Expression and one step intein-mediated purification of biologically active human G-CSF in Escherichia coli.

Recombinant form of granulocyte colony stimulating factor (G-CSF) was first approved by FDA in 1998 for chemotherapy induced neutropenia. However, despite production of its biosimilars, less expensive production of G-CSF could reduce the overall therapeutic cost. The aim of this study was to evaluate the possibility of producing biologically active recombinant G-CSF via a single step purification procedure mediated by a self-cleavable intein. G-CSF was expressed by E. coli BL21 (DE3) through IPTG induction, followed by its purification using pH optimization on a chitin column. Western blotting, ELISA, size exclusion chromatography, circular diachorism, peptide mapping, and in vitro assays were performed to compare the structural similarity and biological activity of the purified G-CSF with Neupogen™. Protein purification was confirmed by revealing a band of approximately 18.8 kDa on SDS-PAGE. Bioactivity and physicochemical assays based on the US pharmacopeia showed almost identical or acceptable ranges of similarities between recombinant G-CSF and Neopogen™. this study, biologically active soluble recombinant G-CSF was successfully produced with high purity without using chaotropic solvents through a one-step procedure. This shorter and more efficient purification procedure can reduce the cost and time of G-CSF production which makes its industrial production more cost-effective and might be also applicable for production of other biopharmaceuticals.

Construction of a Pichia pastoris strain efficiently producing recombinant human granulocyte-colony stimulating factor (rhG-CSF) and study of its biological activity on bone marrow cells.

Non-glycosylated, recombinant human granulocyte colony-stimulating factor (rhG-CSF), produced by Escherichia coli (filgrastim, leukostim) is widely used to treat a number of serious human diseases and aids in the recovery post bone marrow transplantation. Although glycosylation is not required for the manifestation of the biological activity of G-CSF, a number of studies have shown that the carbohydrate residue significantly increases the physicochemical stability of the G-CSF molecule. Therefore, the aim of the present study was to design a Pichia pastoris strain capable of producing glycosylated rhG-CSF, and to study its effects on rat bone marrow cells. The nucleotide sequence of the rhG-CSF gene has been optimized for expression in P. pastoris, synthesized, cloned into the pPICZαA vector and expressed under the control of the AOX promoter in P. pastoris X33. One of the selected clones secreting rhG-CSF, produced 100-120 mg/l of rhG-CSF three days post-induction with methanol. The recombinant cytokine was purified using two-step, ion-exchange chromatography. The final yield of purified G-CSF was 35 mg/L of culture medium. The biological activity of rhG-CSF was examined in rat bone marrow cells. The P. pastoris strain was designed to produce relatively high levels of rhG-CSF. The rhG-CSF protein had a strong stimulating effect on the growth of rat bone marrow cells, which was comparable to that of the commercial drug leukostim, but showed a more persistent effect on granulocyte cells and monocyte sprouts, enabling the enhanced maintenance of the viability of the cells into the 4th day of incubation.

Low-cost customizable microscale toolkit for rapid screening and purification of therapeutic proteins.

Biopharmaceutical separations require tremendous amounts of optimization to achieve acceptable product purity. Typically, large volumes of reagents and biological materials are needed for testing different parameters, thus adding to the expense of biopharmaceutical process development. This study demonstrates a versatile and customizable microscale column (µCol) for biopharmaceutical separations using immobilized metal affinity chromatography (IMAC) as an example application to identify key parameters. µCols have excellent precision, efficiency, and reproducibility, can accommodate any affinity, ion-exchange or size-exclusion-based resin and are compatible with any high-performance liquid chromatography (HPLC) system. µCols reduce reagent amounts, provide comparable purification performance and high-throughput, and are easy to automate compared with current conventional resin columns. We provide a detailed description of the fabrication methods, resin packing methods, and µCol validation experiments using a conventional HPLC system. Finite element modeling using COMSOL Multiphysics was used to validate the experimental performance of the µCols. In this study, µCols were used for improving the purification achieved for granulocyte colony stimulating factor (G-CSF) expressed using a cell-free CHO in vitro translation (IVT) system and were compared to a conventional 1 ml IMAC column. Experimental data revealed comparable purity with a 10-fold reduction in the amount of buffer, resin, and purification time for the µCols compared with conventional columns for similar protein yields.

