Protein Purification Systems

CBB is experienced in conducting 10- and 100-liter E. coli and Pichia pastoris fermentations expressing recombinant proteins. After a typical fermentation, harvested cells are lyzed with a microfluidizer, and solids separated by centrifugation or microfiltration. CBB then purifies the intracellular proteins using several chromatographic methods.

CBB has several protein purification systems. A Gradifrac system is suitable for preliminary trials and an Akta Explorer provides systematic development of purification protocols and scale up under GLP conditions. The Akta explorer system can pump from 10 microliter/min to 100 mL/min while continuously monitoring eluting proteins at three different wavelengths. CBB can monitor pH, temperature, and conductivity to control protein elution.

CBB recently added an AKTA Pilot high-performance, automated liquid chromatography system to its protein purification equipment, which includes an AKTA Explorer. The AKTA Pilot system is well suited for process development, process scale up, and small-scale production; it simplifies the transition from bench to full-scale production. Applications include purifying materials for preclinical testing, clinical trials, diagnostics, and therapeutics. The AKTA Pilot is biocompatible and sanitizable. It meets all GLP and cGMP demands for Phase I - III drug development and full-scale production. Liquid flow rates range from 4 to 800 mL/min with operating pressures to 20 bars.

The Center has several Vantage VA columns, each with 12.5-liter capacity, and several Vantage-L laboratory chromatography columns ranging from 50 mL to 1,000 mL for small-scale process development. For example, CBB routinely conducts Ion Exchange, Hydrophobic Interaction, Affinity, Gel Permeation and Reverse Phase Chromatographic procedures for purifying proteins.

In addition, several ultrafiltration systems are available to develop and support protein purification.

Typically, SDS-PAGE and Western Blot techniques are used to analyze purified proteins. When appropriate, CBB uses 2-D Gel electrophoresis, peptide-mass finger printing, and amino acid sequencing. Example product purifications include several enzymes, an anti-HIV peptide, diagnostic proteins, alpha-synuclein and its variants as well as vaccine proteins. CBB also routinely monitors purified products for endotoxin content.

Finally, CBB can lyophilize protein preparations in bulk or in vials with a computer-controlled Virtis Freeze Dryer.

Back to Top