Production of monoclonal antibodies for breast cancer by HB8696 hybridoma cells using novel perfusion system.

Perfusion culture using spinfilters have been used for the production of health-care products using mammalian cells culture. However, available spinfilters are either highly prone to clog and/or are disposable and hence affects product formation. To address these problems, a novel non-woven Bombyx mori silk screen based spinfilter module for clog-free extended perfusion culture of hybridoma cells has been designed. The module is versatile in nature and reusable, after autoclaving and replacement of used polymeric membrane. Its application for clog-free extended perfusion culture was demonstrated by comparative perfusion experiments of HB8696 cells with stainless-steel spinfilter. HB8696 cells produce monoclonal antibodies (MAbs) 520C9 active against breast cancer oncoprotein. Silk spinfilter was found to be less prone to clog with cells and debris owing to its negatively charged hydrophobic screen compared to the positively charged hydrophilic stainless-steel spinfilter. Therefore, it provides extended cell growth phase and production phase of up to 56 h and 40 h respectively and 57.4% increase in MAb productivity compared to the stainless-steel spinfilter. The effect of different perfusion rates on MAb production was studied and an optimal MAb productivity of 1.6 g L(-1) day(-1) was achieved.

Silk screen based dual spin-filter module for perfusion culture of adherent and non-adherent mammalian cells.

Spin-filters have been primarily used for producing therapeutic proteins from mammalian cells. However, disposability and/or high filter clogging of the existing spin-filter systems affect the process economy and productivity. Hence, to address these drawbacks a reusable dual spin-filter module for perfusion culture of adherent and non-adherent mammalian cells was designed. Two non-woven Bombyx mori silk layers were used as filter screen; the outer layer was conducive to cell attachment whilst the inner was non-conducive. Adherent cells can be cultured either in suspended mode using its inner single module or as monolayer of cells using its dual concentric module. We achieved 30 % higher urokinase productivity as compared to the stainless-steel spin-filter during perfusion experiments of adherent human kidney cells in suspended mode. This was due to the hydrophobic and negatively-charged silk screen that allows clog-free perfusion culture for prolonged periods.

Amino acid supplementation enhances urokinase production by HT-1080 cells.

Medium optimization is an important strategy that can lead to several fold increase in the production of proteins in cell culture. However, the usual methods of medium optimization are complex and time consuming. Urokinase is a widely employed thrombolytic drug for the treatment of stroke. We describe here medium optimization for maximizing urokinase production by HT-1080 cells using supplementation of specific amino acids. The new specifically designed method resulted in 240 % increase in urokinase productivity.

Development of optimal medium for production of commercially important monoclonal antibody 520C9 by hybridoma cell.

Hybridoma HB-8696 produces monoclonal antibody (mAb) 520C9 (mouse IgG(1)), which recognizes breast cancer oncoprotein c-erbB2. The objective of this study was to optimize the medium recipe of HB 8696 cell for production of mAb 520C9. The optimization consisted of two steps: (1) screening of significant nutrients to make subsequent experiments more efficient with less runs and (2) locating their optimal concentrations. 29 variables including essential and non-essential amino acids, glucose, serum and 6 salts, namely NaCl, KCl, CaCl(2), NaH(2)PO(4), MgSO(4) and Na-pyruvate were chosen in screening phase. The Plackett-Burman method was used to screen the variables influencing mAb production. Seven factors namely glucose, serum, asparagine, threonine, serine, NaCl and NaH(2)PO(4) were identified to have a positive influencing role on mAb production with a confidence level >90 % (p < 0.1). Finally, Response surface methodology revealed the optimal level of the variables. The mAb production and average specific mAb production rate were enhanced by 111.05 and 105 %, respectively, compared to control medium.

Step-up/step-down perfusion approach for increased mAb 520C9 production by a hybridoma cell line.

