increased counts of granulocyte-macrophage progenitor cells (GM-CFC) and of granulocytic cells in the femoral marrow and spleen at the time of irradiation. However, postirradiation counts of GM-CFC and granulocytic cells in the marrow of mice pretreated with G-CSF were reduced up to day 18 after irradiation. Interestingly, the D0 values for marrow GM-CFC determined 1 h after in vivo irradiation were 1.98 Gy for controls and 2.47 Gy for mice pretreated with G-CSF, indicating a decreased radiosensitivity of these cells after drug treatment. The inhibitory effects of the pretreatment with G-CSF on the postirradiation granulopoiesis could be attributed to the phenomenon of "rebound quiescence" which can occur after cessation of the treatment with growth factors. Postirradiation recovery of erythropoiesis in the spleen of mice pretreated with G-CSF exhibited a dramatic increase and compensated for the decreased erythropoiesis in the marrow at the time of irradiation. This complexity of the hematopoietic response should be taken into account when administering G-CSF in preirradiation regimens.     

Recombinant human granulocyte colony-stimulating factor enhances superoxide release in human granulocytes stimulated by the chemotactic peptide

Recombinant human granulocyte colony-stimulating factor (G-CSF) by itself was not an effective stimulus for inducing the release of superoxide (O-2) in human granulocytes. However, G-CSF was able to prime human granulocytes, and enhanced O-2 release stimulated by the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). The preincubation with G-CSF for 5-10 min at 37 degrees C was sufficient for priming the cells. The optimal enhancing effect was obtained at 25 ng/ml of G-CSF. The enhancement of O-2 release by G-CSF was observed over the complete range of effective concentrations of FMLP (10(-8)-10(-6) M). These findings indicate that G-CSF is a potent activator of mature granulocyte functions.     

Proliferative effects of purified granulocyte colony-stimulating factor (G-CSF) on normal mouse hemopoietic cells

When granulocyte colony-stimulating factor (G-CSF), purified to homogeneity from mouse lung-conditioned medium, was added to agar cultures of mouse bone marrcw cells, it stimulated the formation of small numbers of granulocytic colonies. At high concentrations of G-CSF, a small proportion of macrophage and granulocyte-macrophage colonies also developed. G-CSF stimulated colony formation by highly enriched progenitor cell populations obtained by fractionation of mouse fetal liver cells using a fluorescence-activated cell sorter, indicating that G-CSF probably acts directly on target progenitor cells. Granulocytic colonies stimulated by G-CSF were small and uniform in size, and at 7 days of culture were composed of highly differentiated cells. Studies using clonal transfer and the delayed addition of other regulators showed that G-CSF could directly stimulate the initial proliferation of a large proportion of the granulocvte-macrophage progenitors in adult marrow and also the survival and/or proliferation of some multipotential, erythroid, and eosinophil progenitors in fetal liver. However, G-CSF was unable to sustain continued proliferation of these cells to result in colony formation. When G-CSF was mixed with purified granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), the combination stimulated the formation by adult marrow cells of more granulocyte-macrophage colonies than either stimulus alone and an overall size increase in all colonies. G-CSF behaves as a predominantly granulopoietic stimulating factor but has some capacity to stimulate the initial proliferation of the same wide range of progenitor cells as that stimulated by GM-CSF.     

Mobilization of human lymphoid progenitors after treatment with granulocyte colony-stimulating factor

Hemopoietic stem and progenitor cells ordinarily residing within bone marrow are released into the circulation following G-CSF administration. Such mobilization has a great clinical impact on hemopoietic stem cell transplantation. Underlying mechanisms are incompletely understood, but may involve G-CSF-induced modulation of chemokines, adhesion molecules, and proteolytic enzymes. We studied G-CSF-induced mobilization of CD34+ CD10+ CD19- Lin- and CD34+ CD10+ CD19+ Lin- cells (early B and pro-B cells, respectively). These mobilized lymphoid populations could differentiate only into B/NK cells or B cells equivalent to their marrow counterparts. Mobilized lymphoid progenitors expressed lymphoid- but not myeloid-related genes including the G-CSF receptor gene, and displayed the same pattern of Ig rearrangement status as their bone marrow counterparts. Decreased expression of VLA-4 and CXCR-4 on mobilized lymphoid progenitors as well as multipotent and myeloid progenitors indicated lineage-independent involvement of these molecules in G-CSF-induced mobilization. The results suggest that by acting through multiple trans-acting signals, G-CSF can mobilize not only myeloid-committed populations but a variety of resident marrow cell populations including lymphoid progenitors.     

Preliminary characterization of bovine beta-lactoglobulin after its conjugation to polyethylene glycol

The major component of the whey fraction of bovine milk, beta-lactoglobulin (betaLG), has been transformed by grafting polyethylene glycol chains either on the thiol group (free and after reduction of the S-S bridges) of the cysteine residues, or on the amino group of the lysine residues and/or of the N-terminal amino acid. Acylation of the protein was achieved at a controlled pH of 7.0 using increasing ratios of activated PEG to betaLG. Transformation of the dimeric form into the monomer occurred at least for the fully pegylated adduct. The number of polymer chains fixed per mole of protein was determined by dosage of the free amino functions still present after reaction. The incidence of pegylation on the secondary structure of betaLG was evaluated using the Fourier Transform Infrared Spectroscopy (FTIR). Denaturation studies with guanidinium hydrochloride (Gu-HCl) by means of spectrofluorimetric measurements, showed an identical behavior of native as well as of pegylated betaLG.The antigenicity of the fully pegylated adduct was examined through antigenic competition towards native betaLG. The pegylated protein exhibited less than 1/100 of the native betaLG inhibition capacity, that could moreover never be complete. This is thus demonstrating the loss of accessibility for at least multiple conformational epitopes through pegylation procedure.Spectrofluorimetric measurements showed that betaLG-N-PEG(7) was still able to bind retinol while no effect on the intrinsic fluorescence could be detected by adding palmitic acid. Whether this last ligand binds or not to pegylated betaLG is discussed. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 40-49, 1997.