The present invention relates to the application of high gradient
magnetic separation (HGMS) to the separation of biological materials, including
cells, organelles and other biological materials. Specifically, this invention
relates to micro columns and micro column systems for high gradient magnetic field
separation of macromolecules and cells.
High gradient magnetic separation (HGMS) refers to a process for selectively
retaining magnetic materials in a chamber or column disposed in a magnetic field.
This technique can also be applied to non-magnetic targets labeled with magnetic
particles. This technique is thoroughly discussed in U.S. Patent Nos. 5,411,863
and 5,385,707, which are hereby incorporated by reference in their entireties.
The material of interest, being either magnetic or coupled to a magnetic
particle, is suspended in a fluid ad applied to the chamber. In the presence of
a magnetic field supplied across the chamber, the material of interest, being magnetic,
is retained in the chamber. Materials which are non-magnetic and do not have magnetic
labels pass through the chamber. The retained materials can then be eluted by changing
the strength of, or by eliminating the magnetic field.
U.S. Patent No. 4,508,625 to Graham (Graham '625), discloses a process
of contacting chelated paramagnetic ions with particles having a negative surface
charge and contained in a carrier liquid to increase the magnetic susceptibility
of the particles. A magnetic field is then applied to the carrier liquid and particles
to separate at least a portion of the particles from the carrier liquid.
U.S. Patent No. 4,666,595 to Graham (Graham '595), discloses an apparatus
for dislodging intact biological cells from a fluid medium by HGMS. The fluid
containing the cells is passed through a flow chamber containing a separation matrix
having interstices through which the fluid passes. The matrix is subjected to a
strong magnetic field during the time that the fluid passes therethrough. At least
some of the cells are thereby magnetically retained by the matrix while the rest
of the fluid passes therethrough.
Graham '595 further discloses a piezoelectric transducer in fluid
communication with the matrix by means of the carrier fluid. When the matrix reaches
its loading capacity for cells, the carrier fluid is replaced by an elutriation
fluid. The piezoelectric transducer is then excited, to generate high frequency
acoustic waves through the fluid in the chamber. The acoustic waves dislodge the
cells (particles) from the matrix and are carried out by the elutriation fluid.
U.S. Patent No. 4,664,796 to Graham et al. (Graham et al. '796) discloses
an HGMS system for separating intact biological cells from a fluid medium. The
system includes a flow chamber containing a separation matrix having interstices
through which the fluid passes, and an associated magnetizing apparatus for coupling
magnetic flux with the matrix. The magnetizing apparatus includes a permanent
magnet having opposing North and South poles, and field guiding pole pieces. The
flux coupler is positioned to pass a strong magnetic field through the matrix during
the time that the carrier fluid passes therethrough to permit capture of the cells
or particles by the matrix.
The flux coupler is positioned so that the magnetic flux is diverted
away from the matrix during the elutriation phase, when the carrier fluid is replaced
by an elutriation fluid, so that the viscous forces of the elutriation fluid exceed
the weakened magnetic attractive forces between the matrix and the cells or particles,
thereby permitting the elutriation fluid to carry away the cells or particles.
Additionally, a piezoelectric transducer may be provided to be used in conjunction
with the diversion of the magnetic flux by the flux coupler during the elutriation
phase, to allow for a slower flow of elutriation fluid.
The matrix is positioned within the flow chamber so as to be subjected
to the full magnetic flux of the magnet when the flow chamber is in a first position,
during separation of the cells from the carrier fluid. When the flow chamber is
rotated approximately 90° from the first position, during the elutriation phase,
the matrix is positioned such that the magnetic flux substantially bypasses the
Graham et al. '795 further discloses the option of using a piezoelectric
transducer in fluid communication with the matrix for use in conjunction with the
positioning of the flux coupler to bypass the strong magnetic field around the
matrix, to allow lower flow rates of the elutriation fluid.
The prior art addresses various methods of HGMS and methods of recapturing
the cell/particles once they have been separated by HGMS. For very small samples,
however, such as those encounter in molecular biology applications, the prior art
is far from ideal for performing HGMS. Very small elution volumes are needed to
efficiently elute very small samples, such as, for example, in the separation of
messenger RNA from total RNA or cell lysates. Larger elution volumes require larger
volumes of enzymes for downstream applications, which become prohibitively expensive
and render the procedure inefficient and unusable. Additionally, small void volumes
are important in situations where chemical reactions are intended to be performed
within the column itself. The present invention is directed to more efficient and
effective use of the HGMS technique for separation of very small samples, especially
for use in clinical and commercial settings.
