High-Throughput Process Development: I-Process Chromatography.

Chromatographic separation serves as "a workhorse" for downstream process development and plays a key role in the removal of product-related, host-cell-related, and process-related impurities. Complex and poorly characterized raw materials and feed material, low feed concentration, product instability, and poor mechanistic understanding of the processes are some of the critical challenges that are faced during the development of a chromatographic step. Traditional process development is performed as a trial-and-error-based evaluation and often leads to a suboptimal process. A high-throughput process development (HTPD) platform involves the integration of miniaturization, automation, and parallelization and provides a systematic approach for time- and resource-efficient chromatographic process development. Creation of such platforms requires the integration of mechanistic knowledge of the process with various statistical tools for data analysis. 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 the HTPD of chromatography steps. It describes the operation of a commercially available device (PreDictor™ plates from GE Healthcare). This device is available in 96-well format with 2 or 6 µL well size. We also 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 0.93). We think that this protocol will be of significant value to those involved in performing the high-throughput process development of the chromatography process.

Integrated continuous processing of proteins expressed as inclusion bodies: GCSF as a case study.

Affordability of biopharmaceuticals continues to be a challenge, particularly in developing economies. This has fuelled advancements in manufacturing that can offer higher productivity and better economics without sacrificing product quality in the form of an integrated continuous manufacturing platform. While platform processes for monoclonal antibodies have existed for more than a decade, development of an integrated continuous manufacturing process for bacterial proteins has received relatively scant attention. In this study, we propose an end-to-end integrated continuous downstream process (from inclusion bodies to unformulated drug substance) for a therapeutic protein expressed in Escherichia coli as inclusion body. The final process consisted of a continuous refolding in a coiled flow inverter reactor directly coupled to a three-column periodic counter-current chromatography for capture of the product followed by a three-column con-current chromatography for polishing. The continuous bioprocessing train was run uninterrupted for 26 h to demonstrate its capability and the resulting output was analyzed for the various critical quality attributes, namely product purity (>99%), high molecular weight impurities (<0.5%), host cell proteins (<100 ppm), and host cell DNA (<10 ppb). All attributes were found to be consistent over the period of operation. The developed assembly offers smaller facility footprint, higher productivity, fewer hold steps, and significantly higher equipment and resin utilization. The complexities of process integration in the context of continuous processing have been highlighted. We hope that the study presented here will promote development of highly efficient, universal, end-to-end, fully continuous platforms for manufacturing of biotherapeutics. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:998-1009, 2017.

Mammalian cell production and purification of progenipoietin, a dual-agonist chimaeric haematopoietic growth factor.

One member of the progenipoietin (ProGP) family of engineered proteins, ProGP-2, is a chimaeric dual cytokine receptor agonist, expressed in mammalian cells, that stimulates both human fetal liver tyrosine kinase-3 (Flt3) and the granulocyte-colony-stimulating-factor (G-CSF) receptor. The production of ProGP-2 on a small and large scale using either anti-(Flt3 ligand) antibody-affinity chromatography, or a combination of (NH4)2SO4 fractionation, anion-exchange chromatography, hydrophobic-interaction chromatography and preparative reverse-phase chromatography is described. ProGP-2 was produced in hollow-fibre reactors containing stably transfected NS0 cells. The productivity of ProGP-2 was initially high, but was found to decrease 3-4-fold over time. When the yield of ProGP-2 decreased, the combination of three conventional chromatography steps was required to meet protein purity similar to that achieved by the anti-(Flt3 ligand) chromatography method. In addition, a protease activity was observed in conditioned media from the hollow-fibre reactors that resulted in increased degradation of ProGP-2 that was removed by hydrophobic-interaction chromatography at higher pH. Together the results demonstrated a method for production and purification of ProGP-2 for additional studies on its haematopoietic activity.

Expression, purification, and characterization of the active immunoglobulin-like domain of human granulocyte-colony-stimulating factor receptor in Escherichia coli.

We succeeded in the expression, purification, and refolding of the immunoglobulin-like (Ig) domain of human granulocyte-colony-stimulating factor (G-CSF) receptor with amino-terminal His-tag in Escherichia coli. The refolded Ig domain bound to a G-CSF affinity column and could be eluted with free G-CSF as a receptor-ligand complex, demonstrating that the Ig domain has the information necessary for binding its ligand, G-CSF. The eluted His-Ig/G-CSF complex could be separated from excess G-CSF by Ni-NTA column chromatography. The yield of this active recombinant His-Ig protein is about 0.72 mg per liter of culture. Its small size and the ease of production make this receptor fragment a useful reagent for the structural analysis of its complex with G-CSF.

Expression and purification of cytokine receptor homology domain of human granulocyte-colony-stimulating factor receptor fusion protein in Escherichia coli.

Direct expression of the cytokine receptor homology (CRH) domain of granulocyte-colony-stimulating factor (G-CSF) receptor is lethal to Escherichia coli. For the efficient and stable production of an active CRH domain in E. coli, we fused the CRH domain with different proteins, such as maltose-binding protein (MalE), glutathione S-transferase, and thioredoxin (Trx). Among these, Trx appeared to be the best in terms of the protein expression level, purification efficiency by affinity chromatography, and binding activity to its ligand, G-CSF. The yield of active Trx-CRH fusion protein increased about 200-fold compared to that of previously reported MalE-CRH fusion.

