Industrialscale Applications

Expanded-bed adsorption is a technique with great potential for initial purification. The number of published industrial scale applications is limited because until recently columns and adsorbents suitable for use in expanded beds were not available. In addition to the three industrial-scale applications described below, an IND (Investigational New Drug) application has been filed with the Food and Drug Administration for a therapeutic recombinant protein in which expanded-bed adsorption is central to the purification process (60).

Purification of Recombinant Human Serum Albumin from Whole Yeast Culture

The number of unit operations in the process for purification of recombinant human serum albumin (rHSA) from yeast was reduced by three when expanded-bed adsorption replaced the conventional packed-bed method (32). In the conventional packed-bed method, three filtration steps were necessary to prepare the yeast culture for the first cation exchange column; when using expanded-bed ad sorption, the unclarified culture could be applied directly to the expanded cation exchanger. By reducing the number of unit operations, the total processing time was deceased and the overall yield was increased. In contrast to the conventional packed-bed process, the expanded bed process could be performed in a totally closed system, and the quality (determined as degree of coloring) of the rHSA was reported to be higher using the expanded-bed process. The process was successfully scaled up (determined by comparing yield of rHSA) from a 50-mm diameter column with 300 mL adsorbent to a 1,000-mm diameter process scale column with 150 L adsorbent. Approximately 2,000 L un-clarified feedstock (1,000 L culture diluted with 1,000 L distilled water) was applied to the process scale column, and the average yield of rHSA from four different runs was 87% (ranging from 82 to 91%). Using a specially made column with a 4-mm inner diameter, the lifetime of the resin was investigated. The results showed that the adsorbent could be reused up to 1,000 times without compromising performance (61).

Purification of Monoclonal Antibodies from Whole Mammalian Cell Culture

A process for recovery of recombinant monoclonal antibodies was directly scaled up from a small lab-scale expanded-bed column containing 70 mL cation exchange adsorbent to a process-scale expanded-bed column to which 12,000 L of cell culture was applied (45). In this single process step, all the cells were removed, the monoclonal antibody was concentrated fivefold, and the yield of antibody obtained was 95%.

Purification of Bovine Serum Albumin from Whole Yeast Culture

In a mimicked purification process, a 4.5% dry weight yeast culture was spiked with bovine serum albumin (2 mg/mL) and used as feedstock. The bovine serum albumin was then recovered from the feedstock using expanded-bed adsorption with an anion exchange adsorbent (27). The process was scaled up from a 25-mm diameter column to a pilot scale column with a 200-mm diameter and finally run in a fully automated system with a production-size column with a 600-mm diameter. The settled bed height was 15 cm throughout all scales, and the feedstock volume was approximately 750 mL in the 25-mm column and 430 L in the 600 mm column. The results from the different scales showed good agreement. The yield of bovine serum albumin was approximately 89% (ranging from 87 to 92%). The wash volume was approximately 11 settled bed volumes, and the volume of the elution peak was approximately 2 settled bed volumes.

Potential Industrial-Scale Applications

The successful use of expanded beds at laboratory and pilot scales has increased significantly during the past few years, giving rise to a number of potential industrial-scale applications. One such example is the purification of the protease inhibitor aprotinin (34), which has good potential as a therapeutic and diagnostic compound. Aprotinin was expressed and secreted from the methylotrophic yeast Hanensula polymorpha, and the initial recovery was performed using 300 mL cation exchange adsorbent in a 50mm diameter expanded-bed column. The only pretreat-ment of the yeast culture was a 1 + 1 dilution with water and an adjustment of pH. The final volume of the feedstock was 6.4 L. The dilution achieved two goals: it lowered the conductivity to favor binding to the ion exchanger, and it reduced the biomass and viscosity of the feedstock so that the bed did not expand too much. The yield of aprotinin was 76%, the purification factor was 4, and the concentration factor was 7.

Another example is the recovery of a recombinant mi-totoxin fusion protein, fibroblast growth factor-saporin (rFGF-2-SAP) (31), which is a possible therapeutic agent for the treatment of diseases characterized by cellular proliferation (e.g., cancer). The fusion protein was expressed in E. coli. After homogenization, it was recovered from the diluted homogenate using 300 mL cation exchanger in a 50-mm diameter expanded-bed column. Homogenate from 300 to 400 g (wet weight) cells was applied to the expanded-bed column. The yield of fusion protein was 65%, and the purification factor was 20. It was also reported that a somewhat modified process was scaled up to handle 8,000 g (wet weight) cells. This process was carried out under GLP (good laboratory practice) conditions, and one such process yielded an average of 1.6 g of protein. The yield and quality of the fusion protein were comparable between the different scales.

Recombinant human nerve growth factor (rhuNGF) produced in Chinese hamster ovary cells is another example of a potential industrial scale application (38). Here, the rhuNGF was recovered using a 25-mm diameter expanded-bed column with cation exchange adsorbent using flow velocities up to 375 cm/h. The yield of rhuNGF was approximately 95% and concentrated approximately 45 to 50 times. During method development, it was found that the dynamic binding capacity increased when the feedstock was applied at 37 °C (normally at 25 °C). The optimal conditions for recovery were determined in this small scale before the process was scaled up 65-fold.

In another example, E. coli was used to express modified exotoxin A from Pseudomonas aeruginosa (29). The exotoxin lacks the enzymatic activity but has retained binding activity. It accumulates in the periplasm of E. coli. After release of the exotoxin by osmotic shock, it was recovered using expanded-bed adsorption with an anion exchange adsorbent. To reduce the viscosity of the extract, it was treated with a DNase prior to application to a pilot-scale expanded-bed column with a 200-mm diameter. Cell extract from 4.5 kg of E. coli was applied to 4.7 L adsorbent at 400 cm/h. Recovery by expanded-bed adsorption gave an exotoxin with a concentration three times greater than that obtained using the conventional packed-bed process and with a slightly higher yield. Furthermore, the entire process was completed in one third of the time.

Humanized immunoglobulin G4 was affinity purified from myeloma cell culture using expanded-bed adsorption at pilot scale (41). A 200-mm diameter expanded-bed column containing 4.7 L protein A adsorbent was used. The cell reactor was connected directly to the expanded bed, and approximately 100 L of cell culture was applied to the column at 37 °C. The purification process (application, wash, and elution) took 2.5 h and 40 g of the antibody was quantitatively recovered. The purity of the antibody was comparable to that obtained by the corresponding packed-bed process.

Other potential large-scale processes include the purification of proteins from milk or whey (46-48).

Brew Your Own Beer

Brew Your Own Beer

Discover How To Become Your Own Brew Master, With Brew Your Own Beer. It takes more than a recipe to make a great beer. Just using the right ingredients doesn't mean your beer will taste like it was meant to. Most of the time it’s the way a beer is made and served that makes it either an exceptional beer or one that gets dumped into the nearest flower pot.

Get My Free Ebook


Post a comment