DISCLOSURE OF THE INVENTION
The present invention provides improvements in high gradient magnetic
separation of materials contained within very small volumes. The present invention
combines the advantages of a binding reaction in suspension (e.g., fast kinetics,
high efficiency) with those of a separation on a column (e.g., purity, simplicity),
while at the same time keeping the elution volume requirements low. Also, a small
void volume is provided for performance of chemical reactions within the column.
The separation techniques may be employed in a continuous process
or sequential processes, with the different steps of the separation being performed
by simply adding different buffers, chemicals, etc., also with potentially different
temperatures, e.g., hot water, etc., into a column. Thus, the complete procedure
is very fast.
The present invention provides a micro separation column having first
and second tubular portions, where the first portion is integral with the second
portion. The first portion has a first cross sectional area which is unequal to
the cross sectional area of the second portion. A matrix which is adapted to selectively
remove at least one component of a mixture as the mixture flows through the tube
is contained in at least part of the first portion and at least part of the second
The matrix contains ferromagnetic material, preferably ferromagnetic
balls or other ferromagnetic particles. The ferromagnetic material may be coated
with a coating which maintains the relative position of the particles with respect
to one another. Preferably, the coating comprises lacquer, and more preferably,
a lacquer as described in at least one of U.S. Patent Nos. 5,691,208; 5,693,539;
5,705,059; and 5,711,871, each of which are hereby incorporated by reference in
their entireties. The ferromagnetic balls or particles preferably have a diameter
or size of at least 100 µm, more preferably greater than about 200 µm and less
than about 2000 µm, still more preferably greater than about 200 µm and less than
about 1000 µm, and most preferably about 280 µm. The matrix (i.e., ferromagnetic
particles and coating) preferably occupies at least about 50 percent of the internal
volume of the first and second portions. The void volume of the column, that is
the interstitial volume which is not occupied by the matrix (i.e., the matrix void
volume) and the volume of the portion of the column that is below the matrix is
preferably less than about 85 µl, more preferably less than about 70 µl, still
more preferably less than about 50 µl, and most preferably about 30 µl. The self-adjusting,
gravitational flow speed is generally greater than about 100 µl/min, more preferably
greater than about 200 µl/min and most preferably greater than about 300 µl/min.
The tube may further comprise a third portion which is integral with
the second portion. The third portion has a third cross sectional area which is
less than the cross sectional area of the second portion. Still further, the tube
may include a fourth portion integral with the third portion. The fourth portion
has an outside dimension (e.g., and outside diameter, but may be an outside dimension
of a structure which is other than circularly shaped in cross-section) which is
less than a respective outside dimension of the third portion. An upper portion
may be provided which is integral with the first portion. The upper portion has
an cross sectional area which is greater than the cross sectional area of the first
Optionally, the micro separation column may include a retainer located
in the second portion adjacent the matrix. Preferably, the retainer is substantially
spherical, and is substantially larger thin the particles that make up the matrix.
Alternatively, the retainer may be a porous mesh or grid or frit.
The tube may be formed from a material such as PCTG, polyethylenes,
polyamids, polypropylenes, acrylics , PET, other plastics which are currently used
for single use laboratory products, and glass, and is preferably formed of a plastic
that will bind to lacquer, most preferably PCTG.
When a spherical retainer is employed, at least one mount preferably
extends into the second portion of the tube for resting the retainer thereon. Preferably,
three mounts are provided for support of the preferred spherically shaped retainer.
Optionally, an upper matrix retainer may be located in the first portion
of the tube, adjacent the matrix. Preferably, the upper matrix retainer comprises
a porous grid or mesh or frit. In addition to ferromagnetic materials, the matrix
may optional include one or more nonmagnetic components, such as glass particles
including spheres, or plastic particles or spheres.
Preferably, the micro separation column of the present invention is
designed to operate by gravity feed, but may alternatively be designed to operate
under a pressure feed.