The hybridoma cell line, HB-8696, produces a monoclonal antibody, 520C9 (mouse IgG(1)) that recognizes the breast cancer oncoprotein, c-erbB2. The effect of perfusion rate (volume of fresh feed/working volume of reactor/day) on cell growth and mAb production was investigated but perfusion at a constant rate and at an arbitrarily increased rate could not maintain exponential cell growth or a higher specific mAb production rate. An optimum step-up/step-down perfusion strategy is therefore proposed for maintaining a steady state production phase at high cell density for ten days. The optimum step-up perfusion could achieve fast cell growth by avoiding any nutrient limited condition and the following optimum step-down perfusion could potentially maintain high live cell density and reduced product dilution as well. The maximum viable cell achieved under optimum perfusion strategy was 2.3 × 10(7) cells/ml which was 19-fold higher than in optimum batch culture. The mAb yield and volumetric productivity were significantly improved to 52 and 50 mg/l day compared to 25 and 3.8 mg/l day in optimum batch, respectively, and could be maintained for up to ten days.

Effect of low culture temperature on urokinase production in hollow fiber reactor.

The effect of culture temperature on urokinase production by HT-1080 cell line was studied in batch culture and hollow fiber reactor. Small-scale t-flask experiments revealed that urokinase production could be enhanced and media utilization could be reduced by lowering the culture temperature in production phase. Urokinase production was scaled up using a hollow fiber perfusion reactor system. Temperature of culture was maintained at the physiological 37 °C during growth phase that extended up to 12 days in hollow fiber bioreactor. Subsequently, in the production phase, culture temperature was lowered to 34 °C. Decrease in culture temperature resulted in a significant increase in urokinase production. Proteolytic degradation and inhibition was also minimized. The medium utilization rate was decreased at lower temperature, and hence, a higher economy of production could be obtained.

External modulation of HT-1080 human fibrosarcoma cells improves urokinase production.

Urokinase was produced in a hollow fiber reactor using HT-1080 human fibrosarcoma cells. External modulation comprised replenishing of the medium in the extracapillary space, reducing the serum concentration in the extracapillary space from 10% to 2% and increasing flow rate of the circulating medium in the intracapillary space from 20 to 80 mL/min, each according to a specific protocol. More than sixfold increase was observed in the cumulative urokinase production for two and three medium replenishing modulations of the extracapillary space. After 15 days of continuous operation, the highest cumulative urokinase obtained was 1.63 x 10(6) PU/mL. SDS-PAGE and zymogram study established that the urokinase obtained was in the high molecular weight range of 54 kDa. The effect of external modulation on cumulative urokinase production was visualized as trajectories with respect to the ratio of lactic acid production rate (LPR) to the glucose uptake rate (GUR). The collective external modulation data showed two separate physiological regions in the cumulative urokinase vs. LPR/GUR plane. The HT-1080 cells exhibited two distinct morphologies in these regions that may be related to acidosis and metastasis. These regions also correspond to low and high urokinase productivity.

Synthesis, regulation and production of urokinase using mammalian cell culture: a comprehensive review.

Urokinase, a serine protease, catalyzes the conversion of plasminogen to plasmin, which is responsible for dissolution of clots in blood vessels. It is an important drug for treatment of thromboembolic disease. Production of urokinase by mammalian cell culture has the following important steps: synthesis, regulation and secretion. Production and accumulation of this product in a bioreactor is a real challenge for biochemical engineers. Considerable information at molecular level needs to be understood for production of urokinase in order to correlate different parameters, which in turn can maximize the productivity. This information will be highlighted in this review. Moreover, urokinase production is a product-inhibited process. Therefore, in situ urokinase separation strategy is required to operate a bioreactor at its maximum urokinase formation rate. Integrated urokinase production and isolation processes developed recently will also be discussed briefly in this review.

Recovery of urokinase from integrated mammalian cell culture cryogel bioreactor and purification of the enzyme using p-aminobenzamidine affinity chromatography.

