Glycolipid membrane anchored recombinant protein production from CHO cells cultGred on porous microcarriers

Recombinant proteins were harvested from Chinese hamster ovary (CHO) cells by a controlled release process, which increased the purity and concentration of the harvested protein. Recombinant human melano-transferrin (p97) was expressed linked to the outer surface of CHO cells by a glycosyl-phosphatidylinositol (GPI) membrane anchor. Cells were grown to confluence in T-flask culture, and the p97 harvested by replacing the growth medium for 30 min with phosphate-buffered saline (PBS) containing 10 mU/mL phosphatidylinositol-phospholipase C (PI-PLC). The GPI anchor was selectively cleaved by PI-PLC. In fresh medium, the CHO cells regained over 95% of their p97 expression within 40 h. The process was repeated for eight harvests. Harvested protein concentrations varied from 1.5 to 3.8 mug/mL due to difficulties in maintaining stable confluent T-flask cultures. Harvesting from cells growing on porous microcarriers was investigated to increase p97 product concentrations and to overcome culture stability problems. Semicontinuous cultures were maintained in spinners for up to 76 days with average bioreactor cell densities of over 10(7) cell/mL. The p97 was harvested at up to 100 mug/mL and 30% purity with protein production remaining stable for 4 harvest cycles. Production of high levels of p97 from CHO cells was maintained at 0.5% serum. (c) 1994 John Wiley & Sons, Inc.     

Membrane anchored protein production from spheroid, porous, and solid microcarrier chinese hamster ovary cell cultures

The influence of the microcarrier type on the performance of a controlled release process used to produce a recombinant glycosyl-phosphatidylinositol anchored protein was investigated. Chinese hamster ovary (CHO) cells expressing the human melanoma tumor antigen (p97) were cultured in 10% serum on Cultispher-GH porous microcarriers and then, for protein production, maintained in 2% serum. Cells were harvested every 48 h and p97 was recovered at 90 mug/mL and 40% purity. Harvested p97 concentrations were increased by harvestingfrom spheroid (241 mug/mL) and smaller porous microcarrier, Cultispher-G (167 mug/mL) cultures. The low total cell specific p97 production of cells cultured on Cultispher-GH was due to necrosis of cells within the beads, decreased p97 expression of the immobilized cells, dilution by the liquid (up to 40% volume) associated with settled beads, and incomplete recovery of p97 from within the beads. Cells cultured on solid microcarriers, Cytodex-1, had the highest cell viability and cell specific p97 production, It is recommended that a two-stage cyclic harvesting process of cells cultured on small Cultispher-G or on Cytodex-1 beads would minimize protein loss and maximize cell specific protein recovery. (c) 1995 John Wiley & Sons Inc.     

ESR studies on membrane fluidity of Chinese hamster ovary cells grown on microcarriers and in suspension

Electron spin resonance (ESR) spin label methods were used to study membrane fluidity of Chinese hamster ovary (CHO) cells grown on microcarriers and in suspension using 5-doxylstearic acid spin label as a probe. CHO cells grown on microcarriers had a more rigid cell membrane compared to CHO cells grown in suspension culture. CHO cells removed from the surface of the microcarriers by either trypsinization, EDTA treatment or osmotic shock had a membrane fluidity similar to that of CHO cells grown in suspension culture. Conversely, when the cells grown in suspension culture were attached and flattened on the surface of the microcarriers the fluidity decreased. Moreover, membrane fluidity of CHO cells grown on microcarriers changed as a function of the population density, whereas that of the cells in suspension did not. This implies that cell adhesion and/or cell-cell interactions influence the fluidity of the cell surface membrane.     

Controlled release process to recover heterologous glycosylphosphatidylinositol membrane anchored proteins from CHO cells

A semicontinuous process has been developed to recover heterologous proteins at increased concentrations and purities. Proteins attached to mammalian cell membranes by glycosylphosphatidylinositol (GPI) anchors can be selectively released into the supernatant by the enzyme phosphatidylinositol-phospholipase C (PI-PLC). Chinese hamster ovary (CHO) cells, genetically engineered to express the GPI anchored, human melanoma antigen (p97), were used as a model system. These cells were grown in protein containing growth medium. During a brief harvesting phase the medium was replaced by phosphate buffered saline (PBS) containing 10 mU/mL of PI-PLC and the GPI anchored protein was cleaved from the cell surface and recovered in soluble form at up to 30% purity. After harvesting, the cells were returned to growth medium where the protein was re-expressed within 40 h. The growth rate, viability, and protein production of cells, repeatedly harvested over a 44-day period, were not adversely affected. This continuous cyclic harvesting process allowed recovery of a heterologous protein at high purity and concentrations and could be applied to the recovery of other GPI anchored proteins and genetically engineered GPI anchored fusion proteins. (c) 1993 John Wiley & Sons, Inc.     