Functional characterization of recombinant human granulocyte colony stimulating factor (hGMCSF) immobilized onto silica nanoparticles.

OBJECTIVES: Granulocyte macrophage colony stimulating factor (GMCSF), an important therapeutic cytokine, was immobilized onto silica nanoparticles. Maintenance of structural integrity and biological performance in immobilized cytokine was assessed to augment its applicability in possible biomedical implications. RESULTS: Following its cloning and expression in E. coli, the recombinant human GMCSF (hGMCSF) was purified as a GST-tagged protein corresponding to a 42 kDa band on SDS-PAGE. The purified cytokine was immobilized onto biocompatible silica nanoparticles (~129.4 nm) by adsorption and the binding was confirmed by dynamic light scattering and infrared spectroscopy. Maximum binding of hGMCSF was at 6.4 µg mg(-1) silica nanoparticles. Efficient release of the cytokine from the nanoparticles with its structural integrity intact was deduced from circular dichroism spectroscopy. hGMCSF-immobilized silica nanoparticles efficiently increased the proliferation of RAW 264.7 macrophage cells with 50 % increase in proliferation at 600 ng hGMCSF µg(-1) silica nanoparticles. CONCLUSIONS: Silica nanoparticles successfully immobilized hGMCSF maintaining its structural integrity. The release of the immobilized cytokine from silica nanoparticles resulted in the increased proliferation of macrophages indicating the potential of the system in future applications.

Use of polymeric membranes for purification of an E. coli expressed biotherapeutic protein.

Polymers have had a significant impact on the field of bioseparations in the past few decades. Most recently, membrane chromatography has emerged as an efficient alternative to the conventional packed-bed chromatography by eliminating the diffusion-related limitations associated with the traditional resin beads. In this article, we examine six membrane adsorbers for purification of granulocyte colony-stimulating factor (GCSF), an Escherichia coli-based biotherapeutic. These adsorbers differ either in their base matrix or in the surface chemistry. The role of interactions between the filter surfaces and the protein molecules in effecting these separations is the focus of the article.

Improved Production and Characterization of Recombinant Human Granulocyte Colony Stimulating Factor from E. coli under Optimized Downstream Processes.

This work reports the upstream and downstream process of recombinant human granulocyte colony stimulating factor (rhG-CSF) expressed in Escherichia coli BL21 (DE3)pLysS. The fed batch mode was selected for the maximum output of biomass (6.4g/L) and purified rhG-CSF (136mg/L) under suitable physicochemical environment. The downstream processing steps viz., recovery, solubilization, refolding and concentration were optimized in this study. The maximum rhG-CSF inclusion bodies recovery yield (97%) was accomplished with frequent homogenization and sonication procedure. An efficient solubilization (96%) of rhG-CSF inclusion bodies were observed with 8M urea at pH 9.5. Refolding efficiency studies showed maximum refolding ⩾86% and ⩾84% at 20°C and pH 9 respectively. The renatured protein solution was concentrated, clarified and partially purified (⩾95%) by the cross flow filtration technique. The concentrated protein was further purified by a single step size exclusion chromatography with ⩾98% purity. The characterization of purified rhG-CSF molecular mass as evidenced by SDS-PAGE, western blot and LC/MS analysis was shown to be 18.8kDa. The secondary structure of rhG-CSF was evaluated by the CD spectroscopic technique based on the helical structural components. The biological activity of the purified rhG-CSF showed a similar activity of cell proliferation with the standard rhG-CSF. Overall, the results demonstrate an optimized downstream process for obtaining high yields of biologically active rhG-CSF.

