阅读说明：本技术 多孔装置和使用方法 (Porous devices and methods of use ) 是由 J·G·蒂尔 J·托玛 W·S·戈登 B·K·拉加万 D·戴尔西奥 于 2021-06-02 设计创作，主要内容包括：本发明公开了多孔装置和使用该多孔装置的过滤方法。(The invention discloses a porous device and a filtration method using the same.)

1. A multi-well device for processing a fluid sample, comprising:

the upper receiving plate, the middle filter plate and the lower collecting plate;

(a) the upper receiving plate comprises a plurality of wells for receiving a fluid sample containing cells, the plurality of wells in the upper receiving plate each comprising a side wall having an inner surface, a bottom end having a downwardly projecting sleeve, and a bottom fluid flow port, wherein the upper receiving plate comprises two pairs of opposing side walls, and each opposing side wall of at least one of the two pairs of opposing side walls of the upper receiving plate comprises a groove having a lower opening for receiving the side wall of the corresponding well at the corresponding side wall on the intermediate filter plate;

(b) the intermediate filter plate comprises a plurality of wells, each of the plurality of wells in the intermediate filter plate comprising a filter for filtering a fluid sample passing through the bottom fluid flow port of the corresponding well in the upper receiving plate;

each filter includes: an upper depth filter layer having an average pore size in the range of about 1 micron to about 20 microns, the upper depth filter layer having an upstream surface and a downstream surface, the upstream surface of the upper depth filter layer providing the upstream surface of the filter; a middle layer comprising a microporous membrane having an average pore size in a range from about 0.4 microns to about 0.8 microns; and a bottom layer having an upstream surface and a downstream surface, comprising a microporous membrane having an average pore size of about 0.2 microns, the downstream surface of the microporous membrane having an average pore size of about 0.2 microns providing the downstream surface of the filter;

wherein the plurality of apertures in the intermediate filter plate each comprise a side wall having an inner surface and located below a downstream surface of the filter, a bottom wall having fluid flow ports, and a rib structure projecting upwardly from the bottom wall, the rib structure having a top surface spaced from the inner surface of the side wall, wherein the filter is sealed in the apertures by compression between an end of the downwardly projecting sleeve of the upper receiving plate and the top surface of the rib structure in the intermediate filter plate;

wherein the bottom wall of each of the plurality of holes in the intermediate filter plate further comprises a downwardly projecting sleeve surrounding the fluid flow port, wherein the space between the downwardly projecting sleeves of adjacent holes of the intermediate filter plate forms a groove, the lower opening of which is for receiving the top end of the corresponding hole in the lower collection plate,

wherein the fluid flow ports in the intermediate filter plate are arranged to allow the filtered fluid sample to flow from the holes in the intermediate filter plate to the corresponding holes in the lower collection plate; and is

Wherein the side walls of the apertures at the side walls of the intermediate filter plate are receivable within corresponding grooves in corresponding opposing side walls of the upper receiving plate;

and

(c) the lower collection plate comprises a plurality of apertures arranged to receive filtered fluid samples through fluid flow ports in corresponding apertures of the intermediate filter plate, the plurality of apertures of the lower collection plate each having a top end and a bottom end, wherein the top end of each of the plurality of apertures in the lower collection plate is receivable in a groove of a downwardly projecting sleeve surrounding the corresponding aperture in the intermediate filter plate.

2. The multi-well device of claim 1, wherein the downwardly projecting sleeves in the upper receiving plate each have an inner surface and an outer surface, and the space between the outer surfaces of adjacent downwardly projecting sleeves in the upper receiving plate forms a groove with a lower opening for receiving the tapered top end portion of the corresponding well in the intermediate filter plate.

3. A method of obtaining a protein from a fluid containing cells, the method comprising:

placing a sample of fluid containing cells in the wells of the upper receiving plate of the multi-well device of claim 1 or 2;

filtering the sample; and

a protein-containing fluid is obtained in the wells of the lower collection plate.

Technical Field

The invention relates to a porous device and a method of use

Background

After culturing the cells, some procedures for obtaining the protein of interest include centrifuging the cell sample in a centrifuge tube, aspirating the supernatant, and separating the protein from other materials in the remaining liquid using a syringe filter.

There is a need for improved devices and methods for obtaining a protein of interest.

Disclosure of Invention

The present invention alleviates at least some of the disadvantages of the prior art. These and other advantages of the present invention will become apparent from the following description.