Production of a membrane-bound proteins,the human gamma-glutamyl transferase,by CHO cells cultivated on microcarriers,in aggregates and in suspension

Recombinant Chinese Hamster Ovary (CHO) cells, engineered for the production of human gamma-glutamyl transferase (GGT), have been grown on Cytodex 1 microcarriers, as aggregates, or as single cells in suspension after adaptation. GGT is a membrane bound enzyme which was not secreted during the culture period. The maximal enzyme activity was found to be directly related to the achieved maximal cell density. Culture of CHO on microcarriers yielded the fastest growth, with a specific growth rate of 0.04 h^–1, the highest cell density (near 1.3×10⁶ cells ml^–1), and the highest enzyme activity around 300 mU ml^–1, which corresponded to a specific cellular level of 20 mU 10^–5 cells. GGT could also be produced by growing CHO cells in suspension as single cells or as aggregates. Under these conditions, however, the specific CHO growth rate was significantly slower and the GGT level per cell was divided by a factor 6. Growing CHO cells without microcarriers also resulted in differences in cell metabolism, with a higher conversion yield of glutamine into ammonia, and a higher cell lysis. The catalytic kinetic constants of the enzyme were found identical for the three culture systems.     

Cultivation of mammalian cells on macroporous microcarriers.

Adherent cells can be cultivated in a stirred-tank bioreactor by attaching to microcarriers. Macroporous microcarriers, with their intraparticle space and surface area for cell growth, can potentially support a higher cell concentration than conventional microcarriers, which support cell growth only on the external surface. Chinese hamster ovary (CHO) cells and green monkey kidney (Vero) cells were cultivated on macroporous microcarriers, Cultispher-G. Cells attached to the microcarriers at a slow rate and grew to a high density. Thin sections of the microcarriers demonstrate that cells were initially on the exterior of the microcarriers and migrated into the interior as cell concentration increased. Vero cells cultivated on these microcarriers were successfully used for the production of vesicular stomatitis virus (VSV).     

Enhanced Production of Human Recombinant Proteins from CHO cells Grown to High Densities in Macroporous Microcarriers

Macroporous microcarriers entrap cells in a mesh network allowing growth to high densities and protect them from high shear forces in stirred bioreactor cultures. We report the growth of Chinese hamster ovary (CHO) cells producing either recombinant human beta-interferon (β-IFN) or recombinant human tissue-plasminogen activator (t-PA) in suspension or embedded in macroporous microcarriers (Cytopore 1 or 2). The microcarriers enhanced the volumetric production of both β-IFN and t-PA by up to 2.5 fold compared to equivalent suspension cultures of CHO cells. Under each condition the cell specific productivity (Q _P) was determined as units of product/cell per day based upon immunological assays. Cells grown in Cytopore 1 microcarriers showed an increase in Q _P with increasing cell densities up to a threshold of >1 × 10⁸ cells/ml. At this point the specific productivity was 2.5 fold higher than equivalent cells grown in suspension but cell densities above this threshold did not enhance Q _P any further. A positive linear correlation (r ² = 0.93) was determined between the specific productivity of each recombinant protein and the corresponding cell density for CHO cells grown in Cytopore 2 cultures. With a cell density range of 25 × 10⁶ to 3 × 10⁸ cells/ml within the microcarriers there was a proportional increase in the specific productivity. The highest specific productivity measured from the microcarrier cultures was ×5 that of suspension cultures. The relationship between specific productivity and cell density within the microcarriers leads to higher yields of recombinant proteins in this culture system. This could be attributed to the environment within the microcarrier matrix that may influence the state of cells that could affect protein synthesis or secretion.     

