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Will dissolved proteins pass through a 0.2 micron filter?

Will dissolved proteins pass through a 0.2 micron filter?



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Given that there may be exceptions, can you usually expect protein to pass through?


Many proteins will pass through a 0.2 micron filter. If the proteins aggregate or if they stick to the filter material because it is charged, they may not.

Barring the above, a 0.2 micron filter will allow proteins up to 200kd to pass through. There are some proteins that are larger.

Editing with some source material.

Nice Presentation on Pore Sizes and Membranes Here

Graphic from that presentation:


Defining a Pore Size and Sterile Filtering 0.2 Micron vs. 0.22 Micron. What’s the difference?

If you were to spend a little time perusing Sterlitech’s selection of membrane disc filters, one thing we’re very proud of might just jump out at you: we have a lot of pore sizes. So many that you might wonder if it’s a little excessive that we carry both 0.2 and 0.22 micron pore sizes. After all, both are used to sterilize fluid passed through them. Can the tiny difference of 0.02 microns really change a filter’s performance characteristics that much?

To answer that question, we must first take a look at one of the methods used to test a filter’s performance: the bubble point test1. Standard tests to verify a filter’s stated pore size usually entail a bubble point test. This test pushes air under pressure through a submerged membrane (either in water or alcohol) to the point where air bubbles first begin to come through the filter membrane2. The largest pore, or pores, in the membrane will bubble first, and the air pressure required to push the bubbles through these pores can be mathematically correlated to pore size. The irregular and tangled nature of the pores of most membrane filters makes it impossible to directly measure the size of an individual pore, so the bubble point test is used to determine the smallest particle that the filter can sieve out of a fluid.

In other words, the stated pore size of filter isn’t literally the size of the pores. It’s a rating of what can’t pass through them. Obviously, when sterilizing solutions, the object is to physically remove bacteria suspended in the solution. Before 0.2 and 0.22 micron filters became standard, it was thought that filters with an absolute rating of 0.45 micron were thought to be sufficient to strain out even the smallest bacteria. However, the discovery of Brevundimonas diminuta, showed that there were still bacteria capable of passing through a 0.45 micron filter in large quantities. After the discovery, researchers and labs competed to create the new filtration standard, arbitrarily defining their filters to be either 0.2 or 0.22 micron in pore size, roughly half the size of the old standard.

What that means is, for the purpose of sterilization, 0.2 micron and 0.22 micron filters are indistinguishable. Their performance is the same, only the difference being the designation of their pore size rating. The real measure of filter’s ability to sterilize fluid is passing the test described in ASTM F838-05, Standard Test Method for Determining Bacterial Retention of Membrane Filters Utilized for Liquid Filtration. Basically, if the filter can retain a minimum of 1 x 107 colony forming units (cfu) per cm2 of a challenge bacteria (usually B.diminuta5), then the filter is suitable to use for sterilization.

Even with the contraction of the standard pore size for sterilization, it turns out that simple size selection is not a sufficient method to fully contain all particles. Jornitz6 et al. showed adsorptive effects also change the manner in which different filter media capture different particles. Influences such as pH, pressure, bacterial load, and the liquid medium itself influence the size of bacteria. However, as long as the filters could capture the required number of challenge bacteria per square centimeter of membrane, then it is a valid sterilizing filter - whatever the stated pore size. Sterlitech Corporation is both a manufacturer and a reseller of different membrane filters, so you will see 0.2 micron and 0.22 micron in our filter offerings but for sterile filtering, both are suitable based on the above information.


Silly question: is DMSO sterile by itself, to add it to cells? - Lab stock bottle could be used? (Aug/22/2005 )

I just wonder, could i use for the purpose of cell freezing DMSO, which is standing on the lab shell, or I should buy a new bottle and to use it only for cell culture, or I could sterilize DMSO?

I don't think there is likely to be much that could grow in DMSO, however, to be on the safe side, you should buy a bottle just for cell culture. Otherwise you can filter sterilise it with a 0.2 micron filter, but it is pretty thick so it can be a bit of an effort.

