Charles H. Schmelzer

Industrial case study: evaluation of a mixed-mode resin for selective capture of a human growth factor recombinantly expressed in E. coli.

Mixed-mode chromatography resins are gaining popularity as effective purification tools for challenging feedstocks. This study presents the development of an industrial application to selectively capture recombinant human vascular endothelial growth factor (rhVEGF) on Capto MMC from an alkaline feedstock. Capto MMC resin contains a ligand that has the potential to participate in ionic, hydrophobic, and hydrogen boding interactions with proteins and is coupled to a highly cross-linked agarose bead matrix. VEGF is a key growth factor involved in angiogenesis and has therapeutic applications for wound healing. In this process, it is expressed in Escherichia coli as inclusion bodies. Solids are harvested from the cell lysate, and the rhVEGF is solubilized and refolded at pH 9.8 in the presence of urea and redox reagents. The unique mixed-mode characteristics of Capto MMC enabled capture of this basic protein with minimal load conditioning and delivered a concentrated pool for downstream processing with >95% yields while reducing host cell protein content to <1.2%. This study explores the impact of loading conditions and residence time on the dynamic binding capacity as well as the development of elution conditions for optimal purification performance. After evaluating various elution buffers, l-arginine HCl was shown to be an effective eluting agent for rhVEGF desorption from the Capto MMC mixed-mode resin since it successfully disrupted the multiple interactions between the resin and rhVEGF. The lab scale effort produced a robust chromatography step that was successfully implemented at commercial manufacturing scale.

Purification and characterization of recombinant human activin B

Recombinant human activin B has been isolated to more than 95% purity from a mammalian kidney cell line. Activin B is a covalently-linked homodimer with an apparent molecular mass of 25.9 kDa (unreduced) and 15.2 kDa (reduced) as determined by SDS-polyacrylamide-gel electrophoresis. On gel filtration in 6 M guanidine hydrochloride, activin B chromatographs with an apparent molecular mass of 11 kDa, whether reduced or not. The amino-terminal sequence of the purified protein is consistent with the expected sequence derived from the beta subunit of inhibin B. The amino acid composition of the purified molecule agrees with the expected theoretical composition of the beta subunit of inhibin B. Activin B has an apparent pI of 4.6 as determined by isoelectric focusing in 6 M urea and 4.7 as determined by chromatofocusing in 6 M urea. The extinction coefficient is 1.8.

Biochemical Characterization of Recombinant Human Nerve Growth Factor

Abstract: Recombinant human nerve growth factor (rhNGF) was expressed and secreted by Chinese hamster ovary cells and purified to homogeneity using ion‐exchange and reversed‐phase (RP) chromatography. The isolated product was shown to be consistent with a 120‐amino‐acid residue polypeptide chain by amino acid composition, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), RP‐HPLC, and mass spectrometry and with an N‐terminal sequence consistent with that expected from the cDNA for human nerve growth factor. By size‐exclusion chromatography, rhNGF behaves like a noncovalent dimer. Limited enzymatic digests of the 120‐residue monomer produced additional species of 118 (trypsin, removal of the C‐terminal Arg119‐Ala120 sequence) and 117 (trypsin plus carboxypeptidase B, removal of the C‐terminal Arg118‐Arg119‐Ala120 sequence) residues. Each of these species was isolated by high‐performance ion‐exchange chromatography and characterized by amino acid and N‐terminal sequence analyses, SDS‐PAGE, RP‐HPLC, and mass spectrometry. All three species were present in the digests as both homodimeric and heterodimeric combinations and found to be equipotent in both the chick dorsal root ganglion cell survival and rat pheochromocytoma neurite extension assays.

Activity and Biospecificity of Proteolyzed Forms and Dimeric Combinations of Recombinant Human and Murine Nerve Growth Factor

Abstract: Purified recombinant human nerve growth factor (rhNGF) and submaxillary gland‐derived murine NGF (muNGF) were characterized by amino acid composition, polyacrylamide gel electrophoresis (PAGE), reversed‐phase HPLC (RP‐HPLC), and high‐performance ion‐exchange chromatography (HPIEC). Limited tryptic digest of the N and C termini of the 120‐residue form of rhNGF produced a species of 109 residues (10–118). The previously observed natural murine analogue of this variant, muNGF lacking the first eight N‐terminal amino acids, was also isolated as a homodimer. Both species were purified using HPIEC and characterized by amino acid analysis, N‐terminal sequence, PAGE, and RP‐HPLC analysis. Each of the four homodimeric species was evaluated in some or all of the following biological assays for NGF: chick dorsal root and sympathetic ganglion assays and rat pheochromocytoma‐12 cell line neurite extension assay. The 118‐residue homodimeric versions of both rhNGF and muNGF displayed equivalent bioactivity, whereas the N terminal‐modified molecules presented activity reduced by 50‐ to 100‐fold. Utilizing HPIEC, we have examined the ability of the monomeric forms of any two of the homogeneous dimeric species of rhNGF to recombine. We have shown that not only can all of the previously described species form dimers by recombination, but an interspecies dimer can be created between muNGF and rhNGF.

