WO2011023354A1

WO2011023354A1 - Column adaptor

Info

Publication number: WO2011023354A1
Application number: PCT/EP2010/005158
Authority: WO
Inventors: Susanne Nath
Original assignee: F. Hoffmann-La Roche Ag
Priority date: 2009-08-25
Filing date: 2010-08-23
Publication date: 2011-03-03

Abstract

The current invention is directed to an adaptor for placing for example a small scale chromatography column above the center of a well of a multi well plate, whereby the adaptor has an outer shape that is congruent to the outer shape of the multi well plate, the adaptor comprises a multitude of holes each hole being above the center of a well of the multi well plate when placed below the adaptor and each hole has a screw thread or a fitting for connecting to a small scale chromatography column, and the adaptor does not extend beyond the shape of the multi well plate for more than two time the wall thickness of the adaptor.

Description

Column Adaptor

Herein is reported an adaptor for placing small scale chromatography columns above the wells of multi well plates in a way to prevent contamination of neighboring wells. The invention comprises the column adaptor and its use in high throughput chromatography resin screening.

Background of the Invention

Different methods are well established and widespread used for protein purification, such as affinity chromatography with microbial proteins (e.g. protein A or protein G affinity chromatography), ion exchange chromatography (e.g. cation exchange (sulfopropyl or carboxymethyl resins), anion exchange (amino ethyl resins) and mixed-mode ion exchange), thiophilic adsorption (e.g. with beta- mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. with phenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g. with Ni(II)- and Cu(II)-affinity material), size exclusion chromatography, and electrophoretical methods (such as gel electrophoresis, capillary electrophoresis) (see e.g. Vijayalakshmi, M.A., Appl. Biochem. Biotech. 75 (1998) 93-102).

The development and optimization of chromatographic methods is a time consuming task due to the fact that a multitude of different combinations of chromatography resins and chromatography conditions have to be evaluated. In order to reduce the required time and resources for evaluating the different chromatography resins a scale down of the process is required.

Exemplary small scale chromatography columns are reported in US 2009/0065415.

DE 197 12 195 reports a method and device for the off-line detection of analytes using MALDI-mass spectrometry. A multiple column chromatography assembly is reported in US 5,603,899. In WO 02/096565 a vacuum manifold for both multi- well plate and individual columns is reported. A modular solid phase extraction plate assembly is reported in WO 00/33960.

Summary of the Invention The first aspect of the current invention is an adaptor, that a) consists of metal and/or plastic, and

b) has a rectangular-U-like cross-section, and

c) has an upper edge point, and

d) has an approximately rectangular outer shape when viewed from the top or the side,

e) comprises a multitude of circular holes, whereby each hole has a screw thread on its interior or is connected to a chromatography column fitting.

In one embodiment the holes of the multitude of circular holes are arranged in a pattern. In a further embodiment a hole is at the upper edge point. In another embodiment the pattern is a linear or diagonal pattern. In still a further embodiment each hole of the multitude of holes is at the intersection of the diagonals of the square formed by the four holes with the smallest distance to the hole. In another embodiment the holes are on the grid points of a grid whereby not on every grid point has to be a hole. In one embodiment a chromatography column is connected to each hole.

In one embodiment the adaptor has a hole in the upper edge point and a) if the diameter of the chromatography column is bigger than the distance of two linearly adjacent grid points, but smaller than or equal to the distance between a first grid point in a first row and a second grid point in a second row of the grid, whereby the first and second row are directly side by side,

and

the first grid point is in a first column and the second grid point is in a second column of the grid,

whereby the first column and the second column are the same,

then

a hole is at every second grid point in each row in each column of the grid, whereby alternately the first grid point and the second grid point of a row have a hole,

or

b) if the diameter of the chromatography column is bigger than the distance of two linearly adjacent grid point, but smaller than or equal to the distance between a first grid point in a first row and a second grid point in a second row of the grid, whereby the first and second row are directly side by side,

and the first grid point is in a first column and the second grid point is in a second column of the grid,

whereby the first column and the second column are directly side by side then

a hole is at every second grid point in each row in every second column of the grid,

or

c) if the diameter of the chromatography column is bigger than the distance of a first grid point in a first row and a second grid point in a second row of the grid, whereby the first and second row are directly side by side,

and

whereby the first column and the second column are directly side by side, but the diameter of the chromatography column is smaller than or equal to the distance of a first grid point in a first row and a second grid point in a second row of the grid,

whereby the first and second row are directly side by side,

and

whereby the first column and the second column are separated by one column, then

a hole is at every fourth grid point in every fourth column of the grid. In one embodiment the chromatography column has an internal diameter of 8 mm and the grid is a 12-by-8 grid. In one embodiment the chromatography column has an internal diameter of 11.3 mm and the grid is a 12-by-8 grid. In a further embodiment the adaptor has a height of at least the height of the fitting of the chromatography column extending through the adaptor plate. Another aspect of the invention is the use of an adaptor according to the invention for the evaluation of column chromatography resins for the purification of a polypeptide. A further aspect is the use of an adaptor according to the invention in the determination of column chromatography conditions for the purification of a polypeptide. Still another aspect of the invention is a method for determining column chromatography condition for the purification of a polypeptide comprising the following steps a) providing an adaptor according to the invention,

