At various stages in life science research and during biological development workflows filtration techniques can be used to reduce particulates or minimize contaminants such as bacteria, mycoplasma, and endotoxin levels.

Clarification and prefiltration techniques can be used for processing particulate-heavy solutions such as post-fermentation broths when bacteria are used to produce recombinant proteins of interest. Filtration through a 0.2 µm membrane device is a common method to sterilize cell culture media, buffers, and reagents. Sensitive tissue culture applications may require substantial removal of mycoplasma, processing the solution through a 0.1 µm pore size filter membrane.

Clarification and prefiltration at lab scale

Filtration is an effective method to reduce particulate contamination in fluids. Filters may be used to clarify high particulate-load solutions such as reagents and buffers that are not required to be sterile. Or they can be used to prolong the life of a final filter of a smaller pore size such as 0.2 µm. A filter used for the clarification or prefiltration of a solution may be manufactured from membranes or made of thicker media (depth media). Depth media typically offer high throughputs and high dirt-holding capacities, capturing contaminants within their structure. Unlike depth media, membranes can be bacteria and particle retentive (pore size dependent), therefore when performing life sciences applications a membrane device will most likely be used for critical and final filtration steps. Some filters may be a combination of filter media types.

Solutions that have insoluble particulate or precipitate can interfere with assays, formulation, or aesthetic appearance of the fluid. Removal of the unwanted particulate can be achieved through filtration. Selection of the pore size and capacity of the filter depends on the end use of the solution and amount of particulate present. Although the final goal may be filtration through a 0.2 µm pore size, some solutions like serum-containing media or post-fermentation broths may be filtered through a large pore size prefilter to remove the excess particulate, debris, colloids, or precipitates that can prematurely clog the final filter. Using a prefilter will manage cost and efficiency of final filtration.

Clarification and prefiltration applications include:

  • Buffers
  • Reagents
  • Serum and serum-containing media
  • Complex growth media
  • Post-fermentation broths
  • Industrial and environmental water samples

Cytiva offers a variety of products for clarification and prefiltration ranging from filter plates and syringe filters for small volumes to capsule filters for many liters at a time. Cytiva also provides bioprocessing filters.

Endotoxin removal

Endotoxin is a complex aggregate of acidic lipopolysaccharides (LPS) and consists of an innermost core of hydrophobic fatty acid groups and a central and outermost region composed of hydrophilic polysaccharides.

In aqueous solutions, endotoxin can exist in various states of aggregation up to 1 MDa. Divalent cations, such as Ca2+ and Mg2+, are found to stabilize the aggregated structure of LPS, whereas detergents help to break down the structure into smaller sub-units. When producing recombinant proteins in E. coli and other gram-negative bacteria, it is often necessary to remove LPS from the final product. This is especially important when carrying out immunological readouts and when developing manufacturing processes. Endotoxin can cause false readings in cell-based assays. There are limits to the amounts of endotoxin allowed in human products.

Cause of endotoxin contamination

Endotoxin is continuously shed from the outer membrane of viable gram-negative bacteria and is released when the bacterial cell dies. Although bacteria are often removed by using a 0.2 μm sterilizing grade filter, LPS itself is difficult to remove or inactivate because it is extremely heat and pH stable. The pyrogenic threshold of an endotoxin reaction is on the order of 1 EU (endotoxin unit ~ 0.1 ng) per kg of body weight. This amount of endotoxin can come from 105 bacterial cells.

Endotoxin removal process

The removal of endotoxin is one of the most difficult downstream processes during protein purification. It is not straightforward to remove endotoxin satisfactorily without excessive loss of product. Because endotoxin is negatively charged at pH above 2, a positively charged membrane surface is an excellent choice to remove endotoxin.

We provide effective filtration solutions for endotoxin removal

When using E. coli or other gram-negative bacteria for recombinant proteins, endotoxin is likely to contaminate the plasmid preparation and the expressed protein. Before conducting additional assays, such as immunologic readouts or developing a manufacturing process, this complex lipopolysaccharide (LPS) should be removed. While intact bacteria can be captured using a 0.2 µm filter, the LPS can be more challenging. An effective method of removal of the negatively charged LPS is to use a substrate with a positive charge. We offer Acrodisc™ Units with Mustang™ E Membrane (Pall™ Life Sciences products) for effective removal of endotoxin. The Acrodisc™ device with positively charged, hydrophilic Mustang™ E membrane is well-suited for the removal of endotoxin from solution due to its highly crosslinked quaternized amine charged surface. This gives very high dynamic capacities under selected conditions for the removal of endotoxin from process feed streams, buffers, and water.

Mycoplasma filtration

Mycoplasma, a common contaminant of cell cultures, is a very small type of bacterium characterized by its lack of a rigid cell wall. The cell wall deficiency results in organisms that have a characteristic elasticity allowing them to form a variety of different shapes ranging from spheres to branched filaments, and sizes varying from 0.3 to 0.8 μm in diameter. This elasticity allows mycoplasma to pass through 0.2 μm (bacterial retentive) filters. In addition, the mycoplasma’s lack of cell wall causes it to be resistant to certain antibiotics, such as penicillin, which interfere with bacterial call wall synthesis.

Why should we be concerned with mycoplasma?

Because of their extremely small size and slow growth rate, the contamination is not readily discernable by routine laboratory methods. Mycoplasmas, however, can have an insidious effect on any cell cultures they infect. Cultures containing one million mammalian cells, for example, may be infected with as many as five hundred million mycoplasmas. The presence of such a large number of contaminants can have drastic effects on the structure and function of the infected host cells.