I don't think there is likely to be much that could grow in DMSO, however, to be on the safe side, you should buy a bottle just for cell culture. Otherwise you can filter sterilise it with a 0.2 micron filter, but it is pretty thick so it can be a bit of an effort.

When I was inexperienced, one of my very mistakes was to filter sterilise DMSO without diluting it. To my horror, the filter dissolved! So, what I do now is to dilute the DMSO (for common lab use should be OK) in my culture media to achieve the final concentration, and then filter sterilise the mixture. Just some advice in case you were planning to filter it straight.

i just sterilised DMSO at 121℃ for 30mins

For filter sterilisation of DMSO, use of nylon membrane filters (0.2 u) are recommended, they are DMSO safe.

We normally sterile filter the 'freezing medium' containing DMSO. This is done by using 0.2Um filter - "STERIFLIP"

I think it is good to filter because, I once found something floating in the freezing medium when I tried to use it as such. Then I checked DMSO - there were some particles floating - just like in FBS!

So far I never heard of any organism growing in pure DMSO.

You don't need to sterilze DMSO and I would not filter the pure solvent. But you should filter the medium/DMSO solution.

The only problem with DMSO I ever had is that an old bottle got 'oxidised' by air oxygen. Then you get problems with the pH. So always check the pH of your medium (colour of indicator).


Function of a micron filter

Filters for intravenous (IV) medication administration are used to remove contaminants from intravenous products. This filtration is intended to protect the patient receiving the medication by filtering out particulate matter, bacteria, and air emboli, protecting the patient from phlebitis due to particulates or infection due to bacteria.

Filters are used with the intravenous administration of many medications. These filters may be contained within an IV line (in-line filter), and are available in a variety of sizes. A 5-micron filter removes large particles that may have been introduced during product preparation, such as glass particles from glass ampules. The 0.22-micron filter is one of the smallest used in patient care, and removes bacteria. There are not currently filters that remove viruses.

Not all intravenous medications should be administered through a filter, and others may require filters of a specific size. The molecules of some medications may be too large to pass through a filter, or may otherwise bind to the filter and be removed. Thus, it is important to consult your institution’s policies for details on which medications require a filter for parenteral administration.


Procedure

Use of MWGF200 For Gel Filtration Chromatography

    Buffer and Resin – It is recommended to use 50 mM Tris-HCl, pH 7.5,with 100 mM KCl as the equilibration buffer with a 90 cm x 1.6 cm Sephacryl ® S-200-HR (Catalog No. S200HR) column at 2–8 °C. For information on resin preparation, column packing, and equilibration contact Technical Service.

1.0 in the peak fraction. Glycerol is added to increase the density of the solution, but its use is optional.

The recommended sample volume is less than 2% of the total gel bed volume. Carefully apply the blue dextran sample to the column (avoid disturbing the gel bed surface) to determine Vo and to check column packing. Immediately after applying the sample, begin collecting fractions of 0.5–1.5% of the total gel bed volume. The flow rate should be

7% of the column volume per hour. Skewing of the blue dextran band represents a fault in the column, although some tailing is normal. The leading peak indicates the void volume. Determine spectrophotometrically the elution volume for blue dextran (Vo for the column) at 280 nm or 610 nm by measuring the volume of effluent collected from the point of sample application to the center of the effluent peak.

Notes: Mixing blue dextran with kit standards or sample proteins is not recommended since many proteins bind to blue dextran.

Prepare protein standards and blue dextran fresh. 3 Occasionally some aggregated protein may appear at the void volume.

The following proteins may be mixed and run together on the columns:
Cytochrome c and β-amylase
Carbonic anhydrase and alcohol dehydrogenase

Apply protein standards to the column using the same sample volume and flow rate as used for the blue dextran sample. The elution of the standard proteins may be followed by absorbance readings at 280 nm. Determine the Ve for the protein standards by measuring the volume of effluent collected from the point of sample application to the center of the effluent peak.


Why is a 0.22 micron pore size considered tight enough to render solutions filtered through it sterile?