A micro separation column according to the present invention includes
first and second tubular portions, with the first portion being integral with the
second portion, and a matrix adapted to selectively remove at least one component
of a mixture as the mixture flows through the tubular portions. The matrix is contained
in at least part of the first portion and at least part of the second portion.
The portion of the matrix which is contained in the first portion accomplishes
a greater removal function than the amount of matrix that is contained in the second
portion. The amount of matrix in the second portion accomplishes a greater flow
resistance function than the amount of matrix contained in the first portion. Preferably,
the overall height of the matrix is less than about 20 mm, more preferably less
than about 15 mm, and most preferably less than about 12 mm. Preferably, the height
of the matrix in the first portion is less than about 10 mm, more preferably less
than about 6 mm.
Further disclosed is a micro separation unit for use in performing
micro separation. The micro separation unit includes a magnetic yoke having at
least one notch formed along a length thereof A pair of magnets is placed within
each notch. Each pair of magnets defines a gap therebetween, which is adapted to
receive a micro separation column therein for performance of micro separation.
Preferably, the yoke is made of steel. Preferably, the yoke includes at least two
notches and more preferably, four.
Each pair of magnets forms a magnetic field in each respective gap
of greater than about 0.2 Tesla, preferably greater than about 0.4 Tesla, more
preferably greater than about 0.5 Tesla, and most preferably greater than about
The micro separation unit further includes a non-fragile covering
encasing the yoke and the magnets. Preferably, the covering is made of polyurethane
rubber. At least one mounting magnet may be further provided within the covering
for magnetically mounting the micro separation unit to a magnetic surface.
A micro column system according to the present invention includes
a micro separation unit comprising a magnetic yoke having at least one notch formed
along a length thereof, and a pair of magnets placed within each of said at least
one notch to form a gap therebetween; and at least one micro separation column,
each comprising: first and second tubular portions, with the first portion being
integral with the second portion, and a matrix adapted to selectively remove at
least one component of a mixture as the mixture flows through the tubular portions.
The matrix is contained in at least part of the first portion and at least part
of the second portion. The part of the matrix contained in the first portion accomplishes
a greater removal function than the amount of matrix contained in the second portion.
The number of micro separation columns equals the number of said gaps contained
in the yoke.
Another aspect of the present invention is related to a separation
and release process for purifying biological material on the micro column. After
retaining the biological material of interest coupled to magnetic particles in
the matrix, the bound material may optionally be dissociated from the magnetic
particles and eluted from the column while the magnetic particles are still magnetically
retained by the matrix. The dissociation may be performed by an adequate change
of buffers, temperature, chemical or enzymatic reaction which dissociates the link
between the magnetic particles and the biological material of interest.
BRIEF DESCRIPTION OF THE DRAWINGS
BEST MODE FOR CARRYING OUT THE INVENTION
- Figure 1 is a sectional view of a prior art column;
- Figure 2 is a sectional view of a preferred embodiment of a micro column according
to the present invention;
- Figure 3 is a sectional view of a micro column according to the present invention;
- Figure 4 is a sectional view of a column, the section being taken perpendicular
to the section shown in Figure 3 at a level indicated by lines IV-IV;
- Figure 5 is a sectional view of a variation of the micro column according to
the present invention;
- Figure 6 is a sectional view showing another variation in the micro column
according to the present invention;
- Figure 7 is a perspective view of the micro column separation system according
to the present invention;
- Figure 8A is a top view of a separation unit according to the present invention;
- Figure 8B is a front view of the separation unit shown in Figure 8A;
- Figure 8C is a top view of the separation unit, with the internal components
shown in phantom lines; and
- Figure 9 shows the composition of drops 1 through 5 (percentage of the mRNA
sample eluted) from Olig(dT) MicroBeads retained in a micro column system, as displayed
on an agarose gel.
The separation of very small samples such as those encountered in
many molecular biology applications, e.g., mRNA, by HGMS calls for the use of very
small elution volumes to efficiently and effectively elute the samples, and for
reaction in a small volume, a small void volume is also required. As an illustration
of the need, a prior art column such as that shown in Figure 1 includes a matrix
1010 of metal spheres of about 280µm size which give a porosity of about 28µm.
The column height of the matrix 1010 is about 20 mm, the void volume of the matrix
1010 is about 70 µl, and the void volume of the column is about 85 µl. The flow
rate through the matrix of spheres is about 400 µl/min.