These potentially negative effects of mycoplasma contamination on cultured cells make it clear that any data derived from mycoplasma-infected cell cultures are of questionable accuracy and should be treated with caution and suspicion, resulting in delays, repeated work, and additional costs.

If not 0.2 μm, then which pore size will effectively reduce mycoplasma?

Mycoplasma can be reduced by filtration with 0.1 μm filters. We manufacture 0.1 μm Supor™ membrane, an inherently hydrophilic polyethersulfone (PES) membrane, that is capable of the reduction of mycoplasma. We incorporate this membrane into many laboratory filter products ideal for tissue and culture applications including Acrodisc™ Sterile Syringe Filters and AcroPak™ capsules (Pall™ Life Sciences products).

Using our Acrodisc™ Sterile Syringe Filters with Supor™ (PES) Membrane (0.1 μm membrane) is your best assurance that mycoplasma will not be introduced into your cultures by serum or serum-containing media.

Sterile membrane filters

Filtering through a 0.2 µm pore size is considered necessary to produce a sterile filtrate. Solutions for filtration can range in volume from just a few milliliters to many liters. Typical fluids may include media with or without serum, buffers, biologic or proteinaceous samples, reagents, or other fluids that need to be free of particulates and meet stringent criteria for microbial contaminants.

A filter recognized as suitable for producing sterile filtrate is 0.2 µm pore size and capable of passing a bacterial challenge to retain a minimum of 107 cfu/cm² of B. dimunita. It should be noted that some samples may benefit from 0.1 µm pore size filtration to remove mycoplasma for sensitive cell culture applications. Sterile filtration is used for many applications:

  • Media with or without serum to support the growth of cell culture
  • Other reagents or solutions with sensitive components that may degrade when exposed to autoclave temperatures
  • No access to an autoclave
  • Filtering supplements for addition to sterile growth media
  • Filtering samples to prevent microbial growth during storage
  • Filtering buffers to remove particulate and prevent microbial growth during storage
  • Filtering a biologic sample for analytical analysis to protect the expensive instrument from microbial contamination
  • Filtering environmental samples for analysis
  • Filtering post-fermentation broths for further purification or analysis

Our sterile membrane filtration solution

We offer a range of sterile filtration products to meet different volume sizes for applications. Our products range from membranes, multi-well filter plates, syringe filters, vacuum devices, and larger capsule filters.

Our AcroPak™ capsule products range in size from 20 cm2 to 1500 cm2 effective filtration area (EFA). Three membrane choices are available in configurations that scale up to products for pilot- and full-scale production.

AcroPak™ Products with Supor™ Membrane (Pall™ Life Sciences products) quickly process difficult-to-filter solutions such as serum, serum supplemented culture media, and viscous or particulate-laden solutions. Supor membrane offers high flow rates and consistently higher total solution throughputs due to higher porosity than most other membranes. It is ideal in situations where rapid filtration or short processing times are essential or where low protein binding is required.

Device

Sample volume*

EFA

AcroPak 20 Filters 100 mL - 5 L 20 cm2
AcroPak 200 Capsules 5 -25 L 200 cm2
AcroPak 500 Capsules 10 -100 L 500 cm2
AcroPak 1000 Capsules 10 -100 L 1000 cm2
AcroPak 1500 Capsules 10 -150 L 1500 cm2
AcroPak™ 400 and 800 Capsules with Fluorodyne™ II Membrane

Offer high flow rates, low adsorptive properties, and low extractables. Well-suited for scale-up and downstream processing applications for biopharmaceutical products. Suitable for applications where customer protocol requires PVDF membrane; double layer sterilizing membrane assures enhanced performance.

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Supor™ EKV Membrane Filters

0.2 micron rated validated sterilizing-grade membrane filters. Utilizing a laid-over-pleat membrane geometry for high flow rates and throughput performance, they are designed for the most cost-effective filtration of a wide range of liquids such as buffers, tissue culture media, and others. A PES membrane ensures very high compatibility over the whole pH range plus very low protein binding to ensure maximal transmission of active ingredients.

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Sterile vacuum filtration

Laboratory vacuum filtration is an easy and convenient way to filter fluids and can be generated using a pump, in-house vacuum lines, or by a faucet aspirator system. Vacuum filter units, also known as bottle top vacuum filters, are popular for sterile media and buffer preparation. We offer a full selection of VacuCap™ and VacuCap PF (Pre Filter) Vacuum Filtration Devices (Pall™ Life Sciences products). They draw fluid directly from mixing flask into the filter unit.

Laboratory vacuum filter units are popular lab essentials to filter media, serum, buffers, and small-batch reagents. Important properties of vacuum filters include:

  • Fast flow rates
  • High throughput
  • No extractables
  • Low binding

Our VacuCap and VacuCap PF Sterile Vacuum Filtration Units meet these important properties plus:

  • Eliminate contamination, risk of spills, and lost time by drawing fluid directly from mixing flask into the filter unit.
  • Use the full filtration potential (up to 5 liters) to fill multiple media bottles
  • Improve safety and ergonomics through eliminating repeated lifting and pouring
  • Improve throughput and flow rates of difficult-to-filter solutions with a built-in prefilter, 0.8/0.2 µm VacuCap PF filter unit
  • Reduce plastic waste and storage space required compared to bottle top vacuum filters.

Learn about Depth Filtration Devices for Cell Cultures, Fermentation Broths, and Cell Lysates.