Lab Filtration & Purification

Consider the following when choosing a lab filter: 

• What volume will be filtered?
• What is your goal? Is it to remove contaminants or concentrate the target? 
• What is the size of the contaminants and target(s)? 
• Is sterility needed? 
• Do I need vacuum, pressure, syringe, peristaltic or other method of action?

Once these points are established, product selection can begin. 
 

Filtration processes can be separated according to mode of action or force behind the separation, e.g. pressure, vacuum, centrifugal, tangential flow filtration (cross flow filtration). 

A filter can be used until the porous media is blocked or fouled significantly enough that the sample cannot pass through, or the flow rate is reduced to an inhibitory amount. Level of usage before blockage depends upon the sample particle loading and pororus material density, porosity, etc. Blocked filters can sometimes be regenerated with membrane flushing, reverse filtration or chemical cleaning, but this comes with other considerations.  

Bacteria can be removed using filters that remove particles at the microscopic level. To ensure all live and intact bacterial cells are removed, a 0.22µm pore size filter should be used. Typically, a 0.22µm cellulose filter, like cellulose acetate, cellulose nitrate or regenerated cellulose membranes, or 0.22µm synthetic membranes, like polyethersulfone or polytetrafluoroethylene filters, are used to remove bacterial cells. If certified as sterilizing grade, 0.22µm pore sized membrane filters will reduce bioburden to a sterility assurance level (SAL) of 10-7, essentially sterilizing the sample that has passed through the membrane filter. 

A 0.45µm pore size filter will remove many larger bacteria and particles to a significant amount. It is estimated that a 0.45µm pore size filter may remove >90% of bacteria. However, to fully remove all bacteria, a 0.22µm pore size filter is recommended. 

A 0.2µm pore size must be used for sample sterilization by filtration. However not all 0.2µm pore size filters are sterilizing grade. To ensure your sample is truly sterilized, the membrane filter must be sterilized in a validated procedure and have a sterility certificate to ensure there are no contaminating bacteria or particles on the downstream side of the membrane. Second, the membrane filter must come with certification to show it has passed the Bacterial Challenge Test (BCT) using the very small Brevundimonas diminuta bacteria, to ensure the membrane filter will retain all cells 0.2µm or greater and ensure a sterile filtered sample.

Mycoplasma are bacteria that lack cell walls. Because of this, they are some of the smallest bacteria so far identified and have malleable structures. A very small 0.1µm membrane filter pore size is needed to remove mycoplasma from samples. These membrane filters are readily available in syringe filter, in-line disc filter and vacuum filter formats. 

Filter papers, such as glass microfiber filters, quartz filters, technical papers and cellulose based depth filters, typically do not come with pore sizes. Instead, retention ratings are provided to give information on the size of particles that may be retained, on or within the filter matrix. These retention ratings are not absolute, unless specified as such, as they are designed to capture and remove larger particles for general clarification applications. 

This depends upon the sample being filtered, possible target molecule / particle of interest and method of filtration. Each membrane can come with its own specifications. For example, regenerated cellulose (RC) has a pH range of 3-14, high solvent resistance, very low non-specific adsorption and high flow rate, making it a very good all-around membrane for many liquid sample applications. Polytetrafluoroethylene (PTFE) has the widest pH range of 1-14, with very high solvent resistance, high flow rate and low non-specific adsorption - however, it is hydrophilic, and so is preferred for solvent or gas applications.  

To isolate target proteins and other molecules from cell culture or cell culture lysate, a filtration step can be used whereby cells, cell debris and larger particles are retained by a 0.2µm or 0.45µm pore sized membrane, while molecules of interest, host cell protein (HCP) and other small cellular products pass through into the filtrate. To reduce membrane fouling and filter blockage, a pre-filtration step is recommended. Prefiltration may be carried out using a glass or cellulose based prefilter with a more porous filter matrix to enable a two-step filtration process. Likewise, filter aids such as pharmaceutical-grade diatomaceous earth can be applied directly within the cell culture to enable a prefiltration matrix layer on top of the 0.2µm membrane layer to prevent blockages.

Proteins are commonly concentrated using ultrafiltration. Product design of ultrafiltration devices for laboratory use consists of ultrafiltration membranes placed within housings specially designed to enable concentration through centrifugal, positive pressure, solvent adsorption and tangential flow separation methods. These methods use controlled forces to pass solutes and non-targeted molecules through a semi-permeable ultrafiltration membrane, into the permeate - while retaining the larger molecule(s) of interest in the retentate. 

There are several purification methods for proteins and other molecules, like viral vectors and exosomes. Preparative chromatographic techniques are ideal options for their high molecule specificities and high separation resolutions, ensuring high yields and molecule purity in lab and process settings. These chromatography methods utilize a range of chemistries, such as ion exchange, affinity ligands, hydrophobic interactions, steric exclusions or a mixture of these methods, known as mixed mod or multimodal chromatography. 

A polishing step is often incorporated into a purification process to remove impurities or unwanted molecule isoforms. It is typically the last stage in a purification process and is needed when the highest level of molecule purity is required. 

Capture purification is where the molecule of interest is specifically targeted by the chromatography chemistry, e.g. Protein A binding ligand to immunoglobulin G (IgG) and captured. Here capacity of binding/capture per sample volume is a priority. A polishing step is employed to target the removal of unwanted molecule isoforms and other impurities, e.g. use of a cation exchange membrane absorber to remove all positively charged impurities, while leaving the negatively charged target molecule to pass through the media. 

Any physical contact with your sample can be a potential source for leachables and extractables (L&E), depending on the aggressiveness and chemical reactiveness of your sample. It is worth assessing what filter material and filter housing is best to avoid L&E. Most membrane materials are 100% pure and have minimal L&E. However, PTFE, Regenerated Cellulose and Polyamide (Nylon) based membranes are well known for their chemical robustness and high purity. Further polypropylene (PP) housing materials also do not contribute any L&E.  For assurance, it's recommended to use devices and membranes that have test data to show no L&E and to check your sample chemical compatibilities.  

Online literature, papers and research forums are a good source of application information. However, for specific advice on filtration methods across a range of application workflows, Sartorius has an extensive range of test data, technical guides and application notes and are available to contact for advice and workflow queries. Further, product samples or demonstrations are often available free of charge to help users confirm the optimal lab filtration processes.