b) providing a multi well plate,

c) providing a chromatography column containing a chromatography material, d) providing a polypeptide,

e) providing a set of column chromatography conditions,

f) connecting the chromatography column to the adaptor,

g) conditioning, i.e. equilibrating and washing, the chromatography column according to the set of column chromatography conditions,

h) combining the adaptor and the multi well plate, whereby each hole of the adaptor is placed above the center of a well of the multi well plate, i) applying the polypeptide to the chromatography column,

j) recovering the polypeptide from the chromatography column according to the set of column chromatography conditions,

k) determining the purity of the recovered polypeptide with respect to each condition of the set of column chromatography conditions,

1) repeating steps f) to k) until all provided chromatography columns containing a chromatography material and/or all provided conditions of the set of column chromatography conditions have been employed in steps g) to j), m) determining the conditions of the set of column chromatography conditions which provides for the best combination of yield and purity of the polypeptide to be the column chromatography condition for the purification of the polypeptide.

Also an aspect as reported herein is a multi-well plate adaptor comprising

a rectangular top plate (101) with longer edges (102, 102a) and shorter edges (103, 103 a) comprising a multitude of holes (108) arranged in rows (109) and columns (110), each hole having a means for taking up a small scale chromatography column, and

rectangular side plates (104, 105, 106, 107) all of the same height (1 12) arranged

a) at and along the edges (102, 102a, 103, 103a) of the top plate (101), b) perpendicular to the top plate (101), and

c) to the same side of the top plate ( 101 ), whereby

the longer edge has a length of from 127.5 mm to 128.2 mm,

the shorter edge has a length of from 85.0 mm to 86.0 mm,

the first hole is at a distance of from 7.2 mm to 11.5 mm from the longer edge and at a distance of from 10.0 mm to 17.1 mm from the shorter edge, and

the holes beside the first hole are arranged as follows

- the minimum distance between two holes in a row or a column is of from 8.0 mm to 10.2 mm and the maximum distance between two holes in a row or a column is of from 9.0 mm to 68.1 mm,

- the minimum distance between a first hole and a second hole in a proximate row and a proximate column is of from 12.7 mm to 26.4 mm and the maximum distance between a first hole and a second hole in a proximate row and a proximate column is of from 12.7 mm to 84.3 mm. Also an aspect as reported herein is the use of the multi-well plate adaptor as reported herein for mounting chromatography columns.

A further aspect is a device comprising a multi-well plate adaptor and a multi-well plate, whereby the multi-well plate is placed below the multi-well plate adaptor, wherein the multi-well plate adaptor comprises - a rectangular top plate (101) with longer edges (102, 102a) and shorter edges (103, 103a) comprising a multitude of holes (108) arranged in rows (109) and columns (110), each hole having a means for taking up a small scale chromatography column, and

- rectangular side plates (104, 105, 106, 107) all of the same height (112) arranged

c) to the same side of the top plate (101),

whereby

- the longer edge has a length of from 127.5 mm to 128.2 mm,

- the shorter edge has a length of from 85.0 mm to 86.0 mm.

Another aspect of the invention is a kit comprising an adaptor according to the invention, a set of chromatography columns, and a set of chromatography materials. Detailed Description of the Invention

The holes of the column adaptor as reported herein are arranged in a way that when a small scale chromatography column is mounted to a hole the mobile phase droplets leaving the column at the outlet will fall into the well of a below placed multi -well-plate without on the one hand contamination of adjacent wells and on the other hand without liquid losses. This can be ensured by maintaining a minimum distance between the outer surface of the droplet which is still attached to the outlet of the column and the wall of the well below.

Herein is reported an adaptor for placing e.g. a small scale chromatography column above the center of a well of a multi well plate, whereby the adaptor has an outer shape that is congruent to the outer shape of the multi well plate, the adaptor comprises a multitude of holes each hole being above the center of a well of the multi well plate when placed below the adaptor and each hole has a screw thread or a chromatography column fitting for connecting to a small scale chromatography column, whereby the adaptor does not extend beyond the shape of the multi well plate for more than two time the wall thickness of the adaptor.

The term "small scale chromatography column" as used in the current invention denotes a chromatography column with at least one of the following parameters a) a bed volume of from 0.2 ml to 10 ml,

b) an internal diameter of from 5 mm to 100 mm,

c) a length of from 10 mm to 100 mm.

In one embodiment a small scale chromatography column has an internal diameter of from 5 mm to 12 mm, a length of from 10 mm to 100 mm, and a volume of from 0.2 ml to 10 ml. The column has in one embodiment a screw thread on its outer surface and in a different embodiment the column has a fitting for connecting to the adaptor at its lower end. The holes of the adaptor according to the current invention comprise accordingly in one embodiment the respective complementary screw thread in order to be connected with the screw thread at the fitting of the column or in a different embodiment the respective complementary fitting in order to be connected with the fitting at the lower end of the column. In all embodiments the column is connected to the adaptor via its lower end in a liquid-tight form.