Bead-to-bead transfer of Vero cells in microcarrier culture

Cell harvesting technique is of considerable importance in the scale-up of microcarrier culture of anchorage-dependent cells. The traditional methods are often time- and labor-consuming and cause physiological damage to the cells. Bead-to-bead cell transfer provides an attractive solution to the scale up process. By intermittent agitation, successful cell transfer was achieved. Significant cell growth was observed where bare beads contacted with confluent ones. Most of the fresh microcarriers reached near confluence four days after addition into the culture medium.     

Simultaneous enhancement of cell proliferation and thermally induced harvest efficiency based on temperature-responsive cationic copolymer-grafted microcarriers

The development of large-scale suspension cell cultures using microcarriers has long been a focus of attention in the fields of pharmacy and biotechnology. Previously, we developed cell-detachable microcarriers based on temperature-responsive poly(N-isopropylacrylamide) (PIPAAm)-grafted beads, on which adhering cells can be noninvasively harvested by only reducing the temperature without the need for proteolytic enzyme treatment. In this study, to improve the cell harvest efficiency from bead surfaces while maintaining cell adhesion and proliferation properties, we prepared temperature-responsive cationic copolymer-grafted beads bearing a copolymer brush consisting of IPAAm, positively charged quaternary amine monomer (3-acrylamidopropyl trimethylammonium chloride; APTAC), and hydrophobic monomer (N-tert-butylacrylamide; tBAAm). The incorporation of positively charged APTAC into the grafted copolymer brush facilitated bead dispersibility in a cell culture system containing Chinese hamster ovary (CHO-K1) cells and consequently allowed for enhanced cell proliferation in the system compared to that of unmodified CMPS and conventional PIPAAm homopolymer-grafted beads. Additionally, P(IPAAm-co-APTAC-co-tBAAm) terpolymer-grafted beads exhibited the most rapid and efficient cell detachment behavior after the temperature was reduced to 20 °C, presumably because the highly hydrated APTAC promoted the overall hydration of the P(IPAAm-co-APTAC-co-tBAAm) chains. Therefore, P(IPAAm-co-APTAC-co-tBAAm) terpolymer-grafted microcarriers are effective in facilitating both cell proliferation and thermally induced cell detachment in a suspension culture system.     

Expression of cell surface GPI-anchored human p97 in baculovirus-infected insect cells

The baculovirus/insect cell system (Autographa californica multiple nuclear polyhedrosis virus/Spodoptera frugiperda Sf9 cells) was used to express the GPI-anchored human melanoma tumor antigen, melanotransferrin or p97. This system served to study the expression and productivity of recombinant GPI-anchored p97 by insect cells. The Sf9 cells expressed a cell surface GPI-anchored form of p97 as well as a soluble form of p97 that did not appear to be derived from the GPI-anchored form of p97. Both recombinant forms, although Endo H resistant, migrated slightly faster ( approximately 88 kDa) than the native p97 ( approximately 95-97 kDa). The insect GPI-anchored p97 was sensitive to PI-PLC, which exposed a detectable cross-reacting determinant. The Sf9 cell surface p97 expression was similar to that of human melanoma (SK-MEL-28) cells, whereas the Sf9 cell specific secretion rate was 10-fold higher. Also Sf9 cells retained considerably higher levels of p97 within the cell. The Sf9 cell surface expression of p97 varied with time after infection, with the maximum expression, which appeared independent of multiplicities of infection greater than 1, occurring at 48 h. After 48 h, levels of cell surface and secreted p97 fell whereas p97 retained within the cell increased, which possibly reflected the lytic nature of the expression system. The successful expression of GPI-anchored human p97 by the baculovirus/insect cell system not only provides a source of p97 for further research but also is the basis of an alternative method for the commercial production of GPI-anchored proteins.     

Bead-to-bead transfer of Vero cells in microcarrier culture

Cell harvesting technique is of considerable importance in the scale-up of microcarrier cultures of anchorage-dependent cells. The traditional methods are often time- and labor-consuming and cause physiological damage to the cells. Bead-to-bead cell transfer provides an attractive solution to the scale up process. By intermittent agitation, successful cell transfer was achieved. Significant cell growth was observed where bare beads contacted with confluent ones. Most of the fresh microcarriers reached near confluence four days after addition into the culture medium. This revised version was published online in July 2006 with corrections to the Cover Date.     

