Low binding liquid retaining and filtering device

Details Low binding liquid retaining and filtering device Low binding liquid retaining and filtering device

2000-07-14

2003-04-08

Fortuna, Ana (Department: 1723)

Liquid purification or separation

Casing divided by membrane into sections having inlet

Each section having inlet

C210S477000, C422S105000, C422S105000, C422S105000

Reexamination Certificate

active

06544417

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to novel plate devices for use in high-throughput screening assays.
BACKGROUND OF THE INVENTION
The binding of small molecules and peptides to protein is a very important parameter to measure in the pharmaceutical industry. As pharmaceutical companies strive to design novel small molecules and peptides to treat various ailments ranging from life threatening diseases including cancer, AIDS, and heart disease to cosmetic complaints such as ACNE, age spots and wrinkles, successful administration of these drugs through the circulatory system is critical. Many drugs that have been shown to be very active in vitro assays have failed to show efficacy in animal models and in people because of the high plasma protein binding exhibited by these compounds. When a molecule is highly bound to proteins in the blood the amount of drug available to diffuse into the target tissue is significantly reduced and the efficacy of the drug will inevitably be poor.
Whether a small molecule binds to plasma proteins or not usually depends on the size of the molecule, the amino acid composition and the tertiary structure of the molecule. When a small molecule binds to plasma proteins the interaction usually is a result of strong ionic and hydrophobic interactions. Because blood contains several hundred proteins there is a high probability that any small molecule will exhibit some level of binding. Determining the level of binding therefore is critical and will directly correlate with efficacy of the molecule. Predicting whether a molecule is going to show high or low protein binding based on molecular structure has proven to be very difficult. The only sure way to determine whether or not a molecule will exhibit high or low protein binding is to test the molecule directly in a protein-binding assay.
The most common method used to measure the level of protein binding exhibited by compounds is equilibrium dialysis assays. In such assays, a set concentration of drug (usually 1 &mgr;M) is added to a set volume of human plasma (usually 3 ml). The mixture is added to dialysis tubing with a molecular weight cut-off of 30 kDa. The mixture is allowed to incubate in a large volume of water (usually 4 litre) for 24 hours at 37° C. Following the incubation the sample is collected and the concentration of drug is calculated. If the compound is completely unbound to protein, the concentration following dialysis would be 0, if 50% bound the concentration would be 0.5 uM, etc. Although equilibrium dialysis has been shown to be accurate and consistent, it is very time consuming and the number of drugs a researcher can test in one assay is dependent on how many 4-litre beakers he/she can set up. Thus, there is a need for a fast, high-throughput assay in the pharmaceutical industry.
The binding of small molecules even to the plastics such as the polypropylene tubes and plates can be a problem. Polypropylene (PP) is currently considered the best type of commercially available plastic plate based on its low non-specific binding properties and solvent resistance. Even the non-specific binding (NSB) to polypropylene can interfere with the calculation of accurate plasma protein binding values.
SUMMARY OF THE INVENTION
This invention is directed to a substrate having at least one substrate well, such as, a filtration device comprising a polymeric material and an additive to which biomolecules exhibit minimal or no binding. In a preferred embodiment, the filtration device includes a filtrate unit-holder plate and comprises a polyolefin, most preferably polypropylene, and Teflon® resin, most preferably about 2.5% Teflon® resin and about 97.5% polypropylene. Said plate contains a plurality of through-holes, each of which is capable of securely holding a filter unit, such as a Microcon-3, 10, 30 or 100® filter unit (see
FIGS. 4
,
5
,
7
and
8
) Said filter unit (see
FIG. 1
) contains a reservoir and a filter membrane, contained within the filter unit-holder plate. The filter unit also contains a base, which is positioned in an opening in each of the filter unit-holder plate through-holes and into a preferably reversible-well collection plate (see
FIGS. 9
,
10
,
11
and
12
). Said “reversible-well collection plate”, also comprises a polymeric material and an additive to which biomolecules exhibit minimal or no binding, preferably polypropylene and Teflon® resin (most preferably, again, at about 2.5% Teflon® resin and about 97.5% polypropylene) and also contains a plurality of wells. The collection plate is designed such that its wells can accommodate either end of the filter unit.
The filter unit-holder plate and collection plates, along with the filter units contained therein, are securely fastened together and used in high-throughput screening assays for the binding of small molecules and peptides to proteins. The design of the plates, and their composition, allows for several advantages in the conducting of such screens, as is described herein.
The filtrate plate device of the present invention consists of a multi-well plate and holder with a unique micro-array format designed to fit 96 Microcon filter units, for example. The holder contains a plurality of through-holes where the filter units are inserted. The ridge of the filter unit lies on top of the through-hole and the bottom (base) of the filter unit passes through. Once the filter units are inserted into the holder, it is placed on top of a multi-well collection plate with the same micro-array format as the holder. The set-up was designed so that several mm of the base of the filter unit protrudes into the wells of the collection plate. This is one way to prevent well to well spill-over during the centrifugation. Alternatively, a spout
60
(
FIG. 14
) could be used to avoid cross-contamination. The holder/plate sandwich can be spun in a tabletop centrifuge with a swinging bucket to collect the filtrate. The plates were designed to allow stacking of the sandwiches on top of one another to allow more plates to be spun at one time.
In terms of method, sample preparation units are inserted into the custom-designed holder which is then placed on top of the collection plate. 200 ul of sample, for example, is added to the filter units in the presence or absence of the small molecule at a concentration of 1 uM. The plates are placed in a swinging bucket rotor, containing microtiter plate holders. The plates are spun at suitable speeds, such as 3000 g for 30 minutes. The free small molecules (typical molecular weight 300-600 Daltons) readily pass through the filter membrane and pass into the collection well. The bound molecules are retained with the plasma proteins, which range in molecular weight from 20,000 to 500,000 Daltons. The collection plate contains the free compound (filtrate) and the filter reservoir contains the bound compound (retentate). The free compound contained in the filtrate can be determined by mass spectrometry, electrospray or a bioassay.
In order to collect the retentate, another collection plate is placed with the wells facing down on top of filter units. The plate is designed to fit tightly around the open end of the filter units. Next, the holder is inverted and spun an additional five minutes to collect the retentate into the second collection plate. The filtrate is then ready to be analyzed for bound compound. Finally, the filter unit-holder and collection plates are designed with V-grooves on the outer exterior of the plates that make them amenable to automation using robotics.


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