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Filtration of Dilute Protein and DNA Solutions
Filtration of Dilute Protein and DNA Solutions
By Kevin A. Seeley, Ph.D., Research Scientist
Introduction
Filtration of Dilute Protein Samples
Filtration of Dilute DNA Samples
Conclusions
Introduction
While heat sterilization is used for many applications, heat may destroy critical cell culture media
components and biomolecules needed for normal cell growth. Consequently, filtration is the method of
choice for the sterilization of cell culture media. Filters capable of ambient sterilization also have been
found useful for the general clarification and purification of cellular lysates as a means to enhance the
recovery of target biomolecules. In both applications, extremely dilute biomolecular components must
pass through the passive sterilizing and purifying barrier without significant loss, alteration, or
contamination. Membrane filters perform best when these issues of “passivity” have been addressed
during product design and manufacture.
The primary function of most submicron-rated filters is to remove or reduce bacteria and fungi that can
contaminate and overgrow a culture. Some reduction, and clarification or purification, can be expected
from any submicron-rated membrane filter. Even among “sterilizing grade” membranes and devices,
performance differences exist in such secondary characteristics as extractables and “binding.” Product
documentation such as pyrogenicity and cell culture certification testing can verify that there are no
extractables from the filter that might affect cell growth. “Binding” has been a more difficult potential
problem to define.
The issue has been confused by binding tests that are not reflective of actual filter usage. Low biomolecule
binding Supor ® membranes are rated and validated as retentive, tested and certified as significantly free
of extractables, and, as the data in this paper will show, do not significantly remove DNA and protein from
dilute samples.
Historically, studies of the binding and transmission of proteins were based on methods where
membrane samples were soaked in more concentrated protein solutions or filtered using recirculation.
Under these conditions, protein binding follows saturation kinetics. While they may indicate the total
binding capacity of a filter, saturation binding methods are not representative of most sterilizing or
clarification applications because they use high protein concentrations and/or extended exposure times.
In such applications, the act of filtration usually occurs within a brief period and fluids pass through the
membrane only once. The amount of protein binding under dynamic filtration conditions is more a
function of binding rate than binding saturation.
In most cases where protein concentrations are above 100 µg/mL, significant loss of protein during
filtration does not occur, as binding sites are quickly saturated. One would expect that protein or DNA
loss due to filtration-membrane binding would be most dramatic and catastrophic when filtering
extremely dilute samples. Based on these considerations, Pall Life Sciences tested the filtration of
extremely dilute protein or DNA solutions under dynamic filtration conditions.
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Filtration of Dilute Protein Samples
We used radioactive iodine-labeled proteins to determine dilute protein binding under typical (dynamic)
filtration conditions. The use of radiolabeled proteins allowed us to detect the binding of trace quantities
of protein while filtering submicrogram concentrations. Preliminary measurements indicated that both
bovine serum albumin (BSA) and IgG respond similarly in terms of filtration membrane binding (data not
shown). Because of its typical behavior as a globular protein, labeled BSA was chosen as an indicator
polypeptide to quantitate membrane binding under dynamic filtration conditions. BioTrace™ NT
(nitrocellulose) and Biodyne® B (positively-charged nylon) membranes are designed to bind
biomolecules, and were included as positive controls for biomolecule binding during filtration.
Labeled BSA (ICN Biochemical) was diluted in phosphate buffered saline (PBS) to a concentration of 0.34
µg/mL. An aliquot of 5.0 mL was filtered giving a total challenge of 1.7 µg BSA. Membrane samples were
placed in a 13 mm Swinney device (Pall Life Sciences Part No. 4317) attached to a 10 mL Luer-Lok*
syringe. A 5.0 mL sample of dilute protein in PBS was drawn into a 10 mL syringe together with 1.0 mL of
air and was filtered at 0.5 mL/second at approximately 20 psi. The air was used to give a 1.0 mL purge to
chase out residual protein sample from the membranes and housing. Following filtration and air purge,
samples were counted without a wash, or had a 1.0 mL PBS wash together with a second 1.0 mL air purge
to completely remove residual sample fluid.
Membrane discs from triplicate samples were then placed directly in 3.0 mL scintillation cocktail and
counted in a multilabel counter (PerkinElmer, Boston, MA).
The data in Table 1 show that the second PBS wash resulted in a slight reduction in counts; however, that
reduction is not statistically significant. The reduction in counts resulting from the second wash is more
likely due to the complete removal of residual liquid than a result of the elution of bound protein. Based
on this observation, it was decided that all further binding assays would include a 1.0 mL buffer rinse.