A simple reduction in the column height of the matrix 1010, while
serving to reduce the volume of the same, is not effective in processing the small
samples referred to since the resultant flow rate through the matrix is too great.
A reduction in the cross sectional area of the matrix increases the probability
of clogging as well as reducing separation speed. A reduction in the height of
the fluid column reduces and possibly eliminates drip formation at the end of the
column, since the pressure head generated must be great enough to overcome the
surface tension at the end of the column where the drips form.
The present invention successfully addresses all of the above-mentioned
potential problems. A preferred embodiment of the present invention 100 is shown
in Figure 2. The micro column 100 is substantially reduced in void volume in comparison
to columns used in the prior art, while maintaining optimal flow speeds, and is
designed for the separation of macromolecules (or cells), that are magnetically
bound via specific biological/chemical interactions, from other molecules (or cells)
in a high gradient magnetic field and for the elution of these molecules/cells
in a small volume. The micro column is made hydrophilic by manufacturing it from
a hydrophilic material such as a hydrophilic plastic, or, more preferably, by coating
the column interiorly with a hydrophilic material, e.g., polyvinyl pyrrolidone.
Alternatively, or in addition thereto, buffers which are poured into the column
may contain one or more surfactants, e.g., SDS.
The matrix 110 includes a first portion 110a having a relatively larger
cross sectional area than that of a second portion 110b. The column 100 includes
a relatively large volume reservoir 112 into which a sample to be separated is
poured. The reservoir 112 funnels 114 into a smaller cross sectional area first
portion 116 of the column that houses the first portion 110a of the matrix. The
first portion narrows down to an even smaller cross sectional area second portion
118 of the column that houses the second portion 110b of the matrix. Although all
of the columns shown in the Figures are of the preferred cylindrical configuration,
the present invention is not to be so limited. For example, the columns may be
formed to have an elliptical cross-section, a square cross section, other geometric
cross-sections or even non-geometric cross-sections. Additionally, the shapes of
the portions do not have to be alike. For example, a first portion might have a
hexagonal cross-section while the second portion might be cylindrical.
The matrix 110 contains ferromagnetic material, preferably balls 120,
but may be other particles which are not spherical, or an integrated three dimensional
mesh having the desired porosity. The ferromagnetic material 120 may be coated
with a coating which maintains the relative position of the particles with respect
to one another. Preferably, the coating is a lacquer. The balls/particles have
a size greater than about 100 µm, preferably greater than about 200 µm ad less
than about 2000 µm, more preferably greater than about 200 µm and less than about
1000 µm, and most preferably about 280 µm. Examples of separation matrices which
are useful for HGMS are more thoroughly described in copending application No.
08/377,744, filed January 23, 1995, as well as U.S. Patent No. 5,411,863, both
of which are hereby incorporated by reference thereto in their entireties. The
matrix preferably occupies at least 50 percent of the internal volume of the first
and second portions.
The column 100 is preferably made of plastics such as polypropylenes,
polyethylenes, acrylics, PET, etc, and, when the matrix is coated with lacquer,
is preferably made of a plastic that will bind with lacquer, most preferably a
resin such as PCTG (polycyclohexadimethylterephtalate modified with Ethylenglycol).
This makes the production of the columns much simpler, since it eliminates a need
to remove excess lacquer after the step of pouring lacquer into the column to coat
the ferromagnetic particles. When the column is made of a material such as polypropylene,
the excess lacquer must be removed from the walls of the column after coating the
ferromagnetic particles. This is a time consuming, tedious step which significantly
increases the cost of production of the columns.
A high gradient magnetic field is generated in the matrix 110 upon
insertion into an external magnetic field. The matrix readily demagnetizes when
it is taken out of the field. The flow rate is lower in the first portion 110a
of the matrix than in the second portion 110b. The first portion 110a of the matrix
primarily performs the separation function, since it is of a larger cross sectional
area and volume that the second portion 110b. The magnetized particles of the matrix
110 retain single superparamagnetic MicroBeads (of an average diameter of 50 nm
/ as specified by Miltenyi Biotec) and material attached to them from a solution
or reaction mixture of variable viscosity, which flows through the column 100,
preferably by gravity. The bound material can be eluted in a small volume. The
second portion 110b primarily performs a flow resistor function, since it is of
a significantly lesser cross-sectional area than the first portion 110a and also
may be formed of smaller sire particles. Of course, the first portion 110a also
performs as a resistive element to some extent. The second portion 110b preferably
functions as a separator somewhat, although it may alternatively be formed entirely
of nonmagnetic particles such as plastic or glass, in which ease, it would function
only as a resistive element.