I work in a biochem. lab and we use 0.22 micron filters to sterilize protein solutions, ensuring little to no microbe growth during storage or prolonged use. Why is this sizing standard? Also, I heard 0.45 micron filters actually provide more microbe clearance due to lower pressures across the filter, which prevent microbes from being forced through the pores. Is there any truth to this?

There are plenty of scholarly articles that state that neither provide complete sterility in filtering. It only depends on the degree of sterility you are looking for, and what microorganisms you have present in the solutions that need to be caught.

Both are enough to filter out most microbes you'll probably encounter in a lab. .22 micron will capture more just based on pore size. I don't think there's any truth to .45 providing better sterility. Filters are designed to prevent microbes from being forced through just by pressure. Plus, there are some organisms (like mycoplasma) that fall between the .45-.22 micron diameter. Iɽ say .45 micron filters are used because they clog less easily and are just easier to push through if used on a syringe.

No method can guarantee 100% sterility. Autoclaving is one of the best, but since you're working with proteins, that method is off the table.


Filtration Resource Library

Filtration is done to achieve one of two things: to clarify and/or sterilize your filtrate, or to analyze the retentate left on the membrane. While there are several different applications that fall under either option, our products are designed to effectively support your entire filtration process and ensure that you achieve the most accurate results.

Laboratory filtration products come in many formats and with varying properties and yet the basics of filtration and many of the filter properties do not change. We have compiled these resources to help you select and use our products. Please contact our support services or your local sales representative for any further assistance you might require.


Filter Sterilisation

The demand for sterile compressed air increases with the adoption of advanced technologies which were unknown a few years ago. The selection of a sterilization filter for a compressed air system can be a difficult task. The production of proteins, vaccines, antibodies, hormones, vitamins and enzymes involves high technology processes which require aseptic and sterile supplies of gases or liquids throughout the manufacturing cycle. The production and packaging of many dairy and food products such as beer, yoghurt, cream and cheese use compressed air or carbon dioxide. The nature of these products makes them susceptible to contamination by micro-organisms held in the compressed air or gas.

Any product that can be contaminated by airborne bacteria must be protected. In the case of food and chemicals produced by fermentation, bacteria would cause serious defects and rejection of the product.

In the fermentation and pharmaceutical industries, compressed air and other gases are used through every stage of the production process from the refining of the raw material to the manufacturing and packaging. Compressed air may be used as a source of energy in a process or as a biomedical aid. Air motors are used for explosion-free mixing of powders, instrumentation and cylinders for the batching of materials.

Process air can be used for aeration of liquids, seed fermentation and laboratory applications. Mixing air into the preparatory chemicals or the final product means that they must be as clean and sterile as the material it serves, so there can be no risk of fouling by solids, liquids or micro-organisms.

Micro-organisms are extremely small and include bacteria, viruses, yeasts, fungus spores and bacteriophages. Bacteria can be from 0.2 micron to 4 micron, viruses less than 0.3 micron and bacteriophages down to 0.04 micron. The filters that have to cope with these organisms have to have a performance rather better than these dimensions. The presence of these living organisms can be a serious problem in process industries, because they are able to multiply in the right conditions.

When selecting compressed air sterilization filters, the follow conditions must be satisfied:

The filter must not allow penetration of any type of micro-organism that could cause contamination.

It must be able to operate reliably for long periods.

The materials of construction must be inert so as not to support biological growth.

It must be easy to install and maintain.

It must be capable of being tested for integrity.

It must be capable of being steam sterilized repeatedly.

The filter must be as small as possible consistent with efficiency so as to reduce problems of installation.

Modern air sterilization filters use pleated hydrophobic binder-free borosilicate microfibres which have an efficiency of better than 99.9999% at 0.01 micron and remain in service for up to 12 months. The element has been constructed using an amalgamation of filter media ( Figure 1 ) the average diameter of the fibre is about 0.5 micron. The polluted air strikes the outer layer of relatively coarse medium (2 micron). This traps the dirt particles before it reaches the microfibre. The borosilicate collects the particles down to 0.01 micron. Any aerosols present are here converted into liquid. In removing micro-organisms, three main mechanisms are present – direct interception, inertial impaction and Brownian motion. The second layer of 2 micron medium is designed to hold the microfibre in place. These two media combined hold back the solid particles. In a dry air stream they will hold back bacteria. As long as the bacteria have no means of multiplying in the medium, they can be held. A filter medium containing a binder material which could act as a nutrient would be unsuitable. The complete construction can be seen in Figure 2 .

