Removal of biological contaminants

Mike Weaver and Dr Simon Cole from Cuno Europe highlight the difficulties in removing biological contaminants from processing waste

Biological contaminants are always difficult to remove cost effectively without causing a detrimental effect to the product. There are many types of biological contaminants but this article addresses the removal of two: nucleic acids and viruses.

Nucleic acids such as DeoxyriboNucleic Acid (DNA) and RiboNucleic Acid (RNA) are replicated in the production of monoclonal antibodies. The cell density in the fermenter has also increased over the years, compounding the problem by increasing the concentration of contaminants in each batch of product. Regulatory bodies such as the FDA and the EMEA demand that these contaminants are removed.

The CBER¹ (Center for Biologics Evaluation and Research), part of the FDA, states that: 'It is suggested that, wherever possible, the final product contain no more than 100pg cellular DNA per dose.'

Viruses are a major concern and the effort and cost to remove these has increased dramatically over the past 10 years. Some viruses such as Human Immunodeficiency Virus (HIV) are well known and are relatively easy to remove when compared with some of the smaller viruses such as Polio (30nm) and Parvovirus B19 (20nm). Regulatory requirements state that at least two viral clearance steps operating by different mechanisms should be employed in processes.

purification processes

The general principle is to remove contaminants as early as possible in the process to prevent contamination and complications to the downstream processes. There is also a requirement to implement a robust viral clearance system. To meet this, different viral clearance methods (removal and inactivation) are employed.

After the cells are grown in the fermenter/bioreactor the product is subsequently separated by a downstream purification process, which can be split into the primary and the secondary purification stages.

Primary purification is where the cells and/or cell debris are removed. The product then enters the secondary purification stage where it is further purified, isolated and formulated. The third and last stage is formulation and filling of the product into containers. Figures 1 and 2 show the primary and secondary purification stages.

An efficient method to clarify and purify the product is by using filters in the process. Depth filters are ideal to remove debris at the initial stages and clarify the product. A prefilter to optimise filter throughput often precedes the membrane filter. Membrane filters provide assurance of microbial reduction and can be integrity tested to assure quality. Filters can also be used to remove specific biological contaminants such as DNA and viruses. Filters will remove debris and biological contaminants by different mechanisms. The predominant mechanisms are:

•Size exclusion: Particles are removed on or near the surface of the filter due to their size;

•Entrapment: Particles are removed by the internal structure of the filter medium. The major mechanisms of particle removal include inertial impaction due to the torturous pathway through the filter, and electrokinetic charge if the filter has one.

Electrokinetic charge offers a substantial benefit as the efficiency of the filter is greatly improved, which means that it can remove much smaller particles, especially those contaminants that are negatively charged such as cell debris, DNA, RNA and some viruses.

An example of a filter that uses all the above mechanisms is the Zeta Plus depth filter medium, which is frequently used in bioprocesses. This filter is composed of cellulose, diatomaceous earth and a resin. The diatomaceous earth provides an enormous surface area (>500m² per gram) for particles to be trapped. The resin provides a positive charge to enhance removal of smaller negatively charged particles. Zeta Plus filters have nominal retention ratings from 100nm to 10mm, depending on the grade.

DNA removal

Table 1 shows data by Dorsey, et al.² This was obtained using calf thymus DNA at a concentration of 10µg/ml. Samples (100ml aliquots) were used to challenge 47mm filter discs at a flow rate of 14-20ml/min (1.2 ; 1.7l/m²/min). The results show significant retention of calf thymus DNA at pH 7.4. This is greatly reduced at pH 9.0, due to the neutralisation of the positive charge on the Zeta Plus medium. This data supports the theory that the filter removes DNA by charge. The majority of mammalian cell and bacterial lysate clarification is performed at neutral pH, where DNA retention is enhanced.

It is always recommended to find the limits of a filter's capability to remove DNA (Figure 3). This means that the filter can be validated to remove DNA consistently under production conditions. Cuno's scientific application support services can assist in these studies. Zeta Plus can be used to remove DNA effectively in bioprocess streams and help to meet regulatory requirements.

Zeta Plus VR (Virus Removal filters) can be used in bioprocess streams to complement other viral removal and inactivation processes. Zeta Plus VR filters retain viruses predominantly by anion exchange adsorption. The recommended position for Zeta Plus VR filter is within the secondary purification stage. At this stage in the downstream process the product is more highly purified, so that the electro-kinetic charge on the medium can be used to its maximum efficiency without competing with other particles for the same charged sites.

Table 2 shows the efficiency of the filters to retain Phi X-174 (a 28nm bacteriophage) in the presence of sodium chloride. The results indicate that the higher ionic strength buffer reduces the ability of the medium to retain Phi X-174, and supports the theory of ion exchange adsorption.

Table 3 shows a second experiment where Zeta Plus VR05 and VR07 media were evaluated to retain Xenotropic Murine Leukemia Virus (XmuLV, 90nm) and Porcine Parvovirus (PPV, 20nm). A major US West Coast biopharmaceutical manufacturer and Bioreliance, a contract laboratory service conducted this experiment. The column eluate solution consisted of a partially purified monoclonal antibody solution in 20mM sodium acetate buffer, pH5, at 20°C. The results support adsorption rather than a size exclusion mechanism for the retention of viruses.

retention rates

Table 4 compares several recognised viral clearance steps and their relationship to Zeta Plus VR media with respect to the mechanism of viral clearance. This demonstrates that Zeta Plus VR filters can be used to complement many methods that are used in existing processes or development of new pharmaceutical drug development programs.

Zeta Plus medium has excellent filtration properties enabling it to remove biological contaminants that are both larger and substantially smaller than its filter retention rating. The use of electrokinetic charge provides significant benefits over filters that are not charged. Cuno's Scientific Application Support Services can assist from the initial concept through laboratory studies and into full-scale production meeting regulatory compliance.