DNA Clearance From Antibody Preparations Using Mustang® Q ...

USD 2278
DNA Clearance From Antibody
Preparations Using Mustang ® Q
Membrane-Model Study
2

DNA Clearance From Antibody Preparations Using
Mustang Q Membrane-Model Study
Introduction
Why Remove DNA
The world market demand for monoclonal
IgG's and recombinant protein is growing
rapidly. Improvements in cell culture techniques
have significantly improved the efficiency of
monoclonal production with concentrations of
monoclonal antibody of over 1g per liter, being
reported in hybridoma supernatants.
However, during the cell growth and the
harvesting steps of the cells, a certain amount
of nucleic acid may contaminate the product
of interest. The nucleic acid contamination
may come from the host cell DNA-either
genomic, vector-or retroviral RNA, which is
considered a greater risk. Fragments of sizes
larger than 2 kilobases are considered to be
potentially carcinogenic, and therefore nucleic
acids have to be removed from the final product.
The Center for Biological Evaluation and
Research (1) in the USA, initially defined the
maximum allowable nucleic acid contamination
as 10 pg per dose. The World Health
Organisation (2) has stated that 100 pg per dose
could be acceptable. More recently, a newer
'Point to consider' from the CBER (3) now states
that "Lot to lot testing for DNA contents in
biological products produced in cell lines
should be performed and lot release
limits established that reflect a level of purity
that can be achieved reasonably and
consistently." This all suggests that a rapid
and cost efficient method which clears nucleic
acid to below acceptable (and ideally
detectable) levels is required.
Why use Membrane Chromatography to Remove DNA
Several methods are available for nucleic with a larger surface area and therefore fewer
acid removal. Among them, we can mention diffusion limitations. At the same time
anion exchange chromatography, which has Mustang Q membrane when compared
proved to be quite effective due to the high to conventional resin based media has
negative charge of nucleic acids, which bind at least a 10 fold higher binding capacity
at high ionic strengths and at pH >2. Under (25 mg DNA / mL of media) combined
these conditions most biotherapeutic proteins with much higher flow rates (more than
do not interact with anion exchange matrices. 10 CV / min).
Therefore, anion exchange (4, 5) as a first step
These units have thus been designed as
is generally considered to be suitable as it can
capsules and cartridges for single use
contribute to reduce the viscosity of the
disposable product. Therefore, neither
protein feed stream. Alternatively, both cation
cleaning nor cleaning validation is necessary,
exchange in a flow through mode and
which is helpful because it can be difficult
nucleases (6, 7) have been used for removal of
or impossible to demonstrate complete
contaminating nucleic acids.
removal of nucleic acids when cleaning
Although conventional anion exchange resin conventional resin media.
chromatography can be used to remove
nucleic acid impurities, DNA is a very large
biomolecule (can be up to 500 nm in diameter
compared to a typical 1-10 nm globular
protein) and it has difficulties diffusing into the
small pores (20-100 nm) of resin beads. In
contrast, Mustang Q anion exchange
membrane has much larger pores (800 nm)
2