FIGURE 1 . Binder-free microfibre media. (ultrafilter)

FIGURE 2 . Construction of a borosilicate filter element. (Zander)

Membrane cartridge filters are extremely flexible and high in tensile strength. Cartridge construction based on a multi-layer combination of filter media support an irrigation mesh. Nylon and polypropylene polymers that have previously been used in coarser grades are now used as membrane filters. Layers of nylon microporous membrane and polypropylene pre-filter are pleated together and supported by an inner core. The end caps are melt sealed in polypropylene. The membrane is of thin nylon having a controlled pore size. Its construction ensures that it cannot release fibres down stream it can be repeatedly autoclaved.

Hollow fibre filter

Hollow fibres were originally developed for dialysis and have now been adapted for compressed air. The filter is arranged with a smaller spread of hole sizes, but with a larger number of membranes. This gives a greater retention ability. Due to a closer pore distribution than conventional membrane filters, the number of pores per unit area of filter is greater, which extends the service life. A closer pore distribution means that the largest pore size is much reduced. A conventional membrane filter has a pore size at least 0.3 micron larger than a hollow fibre filter.

The development of a membrane filter element with a rated pore size of 0.1 micron and a reduced pore distribution means the difference between the retention of viruses and bacteria. The construction of the hollow fibre filter means that it is economical to manufacture smaller elements for laboratory use.

Housings

Sterile filter cartridges must be fitted into a pressure holding housing. The method of sealing is by single or double O-ring seals which allow sealing and movement during steam sterilization or shock loading without breaking the seal. Compressed air sterilization filter housings need to be designed to protect the filter cartridge from stress. Stainless steel housings are the usual choice of material they should be electrolytically polished inside and out, and have no grooves or corners in which micro-organisms can collect. They should incorporate an automatic condensate drain. For economy, sterile filters can be made of galvanised steel housings with stainless steel parts in contact with the compressed air or with a corrosion resistant coating. Aluminium housings with powder coated protection are also available.

Filter selection

Filters are sized according to the flow rate versus pressure drop information supplied by the manufacturer. A sterile filter has an initial pressure drop of about 0.1 bar although the rating can be reduced if necessary to meet the requirements of the process a pressure drop of about 0.2 bar is indicative of dirt collecting in the filter and it must be replaced. A pre-filter should normally be incorporated in the circuit to reduce the load on the sterile filter. Coalescing type pre-filters can sustain a high pressure drop but it is normal to replace the element between 0.3 and 0.6 bar.

A complete filter assembly for producing sterile air will contain a number of separate elements. If the sterile supply is to be taken from a larger system with a variety of applications, an oil lubricated compressor may be the source of air in this case an activated carbon filter will need to be incorporated. If all the air in the process needs to be sterile, then an oil-free compressor is probably the better choice. Figures 3 and 4 show some of the possible choices.


Causes of mycoplasma contamination

The mycoplasmas enter the cell culture through various sources that are difficult to trace. These include the laboratory personnel, the serum, the cell culture media, water baths, incubators, etc. Contamination by humans accounts for the largest source among those mentioned above. The contaminants can spread through dirty clothing, lab wears, human speech near the laminar airflow, the human scalp, sneezing, coughing, etc. Also, a constant in-flow of individuals wherever the cell cultures are kept will increase the risk of contamination.

Mycoplasmas can spread from these sources through cross-contamination and due to poor lab techniques. These include reusing pipettes for multiple cell lines rather than using disposable ones or reusing gloves. The serum is also a source of mycoplasma contamination. Due to its turbid nature, contamination is difficult to be detected in the serum that is most commonly used for every cell culture. Reusing the same bottle of serum again and again for each subculture can enhance the growth of mycoplasmas. The serum is nutrient-rich and also the best source for mycoplasma proliferation. Plus, it is added to the media after autoclaving hence, there is no assurance of the serum being contamination free.