[Recombinant granulocyte-colony stimulating factor (filgrastim): optimization of conditions of isolation and purification from inclusion body].

We developed a unified process platform for two recombinant human GCSF medicines--one with the non-prolonged and the other with prolonged action. This unified technology led to a simpler and cheaper production while introduction of the additional pegylation stage to the technological line eased obtaining of the medicines with different action and allowed to standardize technological process documenting according to GMP requirements.

Expression, Purification, and Biological Evaluation of XTEN-GCSF in a Neutropenic Rat Model.

Granulocyte colony-stimulating factor (GCSF) stimulates the proliferation of neutrophils but it has low serum half-life. Therefore, the present study was done to investigate the effect of XTENylation on biological activity, pharmacokinetics, and pharmacodynamics of GCSF in a neutropenic rat model. XTEN tag was genetically fused to the N-terminal region of GCSF-encoding gene fragment and subcloned into pET28a expression vector. The cytoplasmic expressed recombinant protein was characterized through intrinsic fluorescence spectroscopy (IFS), dynamic light scattering (DLS), and size exclusion chromatography (SEC). In vitro biological activity of the XTEN-GCSF protein was evaluated on NFS60 cell line. Hematopoietic properties and pharmacokinetics were also investigated in a neutropenic rat model. An approximately 140 kDa recombinant protein was detected on SDS-PAGE. Dynamic light scattering and size exclusion chromatography confirmed the increase in hydrodynamic diameter of GCSF molecule after XTENylation. GCSF derivatives showed efficacy in proliferation of NFS60 cell line among which the XTEN-GCSF represented the lowest EC50 value (100.6 pg/ml). Pharmacokinetic studies on neutropenic rats revealed that XTEN polymer could significantly increase protein serum half-life in comparison with the commercially available GCSF molecules. PEGylated and XTENylated GCSF proteins were more effective in stimulation of neutrophils compared to the GCSF molecule alone. XTENylation of GCSF represented promising results in in vitro and in vivo studies. This approach can be a potential alternative to PEGylation strategies for increasing serum half-life of protein.

Cysteine-specific PEGylation of rhG-CSF via selenylsulfide bond.

A new PEGylation reagent enabling selective modification of free thiol groups is described in this article. The reagent was synthesized by attaching linear polyethylene glycol (PEG) N-hydroxysuccinimide to selenocystamine. The reaction was very fast, resulting in over 95% conversion yield. The active group of this new PEG-Se reagent is a diselenide, reacting with thiols via thiol/diselenide exchange reaction. Recombinant human granulocyte colony-stimulating factor (rhG-CSF) with an unpaired cysteine at the position 18 (Cys18) was used as a model protein. It was comparatively PEGylated with the new PEG-Se reagent, as well as with commercially available maleimide (PEG-Mal) and ortho-pyridyl disulfide (PEG-OPSS) PEG reagents. The highest PEGylation yield was obtained with PEG-Mal, followed by PEG-OPSS and PEG-Se. The reaction rates of PEG-Mal and PEG-Se were comparable, while the reaction rate of PEG-OPSS was lower. Purified monoPEGylated rhG-CSF conjugates were characterized and compared. Differences in activity, stability, and in vivo performance were observed, although all conjugates contained a 20 kDa PEG attached to the Cys18. Minor conformational changes were observed in the conjugate prepared with PEG-Mal. These changes were also reflected in low in vitro biological activity and aggregate formation of the maleimide conjugate. The conjugate prepared with PEG-Se had the highest in vitro biological activity, while the conjugate prepared with PEG-OPSS had the best in vivo performance.

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.

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 Pfenex 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.

A high-throughput protein refolding screen in 96-well format combined with design of experiments to optimize the refolding conditions.