An integrated product recovery system was developed to separate urokinase from the cell culture broth of human kidney cells HT1080. Supermacroporous monolithic cryogels provided ideal matrices with respect to surface and flow properties for use as cell culture scaffold as well as for affinity chromatographic capture step of the enzyme in the integrated system. The urokinase was produced continuously in the reactor running for 4 weeks with continuous circulation of 500 ml of culture medium. The enzyme activity in the culture medium reached to 280 Plough units (PU)/mg protein. Cu(II)-iminodiacetic acid (IDA)-polyacrylamide (pAAm) cryogel column was used to capture urokinase by integrating with the gelatin-coupled pAAm-cryogel bioreactor for HT1080 cell culture. After removing the urokinase capture column from the integrated system the bound protein was eluted. The metal affinity capture step gave 4.5-fold purification of the enzyme thus achieving a specific activity of 1300 PU/mg protein. The enzyme eluate from Cu(II)-IDA-pAAm cryogel capture column was further purified on benzamidine-Sepharose affinity column. This step finally led to a homogeneous preparation of different forms of urokinase in two different elution peaks with a best urokinase activity of 13 550 PU/mg of protein. As compared to initial activity in the cell culture broth, about 26.2- and 48.4-fold increase in specific activity was achieved with enzyme yields corresponding to 32% and 35% in two different peak fractions, respectively. Native electrophoresis and SDS-PAGE showed multiple protein bands corresponding to different forms of the urokinase, which were confirmed by Western blotting and zymography.

Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices.

An integrated cell cultivation and protein product separation process was developed using a new type of supermacroporous polyacrylamide gel, called cryogel (pAAm-cryogel) support matrix. Human fibrosarcoma HT1080 and human colon cancer HCT116 cell lines were used to secrete urokinase (an enzyme of immense therapeutic utility) into the culture medium. The secreted protein was isolated from the circulating medium using a chromatographic capture column. A pAAm cryogel support with covalently coupled gelatin (gelatin-pAAm cryogel) was used for the cultivation of anchorage dependent cells in the continuous cell culture mode in 5% carbon dioxide atmosphere. The cells were attached to the matrix within 4-6 h of inoculation and grew as a tissue sheet inside the cryogel matrix. Continuous urokinase secretion into the circulating medium was monitored as a parameter of growth and viability of cells inside the bioreactor. No morphological changes were observed in the cells eluted from the gelatin-cryogel support and re-cultured in T-flask. The gelatin-pAAm cryogel bioreactor was further connected to a pAAm cryogel column carrying Cu(II)-iminodiacetic acid (Cu(II)-IDA)-ligands (Cu(II)-IDA-pAAm cryogel), which had been optimized for the capture of urokinase from the conditioned medium of the cell lines. Thus an automated system was built, which integrated the features of a hollow fiber reactor with a chromatographic protein separation system. The urokinase was continuously captured by the Cu(II)-IDA-pAAm cryogel column and periodically recovered through elution cycles. The urokinase activity increased from 250 PU/mg in the culture fluid to 2,310 PU/mg after recovery from the capture column which gave about ninefold purification of the enzyme. Increased productivity was achieved by operating integrated bioreactor system continuously for 32 days under product inhibition free conditions during which no backpressure or culture contamination was observed. A total 152,600 Plough units of urokinase activity was recovered from 500 mL culture medium using 38 capture columns over a period of 32 days.

Production and purification of urokinase: a comprehensive review.

An increased emphasis on prevention of fatalities due to thrombovascular disorders is broadening opportunities for several cardiovascular agents, especially plasminogen activators, for preventing strokes and heart attacks. Hence, urokinase, as one of the most potent plasminogen activators is attracting a great deal of attention. Developments in cell lines and bioprocess technology have made it possible to produce urokinase from in vitro cell culture. Attempts are now underway to enhance urokinase production from cell culture through media manipulation, bioreactor cultivation, and innovative purification techniques. Downstream processing also poses an intricate problem due to the complexity of cell culture extracts, susceptibility of urokinase to autocatalytic and proteolytic degradation and due to the presence of plasminogen activator inhibitors in the culture media. Hence, enhancing cellular productivity and downstream product recovery continue to be major challenges as discussed in this review. Furthermore, an approach for integrated upstream and downstream processing is needed to develop an economically viable technology. In the present review the emerging trends in urokinase production and purification have been discussed in detail.

Supermacroporous cryogel matrix for integrated protein isolation. Immobilized metal affinity chromatographic purification of urokinase from cell culture broth of a human kidney cell line.