The aerosols that are generated (while talking or pipetting) during working in the laminar airflow are also among the leading causes of contamination. These aerosols generated will enter the culture medium during subculturing processes or from the air if the cell line remains exposed for a longer time. The aerosols are invisible to naked eyes and hence cannot be detected until it leads to contamination.

Another source of the problem is the media, used for subculturing, which is available in both liquid and powdered forms. Due to improper handling or poor lab techniques, mycoplasma can spread through this form of powdered media. Filtration does not ensure complete sterilization as mycoplasma can even escape 0.2-micron filters.

Figure 1: Contamination! Tissue culture media often have indicators included in the media that turns yellow from lowered pH due to microbial contamination. Photo by Kaustubh Kishor Jadhav.

Mycoplasmas can remain in the dry state for a longer time. When they come in contact with a nutrient source, they start proliferating shortly. Once they are present in the laboratory environment, they are difficult to eradicate. You can suppress the growth of mycoplasma but cannot eradicate it completely. When mycoplasmas are seen in the culture, it is a good option to discard the flasks (unless the source of culture is irreplaceable or highly expensive).


Engineering Perspectives in Biotechnology

2.46.4.8 Reaction and Membrane Filtration

The combination of a bioreactor with a membrane filtration system can serve different purposes of either mass recycling into the bioreactor or product recovery from the bioreactor. 3 A large range of microfiltration membranes for the recycling of biological cells and ultrafiltration membranes for the recycling of large-molecular-weight compounds such as enzymes or large coenzymes are available. The membranes can have a symmetric or asymmetric structure and can thereby have a dual function of immobilizing the biocatalyst and of product separation or purely a product recovery function. In the first case, where the membrane serves as biocatalyst support and separation unit, the biocatalyst can be immobilized within the porous membrane or at the membrane surface by different methods such as covalent attachment, ionic interactions, adsorption, entrapment, gel formation, or cross-linking. A variety of different geometries such as flat-sheet, spiral-wound, and tubular structures are available for the membranes, which can then be assembled into the appropriate membrane modules and units for standardized couplings to the feed, retentate, and permeate lines.

The concept of the EMR has been developed and successfully applied by Kula and Wandrey 37 and others. The classical work on the enzymatic reductive amination of α-ketoisocaproate to l -leucine with l -leucine dehydrogenase and simultaneous cofactor regeneration with formate dehydrogenase in a continuously operated membrane reactor 38 has pioneered this integration mode for a large number of industrial bioprocesses. The enzymatic production of amino acids in membrane reactors with simultaneous regeneration of reduced β-nicotinamide adenine dinucleotide (NADH) has been developed to industrial scale 39 at Evonik in Germany. The EMR has been successfully applied in numerous routine productions by other industries (e.g., Tanabe Seiyaku, Sepracor, and Sigma-Aldrich), demonstrating the industrial relevance of this integration of reaction and membrane filtration. 1 The membrane reactors have the advantage of using soluble components (enzymes, substrates, and products), which can be easily replenished, for example, for substrate supply or if more enzyme is needed to keep the bioconversion rate constant because of enzyme deactivation effects. Therefore, attention has to be given to the preparation of enzyme so that the operational stability of the free enzyme is good enough for its use in the EMR.

Extensions of the classical EMR concept include the charged ultrafiltration membrane enzyme reactor, where negatively charged ultrafiltration membranes are used to retain the native cofactor in the reactor, or the use of nanofiltration membranes. For the bioconversion of poorly soluble substrates, an emulsion membrane reactor consisting of a separate chamber with a hydrophilic ultrafiltration membrane for emulsification, an EMR loop with a normal ultrafiltration module, and a circulation pump can be used. 1

Other examples include enantiomerically pure intermediates, anticancer drugs, vitamins, anti-inflammatory compounds, cyclodextrins, and antibiotics. 40


Watch the video: Recenze proteinu EXTRIFIT (August 2022).