A small scale chromatography column comprises (for numbers see Figure 1) a column body (4) to which optionally at it upper end (2) and/or its lower end (3) a closing (6) may be attached, an outlet (5), and a lower end (1). The lower end may comprise a screw thread as shown in Figure 1 cl) or a connector as exemplary shown in Figure 1 c2). The inner diameter of the column is denoted as d, and the outer diameter of the column is denoted as d. The outer diameter d represents the maximum outer diameter of the chromatography column. The column body has a height h which can be the same or different from the height II_R of the chromatography material contained in the column.

The term "multi well plate" as used within the current invention denotes a plate containing equally spaced apart wells each with the same diameter. In different embodiments is the multi well plate a 6 well plate or a 12 well plate or a 24 well plate or a 48 well plate or a 96 well plate. An exemplary 24 well plate is shown in Figure 2 a). As can be seen from Figure 2 is a multi well plate not a square but a rectangle with a longer side and a shorter side. Thus, a multi well plate comprises rows and columns. Within the current application denotes the term "row" the wells arranged in the direction of the longer side and the term "column" the wells arranged in the direction of the shorter side. Thus, a row comprises a higher number of wells than a column.

The term "grid" as used within the current invention denotes a pattern comprising grid points. The grid points are arranged in rows and columns. As can be seen from Figure 2 b) a "grid" as used within the current invention has a rectangular form, in which each row is perpendicular to each column. The term "row" encompasses the grid points in the direction of the longer side of the grid and the term "column" encompasses the grid points in the direction of the shorter side of the grid. Thus, a row comprises a higher number of grid points than a column. The term "X-by-Y grid" as used within the current invention denotes that the grid has X grid points per row, i.e. columns, and a total of Y rows.

In an embodiment the adaptor has a length of from 127 mm to 131 mm, in one embodiment of 127.5 mm to 130.5 mm. In another embodiment the adaptor has a height of from 10 mm to 20 mm, in one embodiment of from 14.5 mm to 15.5 mm. In an embodiment the adaptor has a width of from 85 mm to 89 mm, in one embodiment of from 87.2 mm to 88.5 mm. In an embodiment the adaptor has a wall thickness of from 1 mm to 3 mm, in one embodiment of from 1.5 mm to 2 mm.

In one embodiment the grid is a 3:2 rectangular grid. In one embodiment the grid is a 3-by-2 grid. A 3-by-2 grid corresponds to the well pattern of a 6 well multi well plate. In this embodiment the grid points have a distance of 39 mm to 40 mm in each row and between the rows. In this embodiment the upper edge point is at 24 mm to 30 mm from the shorter side of the adaptor and at 23 mm to 29 mm from the longer side of the adaptor depending on the wall thickness of the adaptor.

In one embodiment the grid is a 4-by-3 grid. A 4-by-3 grid corresponds to the well pattern of a 12 well multi well plate. In this embodiment the grid points have a distance of 25.5 mm to 26.5 mm in each row and between the rows. In this embodiment the upper edge point is at 24 mm to 30 mm from the shorter side of the adaptor and at 16 mm to 22 mm from the longer side of the adaptor.

In one embodiment the grid is a 6-by-4 grid. A 6-by-4 grid corresponds to the well pattern of a 24 well multi well plate. In this embodiment the grid points have a distance of 19 mm to 20 mm in each row and between the rows. In this embodiment the upper edge point is at 17 mm to 23 mm from the shorter side of the adaptor and at 13 mm to 19 mm from the longer side of the adaptor.

In one embodiment the grid is an 8-by-6 grid. An 8-by-6 grid corresponds to the well pattern of a 48 well multi well plate. In this embodiment the grid points have a distance of 12.5 mm to 13.5 mm in each row and between the rows. In this embodiment the upper edge point is at 18 mm to 24 mm from the shorter side of the adaptor and at 10 mm to 16 mm from the longer side of the adaptor.

In one embodiment the grid is a 12-by-8 grid. A 12-by-8 grid corresponds to the well pattern of a 96 well multi well plate. In this embodiment the grid points have a distance of 8.5 mm to 9.5 mm in each row and between the rows. In this embodiment the upper edge point is at 14 mm to 20 mm from the shorter side of the adaptor and at 11 mm to 17 mm from the longer side of the adaptor.