Production of correctly folded and biologically active proteins in Escherichiacoli can be a challenging process. Frequently, proteins are recovered as insoluble inclusion bodies and need to be denatured and refolded into the correct structure. To address this, a refolding screening process based on a 96-well assay format supported by design of experiments (DOE) was developed for identification of optimal refolding conditions. After a first generic screen of 96 different refolding conditions the parameters that produced the best yield were further explored in a focused DOE-based screen. The refolding efficiency and the quality of the refolded protein were analyzed by RP-HPLC and SDS-PAGE. The results were analyzed by the DOE software to identify the optimal concentrations of the critical additives. The optimal refolding conditions suggested by DOE were verified in medium-scale refolding tests, which confirmed the reliability of the predictions. Finally, the refolded protein was purified and its biological activity was tested in vitro. The screen was applied for the refolding of Interleukin 17F (IL-17F), stromal-cell-derived factor-1 (SDF-1α/CXCL12), B cell-attracting chemokine 1 (BCA-1/CXCL13), granulocyte macrophage colony stimulating factor (GM-CSF) and the complement factor C5a. This procedure identified refolding conditions for all the tested proteins. For the proteins where refolding conditions were already available, the optimized conditions identified in the screening process increased the yields between 50% and 100%. Thus, the method described herein is a useful tool to determine the feasibility of refolding and to identify high-yield scalable refolding conditions optimized for each individual protein.

High-Throughput Process Development: II-Membrane Chromatography.

Membrane chromatography is gradually emerging as an alternative to conventional column chromatography. It alleviates some of the major disadvantages associated with the latter, including high-pressure drop across the column bed and dependence on intraparticle diffusion for the transport of solute molecules to their binding sites within the pores of separation media. In the last decade, it has emerged as a method of choice for final polishing of biopharmaceuticals, in particular, monoclonal antibody products. The relevance of such a platform is high in view of the constraints with respect to time and resources that the biopharma industry faces today.This protocol describes the steps involved in performing HTPD of a membrane chromatography step. It describes the operation of a commercially available device (AcroPrep™ Advance filter plate with Mustang S membrane from Pall Corporation). This device is available in 96-well format with a 7 µL membrane in each well. We will discuss the challenges that one faces when performing such experiments as well as possible solutions to alleviate them. Besides describing the operation of the device, the protocol also presents an approach for statistical analysis of the data that are gathered from such a platform. A case study involving the use of the protocol for examining ion-exchange chromatography of the Granulocyte Colony Stimulating Factor (GCSF), a therapeutic product, is briefly discussed. This is intended to demonstrate the usefulness of this protocol in generating data that are representative of the data obtained at the traditional lab scale. The agreement in the data is indeed very significant (regression coefficient 0.9866). We think that this protocol will be of significant value to those involved in performing high-throughput process development of membrane chromatography.

Economic assessment of continuous processing for manufacturing of biotherapeutics.

Continuous processing offers a promising approach to revolutionize biotherapeutics manufacturing as reflected in recent years. The current study offers a comparative economic assessment of batch and continuous processing for the production of biotherapeutic products. Granulocyte-colony stimulating factor (GCSF), a protein expressed in E. coli, and an IgG1 monoclonal antibody, were chosen as representatives of microbial and mammalian derived products for this assessment. Economic indicators-cost of goods (COGs), net present value (NPV), and payback time have been estimated for the assessment. For the case of GCSF, conversion from batch to integrated continuous manufacturing induced a $COGs/g reduction of 83% and 73% at clinical and commercial scales, respectively. For the case of mAb therapeutic, a 68% and 35% reduction in $COGs/g on translation from batch to continuous process was projected for clinical and commercial scales, respectively. Upstream mAb titer was also found to have a significant impact on the process economics. With increasing mAb titer, the $COG/g decreases in both operating modes. With titer increasing from 2 to 8 g/L, the $COG/g of batch process was reduced by 53%, and that of the continuous process was reduced by 63%. Cost savings in both the cases were attributed to increased productivity, efficient equipment and facility utilization, smaller facility footprint, and reduction in utilization of consumables like resin media and buffers actualized by the continuous processing platform. The current study quantifies the economic benefits associated with continuous processing and highlights its potential in reducing the manufacturing cost of biotherapeutics.