A new type of supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing the specific capture of urokinase from conditioned media of human fibrosarcoma cell line HT1080. The affinity adsorbent was designed with the objective of using it as a capture column in an integrated perfusion/protein separation bioreactor setup. A comparative study between the utility of this novel cryogel based matrix and the conventional Sepharose based affinity matrix for the continuous capture of urokinase in an integrated bioreactor system was performed. Cu(II)-ion was coupled to epoxy activated polyacrylamide cryogel and Sepharose using iminodiacetic acid (IDA) as the chelating ligand. About 27-fold purification of urokinase from the conditioned culture media was achieved with Cu(II)-IDA-polyacrylamide cryogel column giving specific activity of about 814 Plough units (PU)/mg protein and enzyme yields of about 80%. High yields (95%) were obtained with Cu(II)-IDA-Sepharose column by virtue of its high binding capacity. However, the adsorbent showed lower selectivity as compared to cryogel matrix giving specific activity of 161 PU/mg protein and purification factor of 5.3. The high porosity, selectivity and reasonably good binding capacity of Cu(II)-IDA-polyacrylamide cryogel column make it a promising option for use as a protein capture column in integrated perfusion/separation processes. The urokinase peak pool from Cu(II)-IDA-polyacrylamide cryogel column could be further resolved into separate fractions for high and low molecular weight forms of urokinase by gel filtration chromatography on Sephacryl S-200. The selectivity of the cryogel based IMAC matrix for urokinase was found to be higher as compared to that of Cu(II)-IDA-Sepharose column.

Urokinase separation from cell culture broth of a human kidney cell line.

A single step ion-exchange chromatography on a sulfo-propyl (SP)- Sepharose column was performed to separate both the high molecular weight (HMW)- and low molecular weight (LMW)- forms of enzymatically active urokinase type plasminogen activator from human kidney (HT1080) cell culture media. The level of urokinase secreted by the cell line reached to about 145 Plough units/ml culture broth within 48 h of cultivation. The conditioned cell culture media was applied directly to the column without any prior concentration steps. Polyacrylamide gel electrophoresis of the column eluates in the presence of sodium dodecyl sulphate showed that the cell line secretes three forms of two-chain high molecular weight (HMW) urokinase of molecular weights (M(r)) 64,000, 60,900 and 55,000. In addition, two low molecular weight (LMW) forms of M(r) 22,000 and 20,000; proteolytic cleavage products of HMW, were also found. The HMW and LMW forms had intrinsic plasminogen dependent proteolytic activity as judged by zymographic analysis. The specific activity of the pooled peak fractions increased (approximately 93-fold) to values as high as 1481 Plough units/ mg protein. Both HMW as well as LMW forms were obtained in significantly high yields.

L (+) lactic acid fermentation and its product polymerization

Lactic acid has been first introduced to us as early as 1780 as a sour component of milk. Ever since we have found its applications in food, pharmaceutical, cosmetic industries etc. Now there are emerging uses as a potential feedstock for the biodegradable polymer industry. The microorganisms being used for lactic acid fermentation, the raw materials reported, the various novel fermentation processes and its processing methods have been reviewed. The properties and applications of lactic acid, its derivatives and polymer have been discussed. The various routes to polymerization and the companies presently involved in lactic acid production have been covered.

Isolation of adh mutant of Lactobacillus rhamnosus for production of L(+) Lactic acid

Lactobacillus rhamnosus, a facultative anaerobe, which produces L (+) lactic acid and ethanol under anaerobic conditions, is used in the present study. An adh- mutant of Lactobacillus rhamnosus MTCC 1408, was developed by chemical mutagenesis, which could produce pure L(+) lactic acid as the only product. Batch fermentation kinetics of the wild type and the mutant strain were studied in glucose-yeast extract medium under conditions of temperature 40ºC and pH 6.2 anaerobically. The biomass yield was similar in both wild type and mutant strains, however lactic acid yield increased by 6.6 percent. A chemically defined media was optimized for supplementation of succinate, acetate and citrate for better biomass formation using single variable optimisation. It was further optimised for varying concentrations of vitamins, amino acids and trace metals by response surface method. The batch biomass yield (0.1g/g) and lactic acid yield (0.88g/g) in the optimised chemically defined media were similar to those obtained in the glucose-yeast extract medium.