The term "above the center of a well" as used within the current invention denotes that a hole of the adaptor or likewise the outlet of a small scale chromatography column is placed at a minimum distance of about 1.85 times the radius of the droplets obtained at the outlet of the small scale chromatography column from the wall of the well. It has been found that this is highly advantageous in order to prevent the droplets at the column outlet from touching the wall of the well and thereby contaminating the content of a neighboring well. The term "outlet" denotes the place at which the mobile phase leaves the small scale chromatography column. In one embodiment is the minimum distance at least 1.85 times the radius of a droplet of a volume of about 25 μl obtained at a column outlet with an outlet diameter of 0.5 mm. The term "congruent" as used within the current application denotes that the adaptor has the same outer shape as a multi well plate when viewed from the top, i.e. when the adaptor is placed on the multi well plate the shape of the multi well plate does not change when viewed from the above or from the below. This does not mean that the adaptor may not extend beyond the dimension of the multi well plate as long as the adaptor has the same outer shape as the multi well plate.

The term "chromatography material" as used within this application denotes a solid material comprising a bulk core material to which chromatographical functional groups are attached, for example by covalent bonds. The bulk core material is understood to be not involved in the chromatographic process, i.e. in the interaction between the substance to be separated and the chromatographical functional groups of the chromatography material. It is merely providing a three dimensional framework to which the chromatographical functional groups are attached and which ensures that the solution containing the substance to be separated can access the chromatographical functional groups. In one embodiment the bulk core material is a solid phase. Thus, in one embodiment the "chromatography material" is a solid phase to which chromatographical functional groups are attached. In one embodiment the "chromatographical functional group" is an ionizable hydrophobic group, or a hydrophobic group, or a complex group in which different chromatographical functional groups are combined in order to bind only a certain type of polypeptide, or a covalently bound charged group.

In the development of a chromatographic separation process for a novel compound different chromatography resins and chromatography conditions have to be evaluated. In order to save time and costs a reduction of the size of the employed chromatography column is envisaged, i.e. a so called down-scaling is employed. The smallest possible equipment is a small scale chromatography column, i.e. a chromatography column with an internal diameter of equal or less than 100 mm. To fully exploit the advantages of such a reduced size a high throughput approach is advantageous. To fully exploit the advantages of such a high throughput approach a maximum number of small scale chromatography columns should be used in one experiment. It has been found that a combination of small scale chromatography column containing the chromatography material and a multi well plate for collecting the eluted mobile phase is well suited for high throughput screening. Depending on the diameter of the employed small scale chromatography column and the well number or well diameter, respectively, of the employed multi well plate the adaptor has a certain pattern of holes according to the current invention. This is advantageous as it has been found by the inventors of the current invention that contamination of neighboring wells can be avoided when the outlet of each of the small scale chromatography columns is placed at a distance from the wall of the beneath placed well of the multi well plate in which the mobile phase passing through the small scale chromatography column shall be collected of at least 1.85 times the radius of the droplets obtained at the outlet of the small scale chromatography column. In one embodiment the diameter of the droplet obtained at the outlet of the small scale chromatography column is of from 20 μm to 6000 μm, in one embodiment of from 200 μm to 5000 μm, an in a further embodiment of from 500 to 4000 μm.

The wells of a multi well plate all have the same diameter. The wells and thereby also the center of the wells of a multi well plate are arranged in rows and columns whereby the rows are perpendicular to the columns. As commercially available multi titer plates have an outer shape in form of a rectangle the number of wells differs between rows and columns. As used within this application denotes the term "row of a multi titer plate" the direction of the multi titer plate with the larger number of wells and the term "column of a multi titer plate" denotes the direction of the multi titer plate with the smaller number of wells. The pattern of the holes on the adaptor according to the invention depends on the outer diameter of the small scale chromatography column and the number of wells of the multi well plate and their position.

This adaptor of the current invention is especially useful when the outer diameter of the small scale chromatography column is bigger than the diameter of the wells of the multi well plate. In this case it is very likely that the droplets at the outlet of the small scale chromatography column will be closer to the wall of the subjacent well as 1.85 times the radius of the droplet. Concomitantly the danger of contaminating neighboring wells and likewise the risk of obtaining false result is increased. If the diameter of the small scale chromatography column is larger than the distance of the centers of two neighboring wells of a multi well plate the number of columns that can be placed above a center of a well of a multi well plate is smaller than the total number of wells of the multi well plate. Therefore in one embodiment the adaptor according to the invention has a hole a) if the diameter of the small scale chromatography column is larger than the diameter of the well of the multi well plate but smaller than or equal to the distance between the center of a first well in a first row of wells of the multi well plate and the center of a second well in a second row of wells of the multi well plate, whereby the first and second row are directly side by side and the first well is in a first column of wells of the multi well plate and the second well is in a second column of wells of the multi well plate, whereby the first column and the second column are the same, above every second well in each row in each column, whereby alternately the first well and the second well of a row are used (see Figure 3);

b) if the diameter of the small scale chromatography column is larger than the diameter of the well of the multi well plate but smaller than or equal to the distance between the center of a first well in a first row of wells of the multi well plate and the center of a second well in a second row of wells of the multi well plate, whereby the first and second row are directly side by side and the first well is in a first column of wells of the multi well plate and the second well is in a second column of wells of the multi well plate, whereby the first column and the second column are directly side by side, above every second well in each row in every second column (see Figure 4);