DNA Clearance From Antibody Preparations Using
Mustang Q Membrane-Model Study
Large DNA molecules
have direct convective
access to Mustang Q
membrane groups
without steric hindrance
Beads 1µm Mustang
slow
fast
slow
slow
fast
slow
fast
fast
slow
fast
fast
fast
fast
fast
fast
fast fast
Figure 1: Comparison of molecular
access of nucleic acids in membranes
and conventional gel media
Solid surface
Area of diffusive flow
Area of convective flow
• 150 Å diameter globular protein
DNA (20,000 bp fragments shown)
What is Mustang Q Media
Mustang Q membrane is made by coating
and tightly crosslinking a highly stable
hydrophilic quarternary amine polymer onto
the surface of polyethersulfone membrane.
The end result is a membrane with an open
nodular structure which has the benefits of a
high surface area and binding capacity for
negatively charged macromolecules, combined
with good mechanical properties, including
800 nm pores to allow for very high flow rates.
Single use Mustang Q units are made of 16
layers of this membrane which are then pleated
and assembled into a ready-to-use capsule
format. The Mustang coin unit is a scaled
down version of the Mustang capsules. It is
ideal for performing scale down experiments,
particularly for validation studies.
The coin unit uses the same 16 layer
Mustang Q membrane that is used in
Mustang Q capsules and cartridges.
Clearance of Calf Thymus DNA from a
Polyclonal IgG Preparation Using the
Mustang Q Media
The goal of the experiments was to find loading
conditions (buffer composition, pH and ionic
strength) under which DNA but not an Ig G
preparation, binds to Mustang Q membrane.
Every monoclonal antibody has a different pI
which will determine its ability to bind to
Mustang Q membrane at any given pH.
Therefore it is difficult to establish a method
suitable for every monoclonal antibody with
Mustang Q membrane, based on just one
example. For this reason we chose a bovine
Ig G preparation which contains many different
Ig G's with a broad spectrum of pI's , therefore
covering many possible eventualities.
Due to its relatively large size and highly
polyanionic nature above pH 2, DNA has a
very high affinity for anion exchange
membranes compared to smaller and more
neutrally charged antibodies. Therefore it was
predicted that binding in slightly acidic and
Clearance of Calf Thymus DNA from a
Polyclonal IgG Preparation Using the
Mustang Q Media
moderate ionic strength-should favor DNA
binding and result in the Ig G fraction having
very little or no interaction with the anionexchange
Mustang Q membrane. To make
separation more challenging and applicable
to industrial applications, the experiments were
carried out using phosphate buffers (as a worst
case scenario) which normally interfere with
anion exchange chromatography due to the
high ionic strength and multiple negative charge
of the phosphate anion.
3

DNA Clearance From Antibody Preparations Using
Mustang Q Membrane-Model Study
Protocols and Results
Three separate experiments were carried out,
using first DNA alone then polyclonal Ig G
alone and finally a combination of both.
For the first experiment (Figure 2a), a solution
of Calf Thymus DNA was made up to a
concentration of 20 µg/mL in 25mM Sodium
Phosphate buffer pH 6.0 + 150 mM NaCl.
A total of 360 mL (7 mg) of this solution was
In the final experiment (Figure 2c), 380 mL of
a bovine Immunoglobulin G solution (4 mg/mL
in phosphate buffer) was spiked with
Calf Thymus DNA (20 µg/mL) and subsequently
loaded on Mustang Q coin using the
conditions described in Experiment 1.
Absorbance at 260 and 280 nm were
continuously monitored. DNA breakthrough
was obtained at 322 mL as judged by an
increase in absorbance at 260 nm. Washing
and elution were carried out according
to Experiment 1 and 14 mL fractions
collected and assayed for DNA content
using our own DNA hybridization testing
method described in the application note
USD 2279 (Figure 3).
loaded on to a Mustang Q coin equilibrated
with 25 mM Sodium Phosphate buffer pH 6.0
+ 150 mM NaCl at 20 CV/min, while monitoring
absorbance at 260 nm. After washing with 40
membranes volumes of equilibration buffer,
bound DNA was eluted by a gradient of
150 to 1000 mM NaCl in 40 CV of equilibration
buffer. Breakthrough was obtained at
approximately 315 mL, as indicated by an
increase in absorbance at 260 nm.
Absorbance (260nm)
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33
Fraction Number
Figure 2a: Calf Thymus DNA
100
90
80
70
60
50
40
30
20
10
0
Conductivity (mS/cm)
This first experiment determined that Calf
Thymus DNA elutes in these conditions above
at a salt concentration greater than 500 mM.
For the second experiment (Figure 2b), a
solution of Bovine Immunoglobulin G was
made up to a concentration of 2 mg/mL in
equilibration buffer. A total of 380 mL (760 mg)
Absorbance (280nm)
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35
100
90
80
70
60
50
40
30
20
10
0
Conductivity (mS/cm)
of this Immunoglobulin G solution was loaded
Fraction Number
on to a Mustang Q coin using exactly the
same method as described for Experiment 1,
and absorbance at 280 nm was continuously
monitored. Washing and elution were carried
out according to Experiment 1.
This second experiment demonstrated that in
the chosen conditions, the chosen bovine
Immunoglobulin G does not bind to Mustang
Q Media.
Experiments 1 and 2 determined the optimum
conditions under which no globulin binds (the
product of interest) while DNA binds (the
impurity) to Mustang Q media.
Figure 2c: Bovine Gamma Globulin
Absorbance
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
AU (260nm)
AU (280nm)
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37
Fraction Number
Figure 2c: Calf Thymus DNA and Bovine Gamma Globulin
Figure 2. Chromatogram of respectively Calf thymus DNA (a) Bovine Ig G solution (b) and Bovine
Ig G solution spiked with Calf Thymus DNA(c) on Mustang Q coin
Adsorption and washing buffer: Phosphate 25 mM pH 6.0 + 150 mM NaCl
Elution buffer: gradient of 150 to 1000 mM NaCl in 40 CV of equilibration buffer
100
90
80
70
60
50
40
30
20
10
0
Conductivity (mS/cm)
4