Scalable inoculation strategies for microcarrier-based animal cell bioprocesses

Scalability is a major demand for high-yield, stable bioprocess systems in animal cell culture-based biopharmaceutical production. Increased yields can be achieved through high-density cell culture, such as in the combination of microcarrier and fluidized bed bioreactor technology. To minimize inocula volume in industrial applications of fluidized bed fermentation systems, it is crucial to increase the bed volume in the reactor during the fermentation process. We tested scale-up strategy for the production of recombinant human arylsulfatase B (ASB) enzyme used in enzyme replacement therapy in patients afflicted with mucopolysaccharidosis type VI (MPS VI). This enzyme was derived from Chinese hamster ovary (CHO) cells cultivated as adherent cell culture on Cytoline macroporous microcarriers (Amersham Biosciences, Uppsala, Sweden) using a Cytopilot Mini fluidized bed bioreactor (FBR; Amersham Biosciences, Vogelbusch, Austria). Both 1:2 expansion (herein referred to as the addition of fresh, not-yet-colonized microcarriers) and 1:6 expansion of the carrier bed were performed successfully; the cells restarted to proliferate for colonizing these newly added carriers; and the stability of the culture was not negatively affected.     

Production and glycosylation of recombinant beta-interferon in suspension and cytopore microcarrier cultures of CHO cells

Microcarriers are suitable for high-density cultures of cells requiring surface attachment and also offer the advantage of easy media removal for product recovery. We have used the macroporous microcarriers Cytopore 1 and 2 for the growth of CHO cells producing recombinant human beta-interferon (beta-IFN) in stirred batch cultures. Although these cells may grow in suspension, in the presence of Cytopore microcarriers they become entrapped in the inner bead matrix where they can be maintained at high densities. Cell growth rates were reduced in microcarrier cultures compared to suspension cultures. However, the beta-IFN yield was up to 3-fold greater as a result of an almost 5-fold higher specific productivity. Maximum productivity was found in cultures containing 1.0 mg/mL of Cytopore 1 or 0.5 mg/mL of Cytopore 2 with a cell/bead ratio of 1029 and 822, respectively. Beta-IFN molecules aggregated in the later stages of all cultures, causing a decrease in response by ELISA. However, the degree of aggregation was significantly less in the microcarrier cultures. The N-linked glycans from beta-IFN were isolated and analyzed by normal phase HPLC. There was no apparent difference in the profile of glycans obtained from each of the suspension and Cytopore culture systems. This suggests that Cytopore microcarriers may be useful in bioprocess development for enhanced recombinant glycoprotein production without affecting the glycosylation profile of the protein.     

Differences in the expression and localization of human melanotransferrin in lepidopteran and dipteran insect cell lines

The ability of several lepidopteran and dipteran insect cell lines to express human melanotransferrin (p97), a glycosyl phosphatidylinositol (GPI)-anchored, iron-binding sialoglycoprotein, was assessed. Spodoptera frugiperda-derived (Sf9) cell lines, transformed with the p97 gene under control of a baculovirus immediate-early promoter, were able to constitutively express the protein and correctly attach it to the outer cell membrane via a GPI anchor as demonstrated by PI-PLC treatment. In contrast, stable constitutive expression could not be demonstrated with cell lines derived from either Drosophila melanogaster (Kc1 or SL2) or Lymantria dispar (Ld652Y) despite the observation that p97 could be detected in transient expression assays. This may indicate that the long-term expression and accumulation of p97 is inhibitory to Drosophila cells, possibly due to improper localization of the protein and resultant competition for cellular iron. In stably transformed Sf9 cells, p97 was expressed on the cell at a maximal level of 0.18 microg/10(6) cells and was secreted at a maximal rate of 9.03 ng/10(6) cells/h. This level was comparable to the amount expressed with the baculovirus system (0.37 microg/10(6) cells and 31.2 ng/10(6) cells/h) and transformed CHO cells (0.88 microg/10(6) cells and 7.8 ng/10(6) cells/h). Deletion of the GPI cleavage/attachment site resulted in an eightfold increase in the secretion rate of p97, when compared to the intact construct suggesting that the rate-limiting step involves processing of the GPI anchor.     