c) if the diameter of the small scale chromatography column is larger than the distance between the center of a first well in a first row of wells of the multi well plate and the center of a second well in a second row of wells of the multi well plate, whereby the first and second row are directly side by side and the first well is in a first column of wells of the multi well plate and the second well is in a second column of wells of wells of the multi well plate, whereby the first column and the second column are directly side by side but smaller than or equal to the distance between the center of a first well in a first row of wells of the multi well plate and the center of a second well in a second row of wells of the multi well plate whereby the first and second row are directly side by side and the first well is in a first column of wells of the multi well plate and the second well is in a second column of wells of the multi well plate, whereby the first column and the second column are separated by one column, above every fourth well in a row in every fourth column (see Figure 5).

The droplet size of a mobile phase droplet leaving the small scale chromatography column outlet is determined by the viscosity of the mobile phase and the diameter of the outlet of the small scale chromatography column. The surface tension at the liquid-air interface defines the maximum droplet size that may form at a given outlet diameter, before gravity induces the droplet tear-off. Without being bound to theory, the volume of the droplets Vd will be larger for fluids having a high surface tension and a low density according to V_d = (2 π a σ)/(g p), whereas a is the opening diameter of the column outlet, σ the surface tension and the p the density of the fluid. Exemplary values are presented in the following Table 1 (data at 25 ⁰C).

Table 1

As can be seen from Table 1 the droplet radius varies between 1.18 mm and 1.77 mm but is about identical for mainly aqueous solutions with about 1.75 mm. The droplet at the outlet of the column will not have a perfect spherical shape but will in fact be distorted along its longitudinal axis, i.e. in line with the column. Thus the maximum radius of the droplet which is still attached to the outlet of the column will be well below the value of about 1.75 mm. It has been found that when the adaptor as reported herein is placed upon a multi well plate the distance between the center of a hole of the column adaptor to the wall of a well of the placed below multi well plate when view from above has to be al least 3.25 mm. This value corresponds to about 1.85 times the droplet radius. Thus, one aspect as reported herein is a multi-well plate adaptor for taking up small scale chromatography columns and for mounting above a multi well plate consisting

- of a rectangular top plate (101) with longer edges (102, 102a) and shorter edges (103, 103 a) comprising a multitude of holes (108) arranged in rows (109) and columns (110), each hole having a means for taking up a small scale chromatography column, and

- of rectangular side plates (104, 105, 106, 107) all of the same height (112) arranged

c) to the same side of the top plate (101), whereby

- the longer edge has a length of from 127.5 mm to 128.2 mm,

- the shorter edge has a length of from 85.0 mm to 86.0 mm,

- the first hole is at a distance of from 7.2 mm to 11.5 mm from the longer edge and at a distance of from 10.0 mm to 17.1 mm from the shorter edge, and

- the holes beside the first hole are arranged as follows

- the minimum distance between two holes in a row or a column is of from 8.0 mm to 10.2 mm and the maximum distance between two holes in a row or a column is of from 9.0 mm to 68.1 mm.

In one embodiment the holes beside the first hole are further arranged such that the minimum distance between a second hole and a third hole, whereby the third hole is in a row directly side-by-side to the row of the second hole and in a column directly side-by-side to the column of the second hole, is of from 12.7 mm to 26.4 mm and the maximum distance between a second hole and a third hole, whereby the third hole is in a row directly side-by-side to the row of the second hole and in a column directly side-by-side to the column of the second hole, is of from 12.7 mm to 84.3 mm.

The term "about" as used herein denotes that the thereafter following value is the center point of a range that is +/- 10 % of the absolute value of the center point.

From the following Table 2 it can be seen that depending on the number of well of a multi-well plate the minimum and maximum possible distance between two holes in rows, in columns, and diagonally varies due to the fact that the distance between the center of a hole of the adaptor to the wall of a well of the placed below multi- well plate when view from above has to be at least 3.25 mm.

Table 2

In Table 3 the characteristics of different multi-well-plates are listed. Table 3

Thus, the holes in the adaptor top plate are arranged as follows: in a basic arrangement the first hole is at the position of the row offset from the shorter edge and of the column offset from the longer edge according to Table 3. The further holes in the rows and columns are at the well center to well center distance, whereby the position of all wells can vary to the shorter edge as well as to the longer edge by a value given as variably of a hole above a well in Table 2.