Purification, crystallization and preliminary X-ray analysis of a complex between granulocyte colony-stimulating factor and its soluble receptor.

Crystals of the complex between granulocyte colony-stimulating factor and its soluble receptor were obtained by a vapour-diffusion method using ammonium sulfate as a precipitant. Addition of 1, 4-dioxane was critical in order to grow the crystals to sufficient sizes. Cryoprotection was essential in order to collect diffraction data at atomic resolution. Two kinds of crystal forms were obtained depending on the cryoprotectants. In a cryosolvent with the same salt concentration as in the crystallization conditions, the crystal belonged to the space group I4(1)22. At higher salt concentrations, the crystal was converted to a different space group P4(1)2(1)2 (P4(3)2(1)2) with the same unit-cell parameters.

Expression and purification of cytokine receptor homology domain of human granulocyte-colony stimulating factor receptor in Escherichia coli.

In an attempt to generate a stable non-glycosylated cytokine receptor homology (CRH) domain (Tyr97-Ala309) of human granulocyte-colony stimulating factor (G-CSF) receptor, two free cysteines in the CRH domain were converted to serine by site-directed mutagenesis. Taking advantage of the tight regulation for the expression of T7 RNA polymerase, the mutated CRH domain was successfully expressed in Escherichia coli (E. coli) with a pelB signal sequence at its NH2-terminus and with a His tag at its COOH-terminus. The processed and secreted CRH domain after solubilization and in vitro refolding retained G-CSF binding activity, and its yield (approximately 40 micrograms/30 ml culture) was more than 100-fold higher than that of the mouse CRH domain expressed by the MalE fusion system in E. coli.

The Src-like tyrosine kinase Hck is activated by granulocyte colony-stimulating factor (G-CSF) and docks to the activated G-CSF receptor.

Activation of the granulocyte colony-stimulating factor receptor (G-CSF-R) leads to tyrosine-phosphorylation of multiple cytoplasmic components. To date, the kinases Jak1, Jak2, Tyk2, Lyn, and Syk have been implicated in this process. However, it is unknown if other kinases might be involved in the diverse responses from the G-CSF-R, which include mitogenesis, survival, differentiation, and functional activation of responsive cells. The hematopoietic cell kinase (Hck) is a member of the Src-family of kinases known to be expressed in cells of the granulocytic lineage. It also interacts with the gp130 subunit of the LIF/IL-6 receptors, which is closely related to the G-CSF-R, and so represents a good candidate for mediating at least some of the downstream signaling from the G-CSF-R. Therefore, we investigated the activation of Hck by the G-CSF-R in intact cells as well as in vitro. These studies revealed recruitment of Hck to activated G-CSF-R, mediated by direct binding via its SH2 domain to multiple phosphotyrosines of the receptor. In addition, we show that Hck becomes activated upon G-CSF treatment and is, in turn, able to phosphorylate the G-CSF-R, indicating a clear functional and physical involvement in G-CSF signaling.

Coexpression of G-CSF with an unglycosylated G-CSF receptor mutant results in secretion of a stable complex.

Previously, we have shown that the entire extracellular domain of the granulocyte-colony stimulating factor receptor (sG-CSFr) produced in Chinese hamster ovary (CHO) cells forms a stable complex with its ligand G-CSF, at a stoichiometry of 2:2. A truncated receptor molecule consisting of the cytokine receptor homology domain and N-terminus Ig-like domain (Ig CRH) behaves quite similarly. Both of these forms of the receptor are highly glycosylated. To address the importance of glycosylation toward receptor activity and stability, and possibly obtain nonglycosylated receptor for crystallization, mutations were made to replace four Asn residues which are N-glycosylated in the truncated receptor. Virtually no receptor was recovered from conditioned media of CHO cells transfected with this mutant construct, although a high-level of mRNA coding for receptor was detected; this mRNA was translated as determined by Western blots of cell lysates. These results indicate that the translated product is apparently not secreted from these cells. Cells transfected with mutant receptor cDNA were cotransfected with a cDNA construct expressing G-CSF in which the single O-glycosylation site was eliminated by mutation. Upon fermentation of the cotransfectants, we observed a large amount of receptor-ligand complex in the conditioned media. The purified unglycosylated complex appeared to be of the same binding stoichiometry and approximate binding affinity as that of complex formed by addition of purified ligand and unmutated receptor. These results show that while glycosylation of sG-CSFr is not necessary for ligand binding, it appears to be crucial in folding and export from the cell.

Swapping between Fas and granulocyte colony-stimulating factor receptor.