Detection of human leukemia inhibitory factor by monoclonal antibody based ELISA

Leukemia inhibitory factor (LIF) is known to exhibit multiple functions by regulating the growth and differentiation of multiple normal cell types as well as malignant cells. To have a better understanding of the role of LIF, it is important to determine the level of LIF in various biological samples by developing an easy, sensitive and LIF specific assay. In this study, we have established a double monoclonal antibody (mAb) based ELISA. Four hybridoma cell lines (D3.14.1, D4.16.9, D25.1.4 and D62.3.2) secreting murine monoclonal antibodies (mAbs) against recombinant human leukemia inhibitory factor (rHuLIF) were produced by immunization of BALB/c mice with rHuLIF and by fusing immune spleen cells with P3X63Ag8U.1 myeloma cells. These mAbs each belong to the IgG1 isotype and have unique isoelectrofocusing point patterns. All four mAbs were shown to have high affinities for rHuLIF (Kd = 7 x 10(-10) to 6 x 10(-11) M) and were able to recognize the native as well as the reduced rHuLIF in an immunoblotting assay. All these mAbs showed no cross-reactivities to IL-1, IL-3, IL-6, TNF-alpha, GCSF and GMCSF. MAb D3.14.1 showed a weak binding to Oncostatin M but not to rMuLIF whereas the other three mAbs D4.16.9, D25.1.4 and D62.3.2 showed cross-reactivity to rMuLIF but not to Oncostatin M. Data obtained from a competitive binding enzyme-linked immunosorbent assay (ELISA) suggested that these four mAbs recognized different epitopes on rHuLIF. Using mAb D4.16.9 as coat antibody and horseradish peroxidase (HRP) conjugated mAb D3.14.1 as the conjugate antibody we established a double mAb based ELISA specific for human LIF which could detect as little as 100 pg/ml and 10 pg/ml of rHuLIF in the absence and in the presence of the ELAST ELISA amplification system, respectively. The addition of serum had very minimal effect on this ELISA.

High-yield expression of human vascular endothelial growth factor VEGF165 in Escherichia coli and purification for therapeutic applications

Vascular endothelial growth factor (VEGF(165)) is a potent mitogen that induces angiogenesis and vascular permeability in vivo and has demonstrated potential in therapeutic applications for accelerating wound healing. An industrial production method that provides high yield as well as high purity, quality, and potency is needed. The process described in this report involves a bacterial expression system capable of producing approximately 9g of rhVEGF per liter of broth and a downstream purification process consisting of protein refolding and three chromatography steps prior to formulation of the drug substance. A high cell density (HCD) fed-batch fermentation process was used to produce rhVEGF in periplasmic inclusion bodies. The inclusion bodies are harvested from the cell lysate and subjected to a single-step protein solubilization and refolding operation to extract the rhVEGF for purification. Overall recovery yields observed during development, including refolding and chromatography, were 30+/-6%. Host cell impurities are consistently cleared below target levels at both laboratory and large-scale demonstrating process robustness. The structure of the refolded and purified rhVEGF was confirmed by mass spectrometry, N-terminal sequencing, and tryptic peptide mapping while product variants were analyzed by multiple HPLC assays. Biological activity was verified by the proliferation of human umbilical vein derived endothelial cells.

High-pressure refolding of human vascular endothelial growth factor (VEGF) recombinantly expressed in bacterial inclusion bodies: Refolding optimization, and feasibility assessment

High‐pressure has been established as an effective technique for refolding proteins at high concentrations. In this study, high hydrostatic pressure (1–3 kbar) was utilized to refold a homodimeric protein from inclusion bodies and the process was evaluated for large‐scale manufacturing feasibility. This research focused on increasing protein concentration while maximizing yield and product quality. Refolding yields of 29–42% were achieved in the absence of urea at 2 kbar and at a protein concentration of 6 g/L. Optimization of the refolding buffer composition via multivariate design of experiments and other process parameters such as refolding pressure, gas sparging, and time under pressure are discussed. Although high‐pressure refolding can be considered a viable technology for manufacturing if the gains are clearly identified, in this particular case, the benefits that the high‐pressure technology offers do not compensate for the drawbacks of implementing new equipment in an existing facility, and unknown impact of scale‐up for this molecule.

Purification and partial characterization of recombinant human differentiation-stimulating factor

Recombinant human differentiation-stimulating factor (rhD-factor) has been isolated to greater than 95% purity from Chinese hamster ovary cells. RhD-factor is a glycoprotein with an apparent molecular weight of 45.6 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On gel filtration in 6 M guanidine-hydrochloride, rhD-factor elutes with an apparent molecular weight of 21.5 kDa; it elutes with an apparent molecular weight of 44.8 kDa under neutral pH (native) conditions. The amino-terminal sequence (12 residues) is consistent with the expected sequence derived from the genomic DNA sequence. Recombinant D-factor is heavily glycosylated with 30% by weight neutral sugar and 12% sialic acid. The ED50 for rhD-factor was 0.25 ng/ml. Trifluoromethanesulfonic acid-deglycosylated rhD-factor has a biological activity comparable to that of the native recombinant protein (ED50 = 0.40 ng/ml). The biological activity of rhD-factor was stable at pH 1 for 40 h, in 6 M guanidine-HCl containing buffers with or without reducing agent, and in 1% SDS. Carboxymethylation of D-factor after reduction totally destroyed biological activity.

Generation of polyclonal antibodies against recombinant human activin A

A goat antiserum to purified recombinant human activin A (rhAct-A), a dimer formed by two beta A-subunits of inhibin, has been produced. The immunoreactivity of the antiserum has been evaluated in an antigen coated enzyme-linked immunosorbent assay, in a radioimmunoassay using iodinated rhAct-A, and by Western blot analysis. The antiserum demonstrated some cross reactivity to inhibin A, a structurally related heterodimer which contains an identical beta A-subunit coupled to a distinct, though similar, alpha subunit. A simple radioimmunoassay for rhAct-A in tissue culture supernatant has been developed with rhAct-A affinity column purified polyclonal antiserum. The assay is precise and sensitive with a range of 0.31-40 ng/ml. The cross reactivity of inhibin A in the RIA is about 4.3%. Despite its cross-reactivity this antiserum will facilitate studies of the physiology of activin A and inhibin A which includes a Western blot analysis where a molecular size distinction is accomplished.