Thus, in certain embodiments

a) the first hole is at a distance of about 1 1.2 mm to the longer edge and about 14.4 mm to the shorter edge, the further holes have a distance in row and column direction of about 9 mm to each other, or

b) the first hole is at a distance of about 10.1 mm to the longer edge and about 18.2 mm to the shorter edge, the further holes have a distance in row and column direction of about 13.1 mm, whereby the position of the individual holes may deviate from this position by at most 2.5 mm in column and/or row direction, or

c) the first hole is at a distance of about 13.8 mm to the longer edge and about 17.5 mm to the shorter edge, the further holes have a distance in row and column direction of about 19.3 mm, whereby the position of the individual holes may deviate from this position by at most 4.9 mm in column and/or row direction, or

d) the first hole is at a distance of about 16.8 mm to the longer edge and about 25.0 mm to the shorter edge, the further holes have a distance in row and column direction of about 26.0 mm, whereby the position of the individual holes may deviate from this position by at most 8.1 mm in column and/or row direction, or

e) the first hole is at a distance of about 23.2 mm to the longer edge and about 24.8 mm to the shorter edge, the further holes have a distance in row and column direction of about 39.1 mm, whereby the position of the individual holes may deviate from this position by at most 14.5 mm in column and/or row direction.

In one embodiment in the multi-well plate adaptor

- the first hole is at a distance of from 11.2 mm from the longer edge and at a distance of from 14.4 mm from the shorter edge, and

- the holes beside the first hole are arranged as follows

- the distance between two holes in a row or a column is about 9.0 mm,

- the distance between a second hole and a third hole, whereby the third hole is in a row directly side-by-side to the row of the second hole and in a column directly side-by-side to the column of the second hole, is about 12.7 mm.

In one embodiment in the multi-well plate adaptor

- the first hole is at a distance of from 7.6 mm from the longer edge and at a distance of from 15.6 mm from the shorter edge, and

- the holes beside the first hole are arranged as follows

- the minimum distance between two holes in a row or a column is about 8.0 and the maximum distance between two holes in a row or a column is about 18.1 mm, and

- the minimum distance between a second hole and a third hole is about 13.4 mm and the maximum distance between a second hole and a third hole is about 23.6 mm.

In one embodiment in the multi-well plate adaptor

- the first hole is at a distance of from 9.0 mm from the longer edge and at a distance of from 12.6 mm from the shorter edge, and

- the holes beside the first hole are arranged as follows - the minimum distance between two holes in a row or a column is about 9.5 mm and the maximum distance between two holes in a row or a column is about 26.1 mm, and

- the minimum distance between a second hole and a third hole is about 17.5 mm and the maximum distance between a second hole and a third hole is about 37.1 mm.

In one embodiment in the multi-well plate adaptor

- the first hole is at a distance of from 8.7 mm from the longer edge and at a distance of from 16.8 mm from the shorter edge, and

- the holes beside the first hole are arranged as follows

- the minimum distance between two holes in a row or a column is about 9.8 and the maximum distance between two holes in a row or a column is about 42.2 mm, and

- the minimum distance between a second hole and a third hole is about 20.6 mm and the maximum distance between a second hole and a third hole is about 53.0 mm.

In one embodiment in the multi-well plate adaptor

- the first hole is at a distance of from 8.7 mm from the longer edge and at a distance of from 10.3 mm from the shorter edge, and

- the holes beside the first hole are arranged as follows

- the minimum distance between two holes in a row or a column is about 10.2 and the maximum distance between two holes in a row or a column is about 68.1 mm, and

- the minimum distance between a second hole and a third hole is about 26.4 mm and the maximum distance between a second hole and a third hole is about 84.3 mm.

In one embodiment the thickness (111) of the top plate (101) is 50 % or less of the height (112) of the side plates. In a further embodiment are the rows parallel to the side plate (104) and the columns are parallel to the side plate (106). In also an embodiment the top plate (101) has a thickness (111) of 25 % to 5 % of the height

(112) of the side plates.

Also an aspect as reported herein is the use of the multi-well plate adaptor, as reported herein for mounting chromatography columns. A further aspect is a device comprising a multi-well plate adaptor and a multi-well plate, whereby the multi-well plate is placed below the multi-well plate adaptor, wherein the multi-well plate adaptor comprises

- a rectangular top plate (101) with longer edges (102, 102a) and shorter edges (103, 103 a) comprising a multitude of holes (108) arranged in rows

(109) and columns (110), each hole having a means for taking up a small scale chromatography column, and

- rectangular side plates (104, 105, 106, 107) all of the same height (1 12) arranged

c) to the same side of the top plate (101),

whereby

- the longer edge has a length of from 127.5 mm to 128.2 mm,

- the shorter edge has a length of from 85.0 mm to 86.0 mm.

In one embodiment the device further comprises one or more chromatography columns mounted to the holes of the top plate of the multi-well plate adaptor on the same side of the top plate.

In another embodiment

a) a chromatography column is mounted to a hole of the top plate of the multi-well plate adaptor, and

b) then further chromatography columns are mounted to each hole of the multi well plate if the outer diameter of the chromatography column is less or equal to the distance of two holes in one row or in one column.