Bioreactor-Based Online Recovery of Human Progenitor Cells with Uncompromised Regenerative Potential: A Bone Tissue Engineering Perspective

The use of a 3D perfusion culture environment for stem cell expansion has been shown to be beneficial for maintenance of the original cell functionality but due to several system inherent characteristics such as the presence of extracellular matrix, the continued development and implementation of 3D perfusion bioreactor technologies is hampered. Therefore, this study developed a methodology for harvesting a progenitor cell population from a 3D open porous culture surface after expansion in a perfusion bioreactor and performed a functional characterization of the expanded cells. An initial screening showed collagenase to be the most interesting reagent to release the cells from the 3D culture surface as it resulted in high yields without compromising cell viability. Subsequently a Design of Experiment approach was used to obtain optimized 3D harvest conditions by assessing the interplay of flow rate, collagenase concentration and incubation time on the harvest efficiency, viability and single cell fraction. Cells that were recovered with the optimized harvest protocol, by perfusing a 880 U/ml collagenase solution for 7 hours at a flow rate of 4 ml/min, were thereafter functionally analyzed for their characteristics as expanded progenitor cell population. As both the in vitro tri-lineage differentiation capacity and the in vivo bone forming potential were maintained after 3D perfusion bioreactor expansion we concluded that the developed seeding, culture and harvest processes did not significantly compromise the viability and potency of the cells and can contribute to the future development of integrated bioprocesses for stem cell expansion.     

Formation of bridges and large cellular clumps in CHO-cell microcarrier cultures: Effects of agitation dimethyl sulfoxide and calf serum

We have investigated conditions that inhibit the tendency of CHO K1 cells to form cellular bridges between microcarriers and dense clumps of cellular overgrowth in microcarrier cultures. Microcarrier aggregation by cellular bridge formation was found to occur only during periods of rapid cell growth. The level of microcarrier aggregation decreased with increasing agitation intensity. Dense masses of cellular overgrowth formed inside bridges connecting the microcarriers and in clumps that protruded off the microcarrier surface. To replace cells that were continuously sheared from the microcarriers, cell growth occurred preferentially in areas of overgrowth after confluent microcarriers were maintained in a serum-free medium. This ultimately led to poor surface coverage as bare spots developed on the microcarrier away from the areas of dense cellular overgrowth. The development of bare spots was inhibited when confluent microcarriers were maintained in medium supplemented with 1% serum. The development of cellular overgrowth was inhibited by dimethyl sulfoxide. Thus, maintaining confluent microcarriers in medium supplemented with 1% dimethyl sulfoxide and 1% calf serum resulted in microcarriers that appeared similar to monolayer cultures. There was also a decrease in bridging in cultures supplemented with either 1% calf serum or 1% dimethyl sulfoxide/1% calf serum compared to serum-free cultures.     

Induction of osteogenic differentiation of osteoblast-like cells MG-63 during cultivation on fibroin microcarriers

We have developed microcarriers made from silk fibroin. Microcarriers can be used as a substrate for cell cultivation and cell delivery during cell-based therapy and for the construction of bioengineered tissue. Fibroin microcarriers were mineralized, which led to the appearance of calcium phosphate crystals on their surface. The ability of mineralized and nonmineralized microcarriers to support osteogenic differentiation of the osteoblast-like cell line MG-63 was estimated by alkaline phosphatase activity, an early marker of bone formation. The experiment showed cells actively proliferating on the surface of both mineralized and nonmodified microcarriers. Culturing MG-63 on the surface of fibroin microcarriers resulted in an increase of alkaline phosphatase activity indicative of osteogenic differentiation of MG-63 cells in the absence of inductors. The level of alkaline phosphatase was higher when mineralized microcarriers were used. Alkaline phosphatase activity of MG-63 cells cultivated using traditional two-dimensional approaches were close to zero. As opposed to conventional monolayer culturing, microcarrier culture cells are in a three-dimensional environment that is closer to physiological conditions. This can have a significant impact on their morphology and functional properties. During this study, we also characterized mechanical properties of porous scaffolds used for microcarriers.     