Thus, glass balls/particles 120' or plastic balls/particles or other
non-ferromagnetic balls or particles may be substituted for some of balls/particles
120 in the first and/or second portions without unduly affecting the separation
capability of the column and matrix, and without affecting the resistive function
of the second portion, see Figure 5. In some instances, all of the balls/particles
120 in the second portion may be so substituted. Preferably, the micro separation
column of the present invention is designed to operate by gravity feed, but may
alternatively be designed to operate under a pressure feed. To permit this, a plunger
160 fits into the reservoir 112 and can be used to flush out the bound material.
In addition, bound material (e.g., cells) can be eluted in a minimum volume by
A porous frit or grid 140 may be positioned adjacent the top end of
the matrix 110, particularly for those embodiments having particles or balls which
are freely displaceable, i.e., not held in place by a lacquer or other binding
agent. The porous frit/grid is preferably made of glass or plastic or metal mesh
and has a pore size greater than or equal to the pore size of the matrix and less
than the particle/ball size of the matrix.
In place of the ball shaped retainer 130, a porous frit or grid 150
may be positioned adjacent the bottom end of the matrix 110, for those embodiments
having particles or balls which are freely displaceable, as well as for those held
in place by a lacquer or other binding agent, see Figure 6. The porous frit or
grid is preferably made of glass or plastic or metal mesh and has a pore size greater
than or equal to the pore size of the matrix and less than the particle/ball size
of the matrix.
When balls 120 are used to form the matrix 110, the ball size is greater
than 100 µm, preferably greater than about 200 µm and less than about 2000 µm,
more preferably greater than about 200 µm and less than about 1000 µm, and most
preferably approximately 280µm. Of course, the size of the balls may be modified
to calibrate or vary a desired rate of flow through the matrix. However, too great
a reduction in the ball size can lead to clogging because of the concurrent reduction
in the pore size in between the balls. On the other hand, too great an increase
in the size of the balls can lead to a flow rate which is unacceptably fast, which
negatively effects the per cent retention of the magnetic particles.
A minimum height of the fluid column (i.e., the height of the fluid
above the tip end of the column) is required to generate sufficient pressure to
overcome the surface tension where drop formation occurs, to ensure a proper flow.
The second portion 110b effectively increases the resistance and allows a lower
overall height of matrix 110 to be used, thereby also reducing the effective volume
of the matrix 110. The overall height of the matrix 110 is less than about 20 mm
and preferably is less than about 15 mm, most preferably less than about 12 mm.
Where small elution volumes are important, the void volume of the column, i.e.
the interstitial area within the matrix that is not occupied by the balls/particles
and the volume of the column extending beneath the matrix, is generally less than
about 85 µl, preferably less than about 70 µl, more preferably less than about
50 µl, and most preferably about 30 µl.
Another factor to be considered in designing a column is the surface
tension that is generated at the end of the column where drops form as the liquid
exits the column. As the column length or height increases, a greater pressure
head is developed to overcome the surface tension. If the surface tension is too
great relative to the pressure head, drop formation at the end of the column will
be compromised and possibly even prevented, thereby halting flow through the column.
Thus, it is necessary to form a third portion 122 of the column, to extend the
length to the end 126. The third portion 122 has a smaller inside cross sectional
area than the second portion 118, as well as a smaller outside dimension (e.g.,
diameter, in the case of a cylindrical portion). The length of the third portion
may vary according to the respective cross sectional areas and the desired flow
Table 1 shows the effect of first, second and third portion cross
sectional areas and heights on flow rate and the correlation between flow rate
and percentage recovery of MicroBeads.
Recovery in correlation to the flow rate.