In a further embodiment

b) a chromatography column is mounted to every second hole in each row and in each column of the top plate starting from the hole of a), whereby alternately the first hole and the second hole of a row is used,

if the diameter of the chromatography column is bigger than the distance of two linearly adjacent holes, but smaller than or equal to the distance between a hole in a first row and a hole in a second row of the top plate, whereby the first and second row are directly side by side, and the first hole is in a first column and the second hole is in a second column of the top plate,

whereby the first column and the second column are directly side-by-side, or

c) a chromatography column is mounted to every second hole in each row in every second column of the top plate starting from the hole of a), if the diameter of the chromatography column is bigger than the distance of two linearly adjacent holes, but smaller than or equal to the distance between a first hole in a first row and a second hole in a second row of the top plate,

whereby the first and second row are separated by one row, and the first hole is in a first column and the second hole is in a second column of the top plate,

whereby the first column and the second column are the same,

or

d) a chromatography column is mounted to every fourth hole in every fourth column of the top plate starting from the hole of a),

if the diameter of the chromatography column is bigger than the distance of a first hole in a first row and a second hole in a second row of the top plate,

whereby the first column and the second column are the same, and the diameter of the chromatography column is smaller than or equal to the distance of a first hole in a first row and a second hole in a second row of the top plate,

whereby the first and second row are directly side by side, and

the first hole is in a first column and the second hole is in a second column of the top plate,

whereby the first column and the second column are separated by one column.

In also an embodiment the chromatography column has an internal diameter of 8 to 11.3 mm. The following example and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. Description of the Figures

Figure 1 Scheme of different small scale chromatography columns.

Figure 2 a) Scheme of a 24 well multi well plate. The Figures denote the columns and the character denotes the rows; b) Scheme of an adaptor according to the invention showing the grid (dotted lines) with the grid points (filled circles), wherein the upper edge point is denoted by an arrow.

Figure 3 a) View from above on a 24 well multi well plate with columns of equal diameter place above the center of every second well in every row; b) determination of the maximum column diameter for this arrangement; c) adaptor according to the invention for this combination of multi well plate and maximum column diameter.

Figure 4 a) View from above on a 24 well multi well plate with columns of equal diameter placed above every second well in every second row; b) determination of the maximum column diameter for this arrangement; c) adaptor according to the invention for this combination of multi well plate and maximum column diameter.

Figure 5 a) View from above on a 24 well multi well plate with columns of equal diameter placed above every forth well in every fourth row; b) determination of the maximum column diameter for this arrangement; c) adaptor according to the invention for this combination of multi well plate and maximum column diameter. Figure 6 View of the adaptor according to the invention for small scale chromatography columns a) from above, b) from the side, c) in a cross section.

Figure 7 Schematic top, side and front view of the adaptor plate as reported herein.

Figure 8 Top and side view of the adaptor plate as reported herein and side view of the adaptor plate as reported herein placed above a multi- well plate with small scale chromatography columns mounted to the adaptor plate and droplets of the liquid phase leaving the small scale chromatography column attached to the outlet of the column. Example

For protein purification (e.g. IgGl monoclonal antibody) the adapter was used for small scale chromatography on Sephadex G25. Therefore the protein in a 10 mM citrate buffer pH 5.0 with a concentration of 14 mg/ml were loaded onto the columns having dimension of 5.0 ml bed volume, a diameter of 11.3 mm and a height of 50 mm.

The chromatography columns were supplied by Atoll. Sephadex chromatography resin was supplied by GE Healthcare.

The adapter was configured as described in Figure 3 c) and placed with the small scale chromatography columns directly above the center of a well of a beneath placed 96 deep well plate.

Equilibration of the columns was performed using a 20 mM phosphate buffer, 30 mM NaCl, pH 6.8. Subsequently the protein was loaded on the column and 1.8 column volumes of 20 mM phosphate buffer, 30 mM NaCl, pH 6.8 were used for elution afterwards. The elution volume was collected in 1.5 - 2 ml fractions in a 96 deep well plate.

All steps of equilibration, protein loading and elution were performed by using a Robotic System (Tecan Evo Freedom 150).

Claims

Patent Claims

1. A multi-well plate adaptor comprising

- a rectangular top plate (101) with longer edges (102, 102a) and shorter edges (103, 103a) comprising a multitude of holes (108) arranged in rows (109) and columns (110), each hole having a means for taking up a small scale chromatography column, and

- rectangular side plates (104, 105, 106, 107) all of the same height (112) arranged a) at and along the edges (102, 102a, 103, 103 a) of the top plate (101),

b) perpendicular to the top plate (101), and

c) to the same side of the top plate (101), whereby

- the longer edge has a length of from 127.5 mm to 128.2 mm, - the shorter edge has a length of from 85.0 mm to 86.0 mm,

- the holes beside the first hole are arranged as follows - the minimum distance between two holes in a row or a column is of from 8.0 mm to 10.2 mm and the maximum distance between two holes in a row or a column is of from 9.0 mm to 68.1 mm.