DNA Clearance From Antibody Preparations Using
Mustang Q Membrane-Model Study
Under the conditions chosen in these
experiments, Calf Thymus DNA binds strongly
to Mustang Q membrane. The hybridization
test demonstrated that no DNA was detected
Fraction 5
until fraction 24 (sensitivity of the assay is
2.5 pg/mL). It supports the fact that there
is no DNA breakthrough detected by P 32
DNA hybridization (Figure 3) until saturation
Fraction 24
is obtained (with binding capacities of
18.5 and 18.3 mg/mL of Mustang Q membrane
for Experiments 1 and 3 respectively).
Fraction 25
At the same time, very little binding of
Immunoglobulin G (< 0.5% of the total) binding
is taking place (as adjudged by absorbance
25
at 280 nm) which demonstrates a very
good recovery of the product of interest.
20
Discussion
The experiments demonstrate very efficient
DNA µg/mL
15
10
Filtrate
Starting Solution
removal of Calf Thymus DNA by Mustang Q
5
membrane in the presence of a bovine Ig G.
This supports the fact that Mustang Q
0
membranes can be used for the efficient
0 1 5 9 13 17 21 25
removal of nucleic acids from both polyclonal
Fraction
and most probably monoclonal antibodies
preparations to below both detectable and
Figure 3: Results of DNA Challenge Test
acceptable levels according to current
FDA guidelines.
5

DNA Clearance From Antibody Preparations Using
Mustang Q Membrane-Model Study
Clearance of Nucleic Acid from a
Monoclonal Cell Culture Supernatant
The aim of this experiment was to demonstrate
that a similar approach to the one described
above could be applied to the use of
Mustang Q membrane for DNA removal from
a very complex solution such as cell culture
supernatant.
Protocols and Results
A Monoclonal Cell Line was grown to a density
of 1 x 10 6 cells per mL in RPMI 1640 media
and the supernatant collected after
centrifugation of the cells. This supernatant
was pre-filtered to 0.2 µm using Supor ® media
and then the pH was readjusted to 6.0 after
addition of 50 mM Sodium Phosphate buffer
pH 6.0. Supernatant (10 mL) was loaded on
to a Mustang Q coin previously equilibrated
with Sodium Phosphate buffer pH 6.0
containing 150 mM NaCl at 10CV / min. The
flow through was collected and assayed for
DNA along with the starting material using the
DNA hybridization method developed by Pall.
During the experiment, absorbance at 280 nm
(protein concentration) and conductivity (salt
concentration) were continuously monitored
as described in Figure 4.
Absorbance (280nm)
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
-0.10
Flow Through
00:00:00 00:05:00 00:10:00 00:15:00 .
Hr:Min:Sec
Fractions
Figure 4: Chromatogram of cell culture supernatant (10 mL) loaded on Mustang Q Coin
Adsorption and washing buffer: Phosphate 25 mM pH 6.0 + 150 mM NaCl
Elution buffer: gradient of 150 to 1000 mM NaCl in 40 CV of equilibration buffer
Flow rate: 10 CV/min
100.0
90.0
80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.00
-10.0
Conductivity (mS/cm)
Discussion
The experiment demonstrated that using the
chosen conditions, Mustang Q membrane
allows for the removal of DNA while the proteins
of interest did not bind on the media. The
hybridization test showed that while the initial
level of contamination was 8 pg of DNA/ mL
of cell culture supernatant, after passage
through the Mustang Q coin this level was
reduced to below detection level (2.5 pg/mL).
These results demonstrate that Mustang Q
membrane chromatography is a very suitable
method for the removal of DNA from complex
solutions, and can even be applied at early
stages of the purification processes on crude
protein solutions.
Pre Mustang Media Flow ThroughFractions (Post Mustang media) Calibration Standards
Figure 5: DNA Hybridization analysis cell culture supernatant loaded on Mustang Q Coin
6