Matrix diameter x height mm
2nd Matrix diameter x height mm
Extension diameter x height mm
Flow rate ml/min
Recovered MicroBeads %
1.9 x 2.7
0.8 x 12
1.9 x 3.5
0.8 x 12
1.9 x 4.5
0.8 x 12
1.9 x 6.0
0.8 x. 12
When using a spherical retainer 130, at least one mount 128 extends
from the top end of the third portion 122 and into the second portion. Each mount
128 is preferably peg-shaped (see also Figure 3). Preferably a set of three mounting
elements 128 (see Figure 4) extend from the third portion into the second portion
and function to support the spherical retainer 130. Retainer 130 is preferably
a ball that is substantially larger than the balls 120 and is sized to prevent
the escape of balls 120 into the third portion during filling of the column 100
with the matrix 110 and all the time when the balls are not held in place with
a lacquer. However, the retainer wall 130 also maintains passages which are at
least as large as the spaces between balls 120 in the matrix 110 so as not to impede
the flow of fluid though the second portion 118 and into the third portion 122.
The distal end of the third section 122 tapers into a tip 126. The
outside dimension (e.g., outside diameter when the tip is the tip of a cylindrical
tube) of the tip 126 is smaller than that of the third section and defines the
preferred drop size of fluid to exit the column. One preferred embodiment has an
outside diameter of about 1.5 mm, but of course, this dimension may be varied by
shaping the end or "nozzle" of the column according to the drop size that is desired.
Another aspect of the invention is related to a separation and release
process for purifying biological material on the column 100. After retaining the
biological material of interest coupled to magnetic particles in the matrix 110,
the bound material may optionally be dissociated from the magnetic particles and
eluted from the column 100 while the magnetic particles are still magnetically
retained by the matrix 110. The dissociation may be performed by an adequate change
of buffers, temperature, chemical or enzymatic reaction which dissociates the link
between the magnetic particles and the biological material of interest. For example,
mRNA may be released form Poly-T conjugated beads by a change of buffer composition
and temperature preferentially above 30°C. Materials bound by antibodies, protein
A or G may be released in the column by changing pH, salt conditions, chemicals
(DTT for SPDP links) or introducing detergents, e.g., SDS or chaotropic agents.
The micro column 100 is designed for use in a micro column HGMS system
according to the present invention. The system 300 includes a separation unit 200
which holds one or more micro columns 100 (four in the preferred embodiment) as
shown in Figure 7. The micro separation unit includes a yoke 210 that forms the
basic framework of the unit and that concentrates the magnetic fields. The yoke
is configured to include a notch 212 in the each area where processing with a micro
column is intended to occur.
A pair of magnets 214 are mounted in each notch 212 so as to form
a narrower gap 216 where the magnetic field of the magnets is focused and where
a micro column is to be received for performing HGMS separation. As noted, in the
preferred embodiment shown in the figures, the yoke 210 connects four pairs of
strong permanent magnets (Figure 8C), that cooperatively produce the magnetic field
needed for four parallel separation processes in four columns. It is reiterated
that, of course, the present invention is in no way to be limited to the configuration
of four micro column stations, as other numbers could just as easily be configured.
Two magnets 218 are preferably connected to the back of the yoke 210
to facilitate attachment or mounting of the unit to a ferromagnetic device such
as a iron stand. Again, a different number of magnets 218 might be used for mounting.
Additionally, other mounting means such as clamps, screws, bolts, etc. could be
alternatively or additionally employed.
The unit thus far described is entirely encased in a non-fragile covering
220. The non-fragile covering protects the internal components of the unit 200
as well as makes the unit more "user friendly" in that it is more pleasant to the
touch (warmer, softer) and is much more easy to clean/sterilize. Preferably, the
covering 220 is a layer of foam of a resin such as a polyurethane rubber, which
protects the unit 200 against corrosion and chemical or mechanical damage. Other
alternative covering materials that serve the same purpose may be employed.
Each gap 216 of the separation unit 200 has a magnetic field that
is greater than 0.2 Tesla, preferably greater than 0.4 Tesla, more preferably greater
than about 0.5 Tesla, and most preferably greater than about 0.6 Tesla. A preferred
embodiment generates magnetic fields in the range of about 0.6 - 0.7 T. Table 2
shows the relationship between the strength of the applied magnetic field and the
amount of MicroBeads that are recovered as a result thereof. The trend is the same,
independent of the type of column used.