2. The multi-well plate adaptor according to claim 1, characterized in that

- it is a 96-well-plate adaptor, - the first hole is at a distance of from 11.2 mm from the longer edge and at a distance of from 14.4 mm from the shorter edge, and

- the holes beside the first hole are arranged as follows - the minimum distance between two holes in a row or a column is about 9.0 and the maximum distance between two holes in a row or a column is about 9.0 mm, and

- the minimum distance between a second hole and a third hole, whereby the third hole is in a row directly side-by-side to the row of the second hole and in a column directly side-by-side to the column of the second hole, is about 12.7 mm and the maximum distance between a second hole and a third hole, whereby the third hole is in a row directly side-by-side to the row of the second hole and in a column directly side-by-side to the column of the second hole, is about 12.7 mm.

3. The multi-well plate adaptor according to any one of the preceding claims, characterized in that the thickness (111) of the top plate (101) is 50 % or less of the height (112) of the side plates.

4. The adaptor according to claim 1, characterized in that the rows are parallel to the side plate (104) and the columns are parallel to the side plate (106).

5. The adaptor according to any one of the preceding claims, characterized in that the top plate (101) has a thickness (111) of 25 % to 5 % of the height (1 12) of the side plates.

6. Use of the multi-well plate adaptor according to any one of the preceding claims for mounting chromatography columns.

7. A device comprising a multi-well plate adaptor and a multi-well plate, whereby the multi-well plate is placed below the multi-well plate adaptor, wherein the multi-well plate adaptor comprises - a rectangular top plate (101) with longer edges (102, 102a) and shorter edges (103, 103 a) comprising a multitude of holes (108) arranged in rows (109) and columns (1 10), each hole having a means for taking up a small scale chromatography column, and

- rectangular side plates (104, 105, 106, 107) all of the same height (112) arranged a) at and along the edges (102, 102a, 103, 103a) of the top plate (101),

b) perpendicular to the top plate (101), and

c) to the same side of the top plate (101), whereby

- the longer edge has a length of from 127.5 mm to 128.2 mm,

- the shorter edge has a length of from 85.0 mm to 86.0 mm.

8. The device according to claim 7, characterized in further comprising one or more chromatography columns mounted to holes of the top plate of the multi- well adaptor on the same side of the top plate opposite to the multi-well plate.

9. The device according to claim 8, characterized in that a) a chromatography column is mounted to a hole of the top plate of the multi-well plate adaptor, and b) further chromatography columns are mounted to each hole of the adaptor plate if the outer diameter of the chromatography column is less or equal to the distance of two holes in one row or in one column.

10. The device according to claim 8, characterized in that a) a chromatography column is mounted to a hole of the top plate of the multi-well plate adaptor, and

b) a chromatography column is mounted to every second hole in each row in each column of the top plate starting from the hole of a), whereby alternately the first hole and the second hole of a row is used,

if the diameter of the chromatography column is bigger than the distance of two linearly adjacent holes, but smaller than or equal to the distance between a hole in a first row and a hole in a second row of the top plate, whereby the first and second row are directly side by side, and

whereby the first column and the second column are directly side-by-side, or c) a chromatography column is mounted to every second hole in each row in every second column of the top plate starting from the hole of a), if the diameter of the chromatography column is bigger than the distance of two linearly adjacent holes, but smaller than or equal to the distance between a first hole in a first row and a second hole in a second row of the top plate,

whereby the first and second row are separated by one row, and

whereby the first column and the second column are the same,

or

whereby the first and second row are directly side by side, and

whereby the first column and the second column are directly side by side, and

the diameter of the chromatography column is smaller than or equal to the distance of a first hole in a first row and a second hole in a second row of the top plate,

whereby the first and second row are directly side by side, and

whereby the first column and the second column are separated by one column.

11. The device according to any one of claims 7 to 10, characterized in that the chromatography column has an internal diameter of 8 to 11.3 mm.

12. Use of the multi-well plate adaptor according to any one of claims 1 to 5 for the evaluation of column chromatography resins for the purification of a polypeptide.

13. Use of the multi-well plate adaptor according to any one of claims 1 to 5 for the determination of column chromatography conditions for the purification of a polypeptide.

14. Method for determining a column chromatography condition for the purification of a polypeptide comprising the following steps a) providing a multi-well plate adaptor according to any one of claims 1 to 5,

b) providing a multi well plate,

c) providing a chromatography column containing a chromatography material,

d) providing a polypeptide,

e) providing a set of column chromatography conditions,

f) mounting the chromatography column to the multi-well plate adaptor, g) conditioning the chromatography column according to the set of column chromatography conditions,

h) mounting the multi-well plate adaptor and the multi well plate, i) applying the polypeptide to the chromatography column,

k) determining the purity of the recovered polypeptide with respect to each condition of the set of column chromatography conditions, 1) repeating steps f) to k) until all provided chromatography columns containing a chromatography material and/or all provided conditions of the set of column chromatography conditions have been employed in steps g) to j),

m) determining the conditions of the set of column chromatography conditions which provides for the best combination of yield and purity of the polypeptide to be the column chromatography condition for the purification of the polypeptide.

15. Kit comprising a) a multi-well plate adaptor according to any one of claims 1 to 5, b) a set of chromatography columns, and

c) a set of chromatography materials.