Embodiments of the present invention provide a multi-well device for processing a fluid sample, comprising: the upper receiving plate, the middle filter plate and the lower collecting plate; (a) the upper receiving plate comprises a plurality of wells for receiving a fluid sample containing cells, the plurality of wells in the upper receiving plate each comprising a side wall having an inner surface, a bottom end having a downwardly projecting sleeve, and a bottom fluid flow port, wherein the upper receiving plate comprises two pairs of opposing side walls, and each opposing side wall of at least one of the two pairs of opposing side walls of the upper receiving plate comprises a groove having a lower opening for receiving the side wall of the corresponding well at the corresponding side wall on the intermediate filter plate; (b) the intermediate filter plate comprises a plurality of wells, each of the plurality of wells in the intermediate filter plate comprising a filter for filtering a fluid sample passing through the bottom fluid flow port of the corresponding well in the upper receiving plate; each filter includes: an upper depth filter layer having an average pore size in the range of about 1 micron to about 20 microns, the upper depth filter layer having an upstream surface and a downstream surface, the upstream surface of the depth filter layer providing the upstream surface of the filter; a middle layer comprising a microporous membrane having an average pore size in a range from about 0.4 microns to about 0.8 microns; and a bottom layer having an upstream surface and a downstream surface, comprising a microporous membrane having an average pore size of about 0.2 microns, the downstream surface of the microporous membrane having an average pore size of about 0.2 microns providing the downstream surface of the filter; wherein the plurality of apertures in the intermediate filter plate each comprise a sidewall having an inner surface and located below a downstream surface of the filter, a bottom wall having fluid flow ports, and a rib structure projecting upwardly from the bottom wall, the rib structure having a top surface spaced from the inner surface of the sidewall, wherein the filter is sealed in the apertures by compression between an end of the downwardly projecting sleeve of the upper receiving plate and the top surface of the rib structure in the intermediate filter plate; wherein the bottom wall of each of the plurality of holes in the intermediate filter plate further comprises a downwardly projecting sleeve surrounding the fluid flow port, wherein the space between the downwardly projecting sleeves of the holes of adjacent intermediate filter plates forms a groove having a lower opening for receiving a top end portion of a corresponding hole in the lower collection plate, wherein the fluid flow port in the intermediate filter plate is arranged to allow the filtered fluid sample to flow from the hole in the intermediate filter plate to the corresponding hole in the lower collection plate; and wherein the side walls of the apertures at the side walls of the intermediate filter plate are receivable within corresponding grooves in corresponding opposing side walls of the upper receiving plate; and (c) the lower collection plate comprises a plurality of wells arranged to receive filtered fluid samples through the fluid flow ports in corresponding wells of the intermediate filter plate, the plurality of wells each having a top end and a bottom end, wherein the top end of each well of the plurality of wells in the lower collection plate is receivable in a groove of a downwardly projecting sleeve surrounding the corresponding well in the intermediate filter plate.

Methods of filtration using embodiments of the porous device are also provided.

Drawings

FIG. 1A is a top view of an assembled multi-well device according to an embodiment of the invention; FIG. 1B is a cross-sectional view of the assembled multi-well device taken along line A-A of FIG. 1A, showing the upper receiving plate, the intermediate filter plate, and the lower collection plate; fig. 1C is an enlarged cross-sectional view showing the holes in the filter plate received in the bottoms of the holes of the upper receiving plate, and also showing the filter sealed between the lower end of the sleeve in the upper receiving plate and the upper surface of the rib structure in the middle filter plate, wherein the ends of the sleeve and the top surface of the rib structure compress the circumference of the filter.

Fig. 2A-2D show various views of the upper receiving plate. FIG. 2A shows a top perspective view, also showing the upper portion of the bore with a generally square aperture; FIG. 2B shows a top view; FIG. 2C shows a bottom view, also showing a lower portion of a bore having a generally annular orifice; fig. 2D shows a cross-sectional view taken along line AA-AA of fig. 2B, further showing an inner surface of the sidewall of each hole and a downwardly inclined sidewall of each hole, wherein the aperture changes from substantially square to substantially annular, each hole comprising a downwardly protruding sleeve to provide a bottom fluid flow port, the sleeve having an inner surface and an outer surface, wherein the inner surface of the sidewall is continuous with the inner surface of the sleeve, and the space between the outer surfaces of adjacent sleeves forms a groove, the lower opening of the groove for receiving the upper end of the hole in the intermediate filter plate, the opposite sidewalls of the upper receiving plate each forming a groove, the lower opening of the groove for receiving the sidewall of the corresponding hole at the corresponding sidewall of the filter plate.