Aqueous Two-Phase-Assisted Precipitation of Proteins: A Platform for Isolation of Process-Related Impurities from Therapeutic Proteins.

Aqueous two-phase systems (ATPS) have been widely and successfully used in the purification of various biological macromolecules such as proteins, nucleic acids, antibiotics, and cell components. Interfacial precipitation of the product often results in lower recovery and selectivity of ATPS. Efficient resolubilization of the interfacial precipitate offers a way to improve the recovery as well as selectivity of ATPS systems.In this protocol, we describe a method for aqueous two-phase-assisted precipitation and resolubilization of the recombinant human Granulocyte Colony Stimulating Factor (GCSF) for its selective isolation from E. coli host cell proteins as well as nucleic acids. This platform purification can be applied to other cytokines as well as most of the hydrophobic proteins that partition into the hydrophobic PEG-rich top phase. Recoveries of up to 100% of the product along with reduction of levels of E. coli host cell proteins (from 250-500 to 10-15 ppm) and of nucleic acids (from 15-20 to 5-15 ng/mL) were observed.

Complete sequencing of the recombinant granulocyte-colony stimulating factor (filgrastim) and detection of biotinylation by mass spectrometry.

Granulocyte-colony stimulating factor stimulates production and antibacterial function of neutrophiles. Therapy using the recombinant protein drug represents a major step forward in oncology. The protein has not been, however, completely sequenced at the protein level and this formed the rationale of the current study. Recombinant G-CSF (filgrastim) was run on two-dimensional gel electrophoresis (2DE), the protein was in-gel digested with trypsin and chymotrypsin, and peptides were analysed on Nano-ESI-LC-MS/MS (high performance ion trap, HCT). Bioinformatic tools used were Mascot v2.2 and Modiro(TM) v1.1 softwares. A single spot was detected on 2DE and peptides resulting from in-gel digestion were unambiguously identified by the MS/MS approach leading to complete sequencing when both searching engines were applied. N-terminal methionine loss, N-terminal methionine oxidation and amidination were observed. Both softwares identified modifications. Complete sequencing by a non-sophisticated and rapid gel-based mass spectrometry approach confirmed the primary structure predicted from nucleic acid sequences. A chemical modification of glutamine 26 with the interim name PentylamineBiotin (Unimod accession number #800) compatible with biotinylation with 5-(biotinamido) pentylamine by the producer was detected by both softwares. Although there is some evidence that biotinylated G-CSF analogues are active, it remains open whether this modification may be responsible for the side effects observed or lead to changes of antigenicity.

Development of an integrated continuous PEGylation and purification Process for granulocyte colony stimulating factor.

PEGylation of therapeutic proteins has long been recognized as a safe and effective approach to enhance pharmacokinetic properties of proteins by increasing the in-vivo half-life and thereby the bioavailability. Despite all the benefits linked to PEGylation, high cost of PEGylated products has hindered accessibility of these products to patients. Continuous processing offers a solution to this predicament with its proven capability to improve economics without sacrificing product quality. In this study, we report the development of an integrated continuous PEGylation and purification process for a therapeutic protein, PEG-GCSF. The methodology to achieve this consisted of developing the batch PEGylation and purification protocols followed by their conversion into an integrated continuous process. A batch process involving rapid and highly productive PEGylation (reaction completion within one hour of reaction time) followed by cation exchange chromatography was developed. Enabling technologies like coiled flow inversion reactor, inline concentrator and counter-current chromatography, were utilized for the successful conversion of the batch process to continuous mode. The final integrated continuous process consisted of continuous PEGylation in a coiled flow inverter reactor followed by four column continuous counter-current cation exchange chromatography. Continuous chromatography was performed in a novel displacement mode, wherein all the multi-PEGylated impurities were removed in the loading flow-through and the pure mono-PEGylated protein was obtained in a single step salt elution. In combination with our previously established GCSF manufacturing train, the end-to-end continuous manufacturing process starting from inclusion bodies to unformulated PEG-GCSF drug substance was successfully run for 12 h. All attributes were found to be consistent over the period of operation with product purity > 99 % and high molecular weight impurities < 0.5 %. We hope that the current study will lay the foundation for implementation of continuous processing as a method to improve manufacturability of PEGylated therapeutic proteins.

