Cross-Reference to Related Applications

This application claims priority to United States provisional patent application number 61/220,614 filed June 26, 2009; the disclosure of which is incorporated herein by reference in its entirety.

Field of the Invention

The present invention relates to the field of chromatography, and more specifically to an improved method for resuspending chromatographic resin in a pre-filled plate. The invention also relates to an apparatus for use with the method.

Background of the Invention

Multiwell plates, also called microtiter plates, have been used for many years in laboratories for the simultaneous analysis of a number of samples. Typical formats include 4, 24, 48, 96 and 384 wells per plate. Initially, these plates had solid bases and liquid samples were pipetted into and out of the wells. Subsequently, plates were provided with wells containing a lower well through hole (known as a "drip" if it is provided with downward protruding lips) pierced through the bottom surface. These microtiter plates allowed the samples to flow through the wells, which permitted larger sample volumes to be processed (since the sample size was no longer limited to the capacity of the well). Later developments of microtiter plates were provided with filter or membrane wells in which each well was provided with a microporous filter or membrane which extended over the cross-section of the well such that all of the sample passing through the well had to pass through the filter or membrane. These microtiter plates are also called microtiter filter plates. A further development of a microtiter plate comprises wells with a lower well through hole or drip and a filter or membrane and which wells are each at least partly filled with a media such as a chromatographic gel or slurry or chromatographic particles. Different screening processes that could be performed on such microtiter plates are for example screening of/for: i) conditions for optimal binding capacity, ii) most efficient wash buffers for washing off impurities from chromatography resin; iii) most efficient eluting solution; iv) selectivity obtained using different ligands, v) best resin either from capacity or purity perspective. Considering the multiwell format of microtiter plates, if processes under different conditions need to be studied there are different possibilities to change conditions on a single plate. For example the concentration of the sample added can be changed, the composition of the buffer in which the sample is dissolved can be changed, the effect of overall time of contact between the sample and a chromatography resin can be studied, or any combinations of the above. High throughput studies of chromatographic separations using microtiter plates filled with chromatography resin proved highly efficient in reducing time and sample requirements necessary for development of large-scale purification processes. One example is the PREDICTOR™ plates (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), which are disposable 96-well filter plates pre-filled with chromatography media. These plates support high throughput process development (HTPD) by allowing parallel screening of chromatographic conditions. The PREDICTOR™ plates can be used to screen different parts of the chromatographic cycle, for example determination of binding, wash, and elution conditions. It has also been used for performing adsorption isotherm studies and quantitative or qualitative time-dependent studies. The chromatographic resins in the pre-filled plate normally will sediment over time to the bottom of the wells. If the plate is shipped or stored upside down, a partially sedimented resin pellet is observed on the upper seal. Either way, the resins need to be resuspended before use. This is ordinarily done by manual "tumbling" that is described in the Instruction Manual for the PREDICTOR™ plates (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). However, this is a manual process which cannot be automated, thus it becomes a rate limiting step if many plates are involved.

Accordingly, there is a need in this field to develop an improved method and apparatus for resuspending the pre-filled plates.

Summary of the Invention

One object of the present invention is to provide an improved method for resuspending sedimented resins in a microtiter plate. The method comprises placing the micro titer plate containing the pre-filled, sedimented resins on one of its sides, and shaking the microtiter plate for efficient resuspension of the sedimented resin pellet. Another object of the invention is to provide a plate holder block for use with the above method, which holds the microtiter plate on the top, and fits on an orbital shaker on its bottom. Thus, it is provided a plate holder block for holding a microtiter plate with the microtiter plate standing on its side, which block comprises a bottom having footprints similar to that of a microtiter plate which fits on an orbital shaker, and a top having notches for securing the microtiter plate on its side.

Further provided is the use of the microtiter plate holder block according to the invention for resuspending sedimented chromatographic resin in a microtiter plate.

One or more of the above-defined objects can be achieved as described in the appended claims. Brief Description of the Drawings

Figure 1 provides a schematic view of a well of a PREDICTOR™ microtiter plate stored in the upright position (left panel) and upside down position (right panel).

Figure 2 is a schematic view of a well of a PREDICTOR™ plate on its side.

Figure 3 illustrates a side view of a PREDICTOR™ plate with lower rim and inward leaning sides (not drawn to scale).

Figure 4 shows a top view of the plate holder according to one embodiment of the invention.

Figure 5 shows a side view of the plate holder block, with two PREDICTOR™ plates secured on its top.

Figure 6 shows a side view of the plate holder block, with a clamping device holding together two PREDICTOR™ plates securely on its top.