Table 1
Post-filtration Flush Does Not Significantly Alter Protein Binding Results
Membrane
With Second Wash CPM in 1000s
Without Second Wash CPM in 1000s
0.45 µm Supor®
17.3 ± 3.1
22.1 ± 1.3
0.45 µm GH Polypro
9.7 ± 2.1
13.8 ± 5.0
0.45 µm Biodyne B
782.4 ± 45.1
820.7 ± 56.2
The data in Figure 1 shows that while the BioTrace NT (nitrocellulose) samples bound greater than 50% of
the available labeled protein, most standard microfiltration membranes bound less than 1.0% of the
extremely dilute protein sample. This data highlights the fact that protein binding studies using static soak
(Table 2) or recircularization do not represent the binding of proteins under typical conditions.
While most microfiltration membranes tested had very low binding, Versapor ® membrane, a generalpurpose clarification membrane, bound significant amounts of protein. Based on this, Versapor
membrane would not be recommended for the filtration of dilute protein samples. The Pall Life Sciences
membranes that bound the lowest amount of labeled protein were GH Polypro (GHP) membranes. These
membranes are extremely passive membranes developed for low-volume analytical sample preparations
where added extractables or sample loss due to binding is unacceptable.
Figure 1
125I-BSA (1.7 µg) was diluted to 5.0 mL in PBS and filtered through a 13 mm disc of the indicated
membrane. Filtration was carried out using a 10 mL syringe at a flow rate of 0.5 mL/second
followed by a 1.0 mL PBS rinse. Binding was determined by comparing the amount of
radioactivity remaining in the membrane (triplicate) to the activity of the starting material.
Nitrocellulose membrane is designed for biomolecule binding and was used as a positive control.
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Filtration of Dilute DNA Samples
DNA molecules are often part of solutions or lysates that require clarification by filtration. To test whether
filtration membranes bind dilute DNA samples, radioactively-labeled PCR products were added to Tris
EDTA (10 mM Tris, 0.1 mM EDTA, pH 7.0) buffer. Samples were filtered using the same general handling
procedures discussed previously for the analysis of protein binding. Biodyne B membrane (Pall Life
Sciences Part No. 60200) is a transfer membrane designed to bind DNA, making it a good choice for a
positive control for binding during filtration.
Figure 2
32P-DNA (500 ng) was diluted to 5.0 mL in Tris-EDTA and filtered through a 13 mm disc of the
indicated membrane. Filtration of the 5.0 mL sample was carried out using a 10 mL syringe at a
flow rate of 0.5 mL/second followed by a 1.0 mL TE rinse. Binding was determined by comparing
the amount of radioactivity remaining in the membrane (triplicate) to the activity of the starting
material. Biodyne B membrane is designed for biomolecule binding and was used as a positive
control.
Double-labeled PCR products were synthesized from primers corresponding to a 400 bp fragment to
pUC18. Free nucleotides and primers were removed from the pooled PCR products by using a 100K
Nanosep® device to concentrate the DNA (see technical article PCR: Before and After). The amount of
radiolabeled DNA present was determined by gel electrophoresis, and specific activity was determined by
counting to be approximately 540 CPM/ng. A 500 ng sample of purified-labeled DNA was diluted to 5.0
mL in TE buffer (10 mM Tris pH 7.0, 1 mM EDTA). A sample containing 5.0 mL of the 100 ng/mL DNA
solution was filtered, air-purged and rinsed with 1.0 mL TE and counted in a multilabel counter
(PerkinElmer, Boston, MA).
As with the protein binding data, the transfer membrane (Biodyne B membrane) controls bound the
majority of the test sample even though the exposure during filtration was brief. Similar results were seen
with other binding membranes (not shown). Supor and GHP membranes bound very low quantities of
DNA, similar to that seen for the competitor’s low-binding membranes. Filtration of dilute DNA solutions
with most low binding microfiltration membranes should not cause significant DNA sample loss.
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Conclusions
Competitive claims relating to protein binding during microfiltration may be based on saturation binding
and not dynamic filtration conditions. The data in Table 2 indicate that large amounts of protein can be
bound when measurements are made under saturation conditions. For example, differences seen
between HT Tuffryn® and Supor membranes are significant when measured under saturation conditions;
but, binding was low for both membranes when measured under dynamic filtration conditions. While
there are some slight differences in the overall binding properties between the low-binding membranes,
the retention of dilute proteins by a non-binding filtration membrane is extremely low when tested under
actual application conditions.
Table 2: Static Soak Shows Protein Binding at Saturation
Membrane
Description
µg/cm²
% Bound
Versapor
0.45 µm Acrylic Copolymer
118
47.2%
Supor
0.45 µm Polyethersulfone
56
22.4%
GH Polypro
0.45 µm Hydrophilic Polypropylene
55
22.0%
HT Tuffryn
0.45 µm Polysulfone
9.3
3.7%
BiodyneA
0.45 µm Amphoteric Nylon 6,6
133
53.2%
A 13 mm disc was soaked for 60 minutes with agitation in a 0.2 mg/mL protein (IgG) solution containing
100,000 counts per minute 125I-IgG. Bound protein was quantified by counting the remaining radioactive
IgG retained on the membrane. (A. Dubitsky)
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