Production of biologically active human myelocytokines in plants.

An effective system for expression of human granulocyte and granulocyte macrophage colony-stimulating factors (hG-CSF and hGM-CSF) in Nicotiana benthamiana plants was developed using viral vector based on tobacco mosaic virus infecting cruciferous plants. The genes of target proteins were cloned into the viral vector driven by actin promoter of Arabidopsis thaliana. The expression vectors were delivered into plant cells by agroinjection. Maximal synthesis rate was detected 5 days after injection and was up to 500 and 300 mg per kg of fresh leaves for hG-CSF and hGM-CSF, respectively. The yield of purified hG-CSF and hGM-CSF was 100 and 50 mg/kg of fresh leaves, respectively. Recombinant plant-made hG-CSF and hGM-CSF stimulated proliferation of murine bone marrow and human erythroleucosis TF-1 cells, respectively, at the same rate as the commercial drugs.

Molecular Cloning, Expression and Purification of G-CSF Isoform D, an Alternative Splice Variant of Human G-CSF.

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of hemopoiesis and granulopoiesis. However, overexpression of G-CSF has been implicated in several important processes in tumor biology such as tumor growth, angiogenesis, and metastasis. Four different mRNA isoforms resulting from alternative splicing have been reported for G-CSF (transcript variants 1, 2, 3 and 4). The mRNAs and protein products of splice variants 1 and 2 have been isolated for the first time, from tumor cell lines. In the present study for the first time we isolated the G-CSF transcript variant 4 encoding G-CSF isoform D from a highly malignant tumor cell line (Mehr80) with overexpression of G-CSF. Both the full-length G-CSF isoform B and G-CSF isoform D were cloned from Mehr80 cell line, overexpressed in Escherichia coli as N-terminal glutathione-S-transferase fusion proteins in the form of inclusion bodies and affinity purified by the batch method using glutathione-Sepharose 4B resin. Both fusion proteins were successfully cloned and expressed. Folded recombinant proteins were solubilized from inclusion bodies using sarkosyl, Triton X-114 and CHAPS and purified. The purity of G-CSF isoforms was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and they were clearly detected in western blot analysis using anti-G-CSF polyclonal antibody. The G-CSF plays various roles in physiological and pathological conditions, however to date, the differential function of G-CSF isoforms remains unknown. Considering the fact that G-CSF isoform D was isolated from a highly malignant tumor cell line with overexpression of G-CSF, the role of this splice variant in tumorigenesis requires further investigation.

A purification method for improving the process yield and quality of recombinant human granulocyte colony-stimulating factor expressed in Escherichia coli and its characterization.

A purification method employing a process-control strategy was developed for improving the yield of rhG-CSF (recombinant human granulocyte colony-stimulating factor). A purity of >/=99% with an overall yield of 2.18 g/l was achieved in the present study. Analysis of the product during purification indicated that detergents removed 72% of LPS (lipopolysaccharides) and 98% of HCPs (host cell proteins) without removing nucleic acid. Cysteine concentration was a key parameter in protein refolding. The bed height and HETP (height equivalent theoretical plates) value in the SEC (size-exclusion chromatography) column was evaluated and its impact on the resolution was studied. Formulation during SEC was found to be crucial for increasing the product yields with saving of time and process costs. The yield obtained in the present study is nearly four times higher than that reported in the literature. The product obtained was found to be acceptable for toxicological studies.