Conclusion
We have demonstrated that anion exchange chromatography using
membrane technology can be an excellent alternative to the use of
conventional gel resins for nucleic acid removal as it allows removal of
nucleic acids below the level requested by regulatory authorities.
Membrane chromatography used in a disposable format significantly
reduces the validation time as both column packing and cleaning validation
will not be required. The pH and salt conditions at which the product of
interest does not bind to the Mustang membrane, but the nucleic acids
do bind, have to be determined in each case. Due to the very high
negative charge of nucleic acid, this can be easily achieved with the vast
majority of monoclonal antibodies and recombinant proteins produced
by mammalian cell culture or any other sources.
Nucleic acid removal was obtained at high flow rates, as typically flow
rates from 10 to 20MV/min have been used, allowing clearance in minutes
rather than in hours. In fact, Mustang Q membranes have a very high
dynamic binding capacity (in the above conditions around
18 mg of DNA were bound per mL of membrane) that is independent of
flow rate due to their convective pores. Resins, on the other hand have
dynamic binding capacities that are very flow rate dependent due to
their numerous diffusive pores. The experiments performed at small
scale suggests that for a typical antibody where the initial contamination
level is around 5 µg/mL in the cell culture supernatant, a Kleenpak TM Nova
10 inch (NP6) capsule could be used to clear 1000 L of cell culture
supernatant in less then 2 hours. This high throughput technology is
ideal for contaminant and impurities removal from biomolecules which
are very susceptible to degradation.
27

References
(1) US FDA. Points to consider in the Production and Testing of New Drugs and Biologics Produced
by Recombinant DNA Technology (1985)
(2) Acceptability of all substrates from production of biologicals. Report of a WHO group.
Technical report Series 757; World Health Organisation, Geneva (1987)
(3) US FDA. Points to consider in the Characterization of Cell Lines used to Produce Biologicals.
FDA, Rockville, MD (1993)
(4) R.D. Sitrin, P. de Phillips, Challenges in developing commercial scale bioseparation and biopurification
processes. International Biotechnology Expo Scientific Conference, Oct. 24, 1990. Oral Presentation
(5) R.W. van Leen, J.G. Bachuis, R. van Beckhoven, H.Burger, L.C.J. Dorssers, R.W.J. Hommes,
P.J. Lemsons; B. Noorclam, N.L/M. Persoon and G. Wagemaker, Production of human
interleukin-3 using industrial microorganisms. Bio/Technology 9, 47-52 (1991)
(6) STREAMLINE ® and Protein A Data file. Pharmacia Biotech Uppsala (1996)
(7) A.L. van Wezel, P. van der Marel, C.P. van Beveren, I. Verma, P.L. Salk and J. Salk,
Detection and elimination of cellular nucleic acids in biologicals produced on continuous cell lines.
Dev. Biol.Standard. 50, 59-69 (1982)
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