Fas belongs to the tumor necrosis factor/nerve growth factor receptor family. The Fas ligand binds to its receptor, Fas, and induces apoptosis in Fas-bearing cells. The granulocyte colony-stimulating factor receptor (G-CSFR) is a member of the hemopoietic growth factor receptor family. G-CSF induces its dimerization and regulates the proliferation and differentiation of neutrophilic granulocytes. We constructed hybrid receptors between Fas and G-CSFR and expressed them in the mouse T cell line WR19L or the mouse myeloid interleukin-3-dependent FDC-P1 cell line. The Fas ligand or an agonistic anti-Fas antibody stimulated proliferation of the FDC-P1 transformants expressing a chimera consisting of the Fas extracellular and G-CSFR cytoplasmic regions. On the other hand, G-CSF could not induce apoptosis in the transformants expressing the chimera consisting of the G-CSFR extracellular and Fas cytoplasmic regions, but these cells were killed by a polyclonal antibody against G-CSFR. These results indicated that receptors belonging to different receptor families can be functionally exchanged and confirm that a homodimer of G-CSFR can transduce the growth signal, whereas Fas must be oligomerized (probably trimerized) to transduce the apoptotic signal.

Dimerization of the extracellular domain of granuloycte-colony stimulating factor receptor by ligand binding: a monovalent ligand induces 2:2 complexes.

Granulocyte-colony stimulating factor (G-CSF) binds to a specific cell surface receptor and induces signals for growth and differentiation in cells of granulocyte hematopoietic lineage. In order to understand how G-CSF binding initiates signals into these cells, we have studied its interactions with the entire extracellular domain of the receptor (sG-CSFR). The sG-CSFR was purified from CHO cell conditioned media with a G-CSF affinity column, resting in a preparation fully competent for ligand binding. However, when sG-CSFR was purified by conventional means, i.e., without affinity chromatography, only about half was competent. Therefore, all studies were carried out using affinity-purified material. The sG-CSFR exhibited a weak self-association into a dimer with a dissociation constant of 200microM in the absence of G-CSF. Addition of G-CSF dimerizes the receptor, with a preferred stoichiometry of 2 G-CSF molecules plus 2 receptors. Unexpectedly, receptor-receptor interactions rather than through two receptors binding to the same G-CSF molecule; i.e., G-CSF is a monovalent ligand. G-CSF binding to the receptor monomer occurs with high affinity. The binding of G-CSF also enhances the receptor-receptor dimerization; when G-CSF is bound to both receptors, dimerization is enhanced 2000-fold, while the interaction of a 1:1 receptor-ligand complex with a second ligand-free receptor is enhanced 80-fold. Thus, the mechanism of receptor dimerization is fundamentally different than that of related cytokine receptors such as growth hormone and erythropoietin receptors. Circular dichroic spectra showed a small but significant conformational change of receptor upon binding G-CSF. This is consistent with the idea that G-CSF binding alters the conformation of the receptor, resulting in an increase in receptor-receptor interactions.

Evidence for the ligand-induced conversion from a dimer to a tetramer of the granulocyte colony-stimulating factor receptor.

An extracellular portion of granulocyte colony-stimulating factor (G-CSF) receptor, which contains an immunoglobulin-like (Ig) domain and cytokine receptor homologous (CRH) region, was secreted into the medium using Trichoplusia ni-Autographa californica nuclear polyhedrosis virus system. The gene product was purified to homogeneity mainly as a dimer (85 kDa) using G-CSF affinity column chromatography and gel filtration HPLC, although the product existed as a monomer (45 kDa) in the medium. Scatchard analyses suggested that only the dimer had high affinity ligand binding (Kd = about 100 pM), which is comparable with the Kd value of the cell surface receptor. The binding of G-CSF to Ig-CRH induced its tetramerization (200-250 kDa). The molecular composition of the tetrameric complex showed a stoichiometry of four ligands bound to four Ig-CRH. These results suggested that the oligomeric mechanism of the G-CSF receptor differs from that reported for growth hormone (GH) receptor, although CD spectrum spectroscopy suggested that the Ig-CRH has a GH receptor-like structure.

[Granulopoietic inhibitor].

The differentiation and proliferation of granulocytes are specifically supported by G-CSF. On the other hand, little is known about the negative modulation of granulopoiesis. We had found that the sera obtained from patients during the recovery phase of hematopoiesis after chemotherapy inhibited the formation of CFU-G, suggesting the modulation of granulopoiesis by negative regulator(s). As the inhibitory effect was specific for CFU-G, the inhibitor in this inhibitory sera was suggested the soluble G-CSF receptor. Several G-CSF receptor fragments were detected in the concentrated fractionated-urine by the immunoblot analysis using the specific antibody against the cytokine receptor homologous (CRH) domain of G-CSF receptor. G-CSF binding protein was successfully obtained from normal human plasma by G-CSF affinity column. Major component was the 90Kd of protein which was thought to be the extracellular domain of G-CSF receptor by the immunoblot analysis. About 90Kd- and 30Kd- bands of the binding-complex formation between G-CSF monomer and the extracellular domain or the fragment of G-CSF receptor were shown in the immunoblot analysis using anti-G-CSF Ab. CFU-G formation was suppressed by the solution of soluble G-CSF receptor fragments, suggesting the competition of G-CSF which bind to granulocyte precursor cells.