Process-Scale Precipitation of Impurities in Mammalian Cell Culture Broth

If certain engineering challenges can be addressed, precipitation may prove to be a valuable tool for antibody purification
Mar 02, 2008


Alternatives to standard chromatography for the purification of MAbs have been analyzed at the industrial scale. This article discusses an alternative use of precipitation to remove cell culture broth impurities, including DNA and host cell protein, while leaving the antibody in the supernatant. It evaluates several types of precipitation agents that are suitable for use in the manufacture of a parenteral therapeutic agent. The applications of this technology at scale are also discussed.

Many current research initiatives are focused on finding alternatives to column chromatography for the purification of monoclonal antibodies (MAbs). The industry-standard capture resin, Protein A, is expensive, has relatively low capacity compared to ion exchange (IEX) resins, and does not tolerate cleaning agents, which limits the resin lifecycle. Attempts to develop alternative capture resins have met with some success, but they generally do not offer the same platform potential as Protein A. An initial purification step with the purification capability and generic nature of Protein A is required, but at lower cost and increased capacity.

In their recent review, Przybycien and colleagues discuss how other industrial applications have used processes without chromatography for years because of the uniqueness of the target molecule.1 The paper classifies the alternative technologies into three distinct areas—bulk, field-based, and adsorptive separation—which are categorized according to their industrial maturity versus resolution potential. Precipitation is described as a mature technology with low resolution potential. However, MAbs have unique properties compared to other components of the broth, which could result in more selective precipitation.

This article will discuss an alternative use of precipitation to remove cell culture broth impurities, including DNA and host cell protein (HCP), while leaving the antibody in the supernatant. It will also present the results of recent experiments determining the efficacy of potential precipitants in impurity removal, and discuss the applications of this technology at scale.

Precipitation of DNA and Protein—Other Applications

A comprehensive search of the literature reveals that precipitants have been used extensively in other industrial applications, and some of these could potentially be adapted for MAb purification. The precipitants fall into two categories—those used to purify DNA and proteins, and those used as a precipitating agent to purify antibodies, which could be optimized to remove impurities while leaving the antibody in the solution.

Several precipitants described in the literature are used to purify DNA in aqueous solutions. These include, but are not limited to, indium chloride, polyaluminum chloride, manganese chloride, and zinc.2–5 In addition, several groups have reported on the use of polyamines, such as spermine and spermidine, for DNA precipitation.6,7 While these techniques are appropriate for precipitation at the laboratory scale as research techniques, they are not suitable for various reasons for industrial-scale manufacture.

There are, however, precipitants referenced in the literature that could be adapted for use in therapeutic MAb manufacture. One such class of compounds is charged polymers, such as polyethyl-eneimine (PEI) and polyacrylic acid (PAA), which have proven effective in precipitating both proteins and nucleic acids because of the interaction between the charges on the proteins and the polymers, creating an insoluble complex.8 The use of PEI for the precipitation of nucleic acids has been studied in detail by Cordes and colleagues and by Dissing and Mattiason.9,10

In addition to charged polymers, cationic detergents such as cetyltrimethyl ammonium bromide (CTAB) and domiphen bromide (DB) have been used to precipitate cellular DNA.11,12 Goerke and colleagues were able to demonstrate the use of DB as a DNA precipitant in the purification of adenovirus.12

Persson, et al., used 6,9-diamino-2-ethoxyacridine lactate (ethacridine or ethodin) as a precipitant for both DNA and HCP from an Escherichia coli homogenate containing antibody fragments.13 The use of ethacridine (a highly aromatic, cationic dye used as an antiseptic) as a precipitant in antibody purification was first demonstrated by Horejsi and colleagues in 1956.14