Fig. 3A-3E show various views of the intermediate filter plate without the filter. FIG. 3A shows a top perspective view, further showing the fluid flow ports and the rib structure having a radial configuration projecting upwardly from the bottom wall of the aperture of the intermediate filter plate; FIG. 3B shows a top view; FIG. 3C shows a bottom view; fig. 3D shows a cross-sectional view of the intermediate filter plate taken along line a-a of fig. 3A, showing a sleeve projecting downwardly from a lower surface of the bottom wall of the aperture of the intermediate filter plate, and a downwardly projecting spout in fluid communication with each fluid flow port, wherein each sleeve is spaced apart from the centrally located spout, and the spaces between the outer surfaces of adjacent sleeves form a groove, the lower opening of the groove for receiving the upper end of the aperture in the lower collector plate, and the upper end of the aperture is arranged to be received in the groove between the downwardly projecting sleeves in the corresponding aperture of the upper receiver plate; FIG. 3E illustrates a cross-sectional view of the hole taken along line B-B of FIG. 3A, showing the bottom wall having a rib structure with a top surface spaced from the inner surface of the sidewall, the bottom wall sloping downwardly toward the fluid flow port, the downwardly facing spout communicating with the fluid flow port;

fig. 4A-4C show various views of the lower collection plate. FIG. 4A shows a top view;

FIG. 4B shows a bottom view; fig. 4C shows a cross-sectional view taken along line a-a of fig. 4A, further showing the upper ends of the holes in the lower collection plate, which holes fit in corresponding grooves of the intermediate filter plate.

Fig. 5 is a schematic cross-sectional view of the wells of each of the three plates in the assembled device, showing (left) the wells before addition of the cell-containing fluid sample, and showing (right) the wells with cells in the upper receiving plate and the wells with filtrate in the lower collecting plate.

Detailed Description

According to an embodiment of the present invention, there is provided a multi-well device for processing a fluid sample, comprising: the upper receiving plate, the middle filter plate and the lower collecting plate; (a) the upper receiving plate comprises a plurality of wells for receiving a fluid sample containing cells, the plurality of wells in the upper receiving plate each comprising a side wall having an inner surface, a bottom end having a downwardly projecting sleeve, and a bottom fluid flow port, wherein the upper receiving plate comprises two pairs of opposing side walls, and each opposing side wall of at least one of the two pairs of opposing side walls of the upper receiving plate comprises a groove having a lower opening for receiving the side wall of the corresponding well at the corresponding side wall on the intermediate filter plate; (b) the intermediate filter plate comprises a plurality of wells, each of the plurality of wells in the intermediate filter plate comprising a filter for filtering a fluid sample passing through the bottom fluid flow port of the corresponding well in the upper receiving plate; each filter includes: an upper depth filter layer having an average pore size in the range of about 1 micron to about 20 microns, the upper depth filter layer having an upstream surface and a downstream surface, the upstream surface of the depth filter layer providing the upstream surface of the filter; a middle layer comprising a microporous membrane having an average pore size in a range from about 0.4 microns to about 0.8 microns; and a bottom layer having an upstream surface and a downstream surface, comprising a microporous membrane having an average pore size of about 0.2 microns, the downstream surface of the microporous membrane having an average pore size of about 0.2 microns providing the downstream surface of the filter; wherein the plurality of apertures in the intermediate filter plate each comprise a sidewall having an inner surface and located below a downstream surface of the filter, a bottom wall having fluid flow ports, and a rib structure projecting upwardly from the bottom wall, the rib structure having a top surface spaced from the inner surface of the sidewall, wherein the filter is sealed in the apertures by compression between an end of the downwardly projecting sleeve of the upper receiving plate and the top surface of the rib structure in the intermediate filter plate; wherein the bottom wall of each of the plurality of holes in the intermediate filter plate further comprises a downwardly projecting sleeve surrounding the fluid flow port, wherein the space between the downwardly projecting sleeves of the holes of adjacent intermediate filter plates forms a groove having a lower opening for receiving a top end portion of a corresponding hole in the lower collection plate, wherein the fluid flow port in the intermediate filter plate is arranged to allow the filtered fluid sample to flow from the hole in the intermediate filter plate to the corresponding hole in the lower collection plate; and wherein the side walls of the apertures at the side walls of the intermediate filter plate are receivable within corresponding grooves in corresponding opposing side walls of the upper receiving plate; and (c) the lower collection plate comprises a plurality of wells arranged to receive filtered fluid samples through the fluid flow ports in corresponding wells of the intermediate filter plate, the plurality of wells each having a top end and a bottom end, wherein the top end of each well of the plurality of wells in the lower collection plate is receivable in a groove of a downwardly projecting sleeve surrounding the corresponding well in the intermediate filter plate.