Recovery of MicroBeads in correlation to the strength of the magnetic field.
Magnetic field (Tesla)
As shown in Figures 8A ad 8B, covering 220 forms bevels 222 at the
top and bottom of each of the gaps 216. The bevels are designed to mate with the
funneling portion 114 of the micro column, which further stabilizes the micro column
in a vertical position within gap 216. The bevels 222 are formed at the top and
bottom of each gap 216 to render the unit 200 symmetrical about its horizontal
axis. Thus, the top and bottom of the unit are identical and it is therefor impossible
for a user to employ the unit "upside down". As shown in Figure 8B, the angle of
the bevel 222 is preferably about 90°, but this angle can of course vary according
to the slope of the funneling of a micro column to be held in the gap and bevel.
Example 1 - To achieve a small elution volume (<50 µl) the
part of the micro column filled with matrix had a total volume of 52 mm3
leaving space for 22 µl of fluid (matrix volume) when standard ferromagnetic material
was used (iron balls of an average diameter of 280 µm). Together with the volume
in the portion 122 of the column, the void volume of the column that was relevant
for the elution was 29 µl.
To ensure that more than 90% of the MicroBeads applied to the column
(in a buffer containing detergent), (in a magnetic field of 0.6 - 0.7 T) were retained
at a matrix of a height of 11 mm, the flow rate of the MicroBead suspension had
to be regulated. For this reason the matrix was bipartite. The lower 6 mm part
of the matrix (i.e., 110b) had a inside diameter of only 1.9 mm which had severe
impact on the flow rate whereas the upper 5 mm of the matrix (i.e., 110a) had a
larger diameter of 3 mm to decrease the probability of clogging of the column.
The matrix was delimited at the bottom by a steel ball (i.e., 130)
of 1.6 mm diameter. Below this the inner cross sectional area of the tube (i.e.,
122) was reduced to 0.8 mm. The steel ball was positioned on three bridges (i.e.,
mounts 128) that kept it from closing the tube. The steel ball prevented the ferromagnetic
material from slipping out during the filling process.
To make sure that the column allowed drop formation by gravity when
the buffer was applied on top of the matrix, the total height of the part of the
column filled with buffer was empirically determined to be 24 mm. For that reason
the column was extended beyond the matrix area by a tube 122 with a length of 12
mm and a diameter of 0.8 mm.
The matrix plus bottom extension had a calculated void volume of 29
µl. To achieve a minimal elution volume the first fraction of buffer that flowed
from the column during such an elution (an amount of buffer that comes close to
the void volume) could be skipped since it would not contain any of the eluted
material. The buffer drop size is designed to be smaller than about 80% of the
void volume of the column so that the first drop can be thrown out. For this reason
the drop size of (detergent-free) buffer was defined to be approximately 24 µl.
This was achieved by adjusting the diameter of the bottom tip of the column to
In addition, the controlled drop size led to a defined elution volume.
Drops 2 and 3 contained >80% of the eluted material (see Figure 9) and drops
2-4 contained >90% of the eluted material.
The micro columns 100 placed in the separation unit 200 described
above can bind at least 2 mg of MicroBeads as determined by optical density of
the MicroBeads at a wavelength of 450 nm (Table 1). About 90 to 98% of 0.1 - 2
mg basic MicroBeads (Miltenyi Biotec GmBH) applied to the column are retained in
the magnetic field as determined by optical density of the MicroBeads at a wavelength
of 450 nm (Table 1).
Since the flow rate is primarily maintained by the 1.9 mm diameter
part of the matrix it is easy to reduce or enhance the flow rate by changing the
diameter of the balls. The flow rate of buffer (containing detergent, 1% SDS) in
a column with a standard matrix (280 µm balls) is 300 µl/min. The flow rate of
a column with balls of an average diameter of 230 µm is 200 µl/min. The average
flow rate of automatically produced columns with a matrix of 280 µm balls is 320
+/- 100 µl. The average drop size of water is 23.9 µl.
For may applications it is advantageous to elute the bound material
from the MicroBeads while the MicroBeads are still bound to the matrix in the magnetic
field. In this case the material is eluted by adding a different buffer that breaks
the chemical interactions between the retained molecule and the catching agent.