Detailed Description of the Invention Currently, manual tumbling is used for resuspending resin particles in a

PREDICTOR™ microtiter plate. As discussed in the background section, this cannot be easily automated and quickly becomes a rate- limiting step if multiple plates need to be processed. As shown below in the Examples, shaking the plate in an orbital shaker (with the plate in either upright or upside down position) also failed to provide consistent resuspension of sedimented resin, although resuspension of a plate stored in the upright position appears to be easier than that stored in a upside down position (where the pellet is situated on top of the cover foil). The reason could be insufficient mixing in the upside down position, if the tip of the vortex formed by the orbital shaker does not reach the sedimented "pellet". In addition, any pellet remaining on the upper foil is removed with the foil, reducing the amount of resin in the well.

Figure 1 shows cross-sectional diagrams of a single, representative well of a microtiter plate. The representative well contains sedimented resin particles and a buffer. In Figure 1, the plate is either placed in upright position (left panel) or upside down position (right panel). Figure 2 illustrates a well of a microtiter plate when the microtiter plate is placed on its side. Here as in Figure 1, the well contains resin particles and buffer. In this hypothetical well, the microtiter plate had been stored in an upside down position, so the resin particles had sedimented near the top foil. It is discovered that resuspension of the sedimented resin pellet is best achieved when the microtiter plates is shaken standing on its side. Efficient resuspension is achieved regardless of how the plate had been stored. This method is generally applicable not only for PREDICTOR™ plates but for all microtiter plates containing pre-filled resin. Further, the separation resin could be a chromatography media and it can also be called for example a solid phase, matrix, gel or adsorber. The present invention thus in one aspect relates to an improved method for the resuspension of sedimented resins in a microtiter plate. The method comprises placing the microtiter plate containing the pre-filled, sedimented resins on one of its sides, and shaking the microtiter plate to allow for efficient resuspension of the sedimented resin pellet. In one embodiment, the microtiter plate is secured on a microtiter plate shaker, and shaking is performed with the microtiter plate standing on its side. In a preferred embodiment, the microtiter plate is placed on one of its sides on a plate holder block which is described in detail below, and the plate holder block is secured on the shaker for shaking. Optimal conditions for shaking are easily determined by simple experimentation, following the principles of examples provided below. For a PREDICTOR™ plate stored upside down, a five minute shake at minimally 1,100 rpm proves sufficient.

The current plate shakers (such as IKA MTS 2/4 digital and IKA Schuttler MTS 2) are not designed for holding a microtiter plate on its side. Thus, in another aspect of the invention, it is provided a plate holder block for use with the above method. The plate holder block is designed such that it holds microtiter plates on its top, and fits on an orbital shaker on its bottom. Thus, the plate holder block comprises a bottom having footprints similar to that of a microtiter plate which fits on an orbital shaker, and a top having notches for securing the microtiter plate on its side. As an example, the following illustrates a plate holder block useful for shaking a PREDICTOR™ plate. Slight variations can be easily made to accommodate other microtiter plates. Figure 3 illustrates a side view of a PREDICTOR™ plate with lower rim and inward leaning sides (not drawn to scale). An example of a plate holder block contains three notches on the top, each fitting the rim of a PREDICTOR™ plate (Figure 4). The two outer notches are used for holding two plates simultaneously (wells facing inwards) while the third notch is used if only one plate is to be resuspended. The third notch is off- center to put the center of gravity of the plate in the middle of the apparatus. It is envisioned that variations of the design (e.g., two notches or a single notch or multiple notches) also work and are within the embodiments of the invention.

The upper face of the plate holder apparatus is sloping to compensate for the slightly inward leaning sides of the PREDICTOR™ plates so that they are held in a vertical position (Figure 5). Figure 5 shows a side view of the plate holder block, with two PREDICTOR™ plates secured on its top.

The plates are held securely in place on the plate holder by a clamping device (not shown), in its simplest form a rubber band. The plate holder needs to have a corresponding groove to accommodate the clamping device and still allow flat mounting on the orbital shaker (Figure 4). An exemplary clamp device is shown in Figure 6, with the plate holder block and two PREDICTOR™ plates shown in Figure 5.

The materials for manufacturing the plate holder block will typically be polymeric, since these materials lend themselves to mass manufacturing techniques. Various methods in the art can be used to confirm that selected polymers possess the desired properties.

Polymeric materials can particularly facilitate manufacturing by molding methods known in the art and developed in the future, such as insert or injection molding.

Furthermore, this invention covers the use of a microtiter plate holder block according to the invention for resuspending sedimented chromatographic resin in a microtiter plate.

