February 27, 2024

Culture Fluid & Lysate

Bioprocessing aids are used as direct additives during clarification (centrifugation or filtration) prior to chromatography. They improve the flocculation process, remove pyrogens, color bodies, and acidic impurities (nucleic acids and acidic proteins) that can foul chromatography columns. This paper describes three examples of the uses of Biocryl bioprocessing aids: a study of the clarification of pseudomonas cell lysate using bioprocessing aids as an alternative to heat treatment following high-speed centrifugation; a study of the effect of pre-chromatography polishing on peptide purification from a recombinant strain of yeast; and DNA removal in the purification of monoclonal antibodies produced in tissue culture.

High-Performance Liquid Chromatography Color Body High-Speed Centrifugation High-Performance Liquid Chromatography Chromatogram Peptide Unit

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INTRODUCTION

It is well known that genetic engineering and molecular biology have become enabling technologies in the development of a biochemical manufacturing industry capable of producing therapeutic and diagnostic proteins. However, with every process stream, one is faced with the need to purify a product that may be present in only minute quantities from an often intractable culture fluid or lysate. It is only through efficient downstream processing that one can scale up to volumes that make the production and purification of these proteins commercially viable. Enhancements in the early processing steps by removal of nucleic acids, pyrogens, particulate materials, and contaminating proteins will ultimately improve the high resolution and very costly separation procedures employed for production chromatography to purify proteins.

The removal of contaminants from a variety of processing streams by flocculation has been reported in the literature by a number of investigators. A review article by Bell et al describes a number of materials used for protein precipitation. Kendrick provides a thorough description of the use of polyethyleneimine (PEI), and Agerkvist, et al describes the use of chitosan (deacetylated chitin) as a flocculating agent. These investigators have shown that water-soluble, cationic polymers of high molecular weight and charge density are effective for the flocculation of proteins. However, lendrisak points out that the use of acidic polyelectrolytes requires a low operating pH which may denature many enzymes. Chitosan must be solubilized before use and held at low pH to be maintained in solution.

lendrisak provides some examples of PEl removing only contaminants from a crude broth and leaving the target protein in the supernatant following centrifugation. However, protein fractionation with PEl often requires recovery of the desired product from a PEl pellet by extraction with buffers at higher salt concentrations. This approach adds centrifugation and redispersion steps to the purification strategy and may become cumbersome in large scale applications.

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