One example for the separation of macromolecules is the isolation of mRNA from
crude cell extract via the specific interaction of oligo(dT) coupled to MicroBeads
with the poly A tail of the mRNA. (Approximately 0.01% of the total cell mass is
1 x 107 cultured hybridoma cells were washed in PBS, the
pellet was resuspended and lysed in 1 ml of a lysis/binding buffer (0.1 M Tris/HCL
pH 8.1, 1% SDS, 0.2M LiCl, 10 mM EDTA, 5mM DDT. The SDS completely inactivates
the activity of cellular RNAases, which are set free by the lysis.)
To strongly reduce the high viscosity of the lysate, caused by genomic
DNA, it was centrifuged through a porous matrix (2 min. at 13000 x g through three
layers of blotting paper placed on a porous polypropylene filter. This procedure
does not interfere with the integrity of the mRNA.)
50 µl of oligo(dT) MicroBeads were added to the lysate and the lysate
was mixed. (For the hybridization of mRNA to oligo(dT) MicroBeads no additional
incubation is necessary).
A column placed in the magnet was prepared by adding 100 µl of lysis/binding
buffer. The lysate was added. After it had flowed through the matrix, two 250 µl
aliquots of lysis/binding buffer were added, to wash away all unbound material
(proteins, DNA) and four 250 µl aliquots of wash buffer (50 mM Tris/HCL pH 7.5,
25 mM NaCl, 1 mM EDTA) were added, to wash away all unspecifically bound material
To elute the mRNA from the MicroBeads, 200 µl of 65°C elution buffer
(1 mM EDTA) was added. Drops 1 through 5 were collected in separate tubes and
analyzed on a 0.8% agarose gel stained with Ethidiumbromide (see Figure 9).
Example 2 - Immunomagnetic isolation of protein with Protein G MicroBeads
Percent recovery of approx. 100 µg of MicroBeads of different batches applied
to different columns. a) diameter of matrix balls: 230 µm
Percent recovery of approx. 2 mg. of MicroBeads of batch B applied to column 1.
b) diameter of matrix balls: 280 µm
Column 1 97.8
Another example for the separation of macromolecules is the isolation
of protein from crude cell extract via antibodies, that bind to the protein and
are then caught by protein G coupled to magnetic MicroBeads.
1 x 107 mouse liver cells were lysed in 1 ml of a lysis
buffer, that left the nuclei intact (150 mM NaCl, 1% Triton x 100, 50 mM Tris pH
8.1). The nuclei were removed by centrifugation. The supernatant was spiked with
100 ng of Phycoerythrin. It was then mixed with 1 µg of a monoclonal anti Phycoerythrin
antibody and incubated at 6 °C for 5 - 30 min. 10 µl of Protein G MicroBeads (carrying
0.5 µg recombinant Protein G) were added, the reaction mixture was briefly mixed
and incubated for an additional 5 - 30 min. at 6 °C.
A Micro-column was placed in the described magnetic separator and
prepared by washing with 100 µl of lysis buffer. The reaction mixture was applied
onto the column. After the reaction mixture had completely flowed through the column,
the column was washed by adding 3 x 125 µl lysis buffer ad 4x with 125 µl PBS.
For elution the column was left in the magnetic separator and the
buffer was exchanged by adding 50 µl of an SDS gel sample buffer (containing 1%
SDS). The buffer was incubated in the column for 3 min. to dissolve the immunomagnetic
complexes. Then the elution proceeded by adding 75 µl of sample buffer and collecting
the drops (2-4), which contained the antigen and the antibody eluted from the column.
Due to the surfactant (SDS) the drops have an average volume of 15 µl, thus the
total elution volume is 45 µl.
The separation was analyzed on an SDS Polyacrylamide gel, the results
of which are shown in Figure 10. Proteins were made visible by silver staining.
"A" and "B" in Figure 10 represent eluants of two independent isolations. "C" represents
a size marker. "D" represents the anti Phycoerythrin antibody and "E" represents
the Phycoerythrin. "F" represents the flow through of one separation.
This method of immunoaffinity purification can be performed in less
than an hour. It omits the centrifugation steps and long incubation periods, typical
for standard immunoprecipitation protocols. In addition it yields very high purities.
With the highly sensitive silver staining procedure nearly only the antibody and
the antigen is detectable on the SDS-PAGE shown.