Plant protein hydrolysates support CHO-320 cells proliferation and recombinant IFN-γ production in suspension and inside microcarriers in protein-free media

We have recently developed a protein-free medium (PFS) able to support the growth of Chinese hamster ovary (CHO) cells in suspension. Upon further supplementation with some plant protein hydrolysates, medium performances reached what could be observed in serum-containing media [Burteau et al. In Vitro Cell. Dev. Biol.-Anim. 39 (2003) 291]. Now, we describe the use of rice and wheat protein hydrolysates, as non-nutritional additives to the culture medium to support productivity and cell growth in suspension or in microcarriers. When CHO-320 cells secreting recombinant interferon-gamma (IFN-γ) were cultivated in suspension in a bioreactor with our PFS supplemented with wheat hydrolysates, the maximum cell density increased by 25% and the IFN-γ secretion by 60% compared to the control PFS. A small-scale perfusion system consisting of CHO-320 cells growing on and inside fibrous microcarriers under discontinuous operation was first developed. Under these conditions, rice protein hydrolysates stimulated recombinant IFN-γ secretion by 30% compared to the control PFS. At the bioreactorscale, similar results were obtained but when compared to shake-flasks studies, nutrients, oxygen or toxic by-products gradients inside the microcarriers seemed to be the main limitation of the system. An increase of the perfusion rate to maintain glucose concentration over 5.5 mM and dissolved oxygen (DO) at 60% was able to stimulate the production of IFN-γ to a level of 6.6 μg h⁻¹ g⁻¹ of microcarriers after 160 h when a cellular density of about 4 × 10⁸ cell g⁻¹ of carriers was reached.     

Calcium-alginate gel bead cross-linked with gelatin as microcarrier for anchorage-dependent cell culture

Valuable products obtainedfrom the cultivation of anchorage-dependent mammalian cells require large-scale processes to obtain commercially useful quantities. It is generally accepted that suspension culture is the ideal mode of operation. Because anchorage-dependent cells need surfaces to be able to attach and spread, the incorporation of microcarriers to suspension culture is indispensable. Since the dextran-based microcarrier wasfirst introduced, many different types of microcarriers have been developed and commercialized. In this study, alginate-based microcarriers were made in the following order: (i) calcium-alginate gel beads prepared by dropping a blend of sodium alginate and propylene glycol alginate (PGA) into calcium chloride solution, (ii) the PGA section of gel beads cross-linked with gelatin in alkaline solution (i.e., via the transacylation reaction between the ester group of PGA and amino group of gelatin), and (iii) gelatin membrane around the beads further cross-linked by glutaraldehyde. The glutaraldehyde-treated gelatintransacylated PGA/alginate microcarrier showed superior features in high stability under phosphate-containing solution, density close to that of culture medium, and transparency. Moreover, the Chinese hamster ovary CHO-KI and amphotropic retrovirus producer PA317 cells cultivated on the newly synthesized microcarriers exhibited similar growth kinetics of these two types of cell lines cultured on commercial polystyrene microcarriers. However, cell morphology was easily monitored on the transparent microcarriers made in this study.     

磷脂酰肌醇特异性磷脂酶C的异源表达和应用

旨在研究大肠杆菌产磷脂酰肌醇特异性磷脂酶C(PI-PLC)的发酵表达和分离纯化,探究PI-PLC酶切GPI锚定蛋白的效果。依据NCBI数据库中蜡样芽孢杆菌的PI-PLC的基因序列,按照大肠杆菌的密码子偏好性进行密码子优化,合成相应基因序列并构建基因表达载体pGEX-6P-1-PI-PLC。将重组质粒转入受体菌E.coli BL21(DE3)中,通过加入异丙基硫代-β-D-半乳糖苷(IPTG)诱导目的基因PI-PLC表达。经检测,含有GST标签的PI-PLC融合蛋白以可溶蛋白形式存在于菌体破碎上清,分子量约为61 kDa,与预期相符。初步优化诱导表达条件后,发现最佳诱导表达条件为:以接种量5%接种体积分数接种,待菌体生长至OD600nm达到0.5,在16℃条件下以0.3 mmol/L浓度IPTG诱导24 h。利用GST标签对蛋白进行纯化,纯化后的PI-PLC质量浓度为0.52 mg/mL,比酶活为1322.5 U/mg。利用PI-PLC酶液对哺乳动物细胞表面的模式GPI锚定蛋白CD59进行酶切,酶切作用显著。因此,下一步可以将PI-PLC融合蛋白应用于细胞生物学中对GPI-APs的研究和鉴定。