Below, the present invention will be described by way of examples, which are provided for illustrative purposes only and accordingly are not to be construed as limiting the scope of the present invention as defined by the appended claims. All references given below and elsewhere in this application are hereby included herein by reference.

Examples

The aim of this study was to investigate if the resin pellets formed due to sedimentation during storage and transportation of the plates can be effectively re- suspended. During transportation, the plates could be placed upright or upside down. If placed upside down, the gel would sediment against the upper foil. Resuspension of both were tested using different mixing methods. Experimental Apparatus Vacuum manifold UV plate

Filter plates (for blank and max UV calculations) End-over-end rotation device, Labinco PREDICTOR™ 96-well plate Micro plate shaker IKA ^® MTS 2/4 digital Spectrophotometer SpectraMax ^® Plus Collection plate

Resins, Proteins and Chemicals CAPTO™ S: Lot 310505 CAPTO™ Q: Lot 10011971

BSA: Sigma A7030 batch # 104K0459 α- chymotrypsin: Fluka 27270 lot S33549036

Tris: Merck 1.08382.0500 lot 8382X011 611

Buffer:

CAPTO™ Q: 50 mM Tris pH 8,0 (Dissolve 6.057 Tris in approx 900 ml of pure water. Titrate to pH 8.00 with HCl. Make up volume to 1000.0 ml with MILLI-Q ^® water.) CAPTO™ S : 5OmM Acetate pH 4.25 Methods

PREDICTOR™ plates were filled with CAPTO™ Q or CAPTO™ S resins. The plates were filled with a buffer containing an amount of particular resin as indicated below, to a volume of either 200 μl or 500 μl, respectively. To test the effect of various resuspension methods, the plates were kept three days to a week to allow sedimentation, with some plates kept in an upright position while others kept upside down.

The plates were then subjected to various resuspension methods. After the resuspension step, the plates were subjected to visual inspection for any indications of resin pellet remaining on the top seal. This was done by removing the upper foil to observe if there was any resin left on the foil.

Certain plates were further tested to show that when plates passed visual inspection, protein binding capacity of the resin was within the expected range. The storage solution was removed by vacuum filtration; the plates were equilibrated by adding 200μl loading buffer /well three times. After each vacuum filtration the bottom of the plate was blotted on a soft paper. Protein solution was applied to each well and incubated on a micro plate shaker at 1100 rpm for 1 hour. The flow-through was recovered on a UV-plate, by vacuum filtration. One blank and one max UV-plate were also prepared by filling a UV-plate with loading buffer for the blank plate and protein solution for the max plate. The protein concentration was measured using a Spectrophotometer SpectraMax ^® Plus.

Results

Our preliminary tests showed that the most difficult sample plates to resuspend were those that had been stored upside down. The following experiments were therefore performed using sample plates which had been left upside down for a period of time (3 days to 1 week). We first tested whether the amount of resin and buffer used would have any effect on resuspension. PREDICTOR™ plates containing 2 μl or 20 μl CAPTO™ Q or CAPTO™ S were tested. The final buffer volumes in each well are 200 μl or 500 μl, respectively. The plates were either resuspended on a shaker in an upright position, or by manual tumbling (see Instruction Manual for PREDICTOR™ plates, GE Healthcare Bio- Sciences AB). The results were compared to reference plates, which were left standing upright unmoved on the lab bench. A number of the samples failed visual inspection, especially when 20μl resin was used. The protein binding capacity of the resin that passed visual inspection was tested, and the results were within the range expected. This suggests that visual inspection is a trusted method for an indication of complete resuspension of the resin. The result is presented in Table 1. The tests showed that the method of manual tumbling worked well, while different kinds of shaking procedures, whether having the plate upright, upside down or a combination of both, did not always work. On many occasions, sedimented resin pellet was left on the upper foil when the foil was removed from the plate. No difference was seen between the different resins, or due to the amount of resin or the amount of buffer in each well.

However, it is discovered that when put on its side, a short, five-minute shake at 1,100 rpm completely resuspended the resin pellets (Table T). No resin pellet was observed on the upper foil. Thus shaking the plate on its side works consistently and works as well as manual tumbling. Table 2: a five-minute shake at 1,100 rpm, on its side, completely resuspends the resin pellet.

The above examples illustrate specific aspects of the present invention and are not intended to limit the scope thereof in any respect and should not be so construed. Those skilled in the art having the benefit of the teachings of the present invention as set forth above, can effect numerous modifications thereto. These modifications are to be construed as being encompassed within the scope of the present invention as set forth in the appended claims.