Preferably, the downwardly protruding sleeves in the upper receiving plate each have an inner surface and an outer surface, and the space between the outer surfaces of adjacent downwardly protruding sleeves in the upper receiving plate forms a groove, the lower opening of which is for receiving the conical top end of the corresponding hole in the intermediate filter plate.

Methods of filtration using embodiments of the porous device are also provided.

In one embodiment, a method for obtaining a protein from a fluid containing cells is provided, the method comprising: placing a fluid sample containing cells in wells of an upper receiving plate of an embodiment of a multi-well device; filtering the sample; and obtaining a protein-containing fluid in the wells of the lower collection plate.

Advantageously, the protein can be obtained in a less labor intensive manner and in a shorter time. In addition, the multi-wall device is used for replacing a plurality of centrifuge tubes, pipette tips, sterile filters and syringes, and is more environment-friendly. Furthermore, the use of an upper receiving plate having an upper square or generally square aperture and a lower annular or generally annular aperture allows for a greater volume of sample to be filtered while still sealing the filter in the well.

Each plate (typically in a rectangular array) includes a plurality of wells, 24 wells in the illustrated embodiment, but the plate may include a greater number of wells, such as 94 wells, or 384 wells, or more wells than 384 wells, or less than 24 wells. Typically, the apertures are arranged in a two-dimensional configuration. The wells are generally cylindrical except for the upper portion of the well in the upper receiving plate, and have fluid-tight walls and a depth and width for the desired use and amount of fluid to be sampled.

Each aperture in the filter panel comprises a filter having three layers or filter elements, and in some embodiments, the filter is comprised of three layers or filter elements. The filter is located at the bottom of the well. As will be described in greater detail below, the upper receiving plate and the filter plate include structures that contact portions of the respective top and bottom surfaces of the filter such that the filter is sealed in the pores without fluid bypass.

Each of the components of the present invention will now be described in greater detail below, with like components having like reference numerals.

According to the illustrated embodiment, particularly shown in fig. 1B and 5, the porous device 1000 comprises: an upper receiving plate 100 including a plurality of holes 101; an intermediate filter plate 200 comprising a plurality of pores 201, each pore comprising a filter 250; and a lower collection plate 300 comprising a plurality of apertures 301.

An illustrative embodiment of the upper receiving plate 100 is shown in greater detail in fig. 1A-1C, 2A-2D. The illustrated upper receiving plate has an upper portion 100A and a lower portion 100B, two pairs of opposing sidewalls 110 and 120, and a plurality of wells 101 for receiving a fluid sample containing cells. The bore 101 has a sidewall 102 (which has an inner surface 102A), a top end 130A, a bottom end 130B, a bottom wall 103, a downwardly projecting sleeve 104 with a receiving plate bottom fluid flow port 105. Each hole has an orifice 125. The downwardly projecting sleeve 104 has an inner surface 104A continuous with the inner surface 102A, an outer surface 104B, and an end 104C (see fig. 1C). As will be discussed in more detail below, the sleeve end 104C compresses the upper surface of the filter in the bore of the intermediate filter plate to help seal the filter in the bore.

Preferably, the space between the outer surfaces 104B of adjacent downwardly protruding sleeves 104 in the receiving plate forms a groove 109' having a lower opening for receiving the top end portion 230A of a corresponding hole 201 in the intermediate filter plate 200 (see fig. 1B and 1C). Using fig. 1C as a reference, preferably tip 230A received in recess 109' is tapered, and in some embodiments, a lower portion of outer surface 104B is indented, and tapered tip 230 pushes adjacent outer surfaces apart and beyond the indentation when received into the upper receiving plate.

Preferably, as shown particularly in fig. 2A, the upper orifice 125A of the bore has a square configuration, while the lower orifice 125B has an annular configuration.

At least one pair of opposing sidewalls (illustrated as opposing sidewalls 110) includes a groove 109 at the lower portion 100B having a lower opening for receiving a sidewall 202 of a corresponding hole at a corresponding sidewall 210 on the intermediate filter plate 200 (see fig. 1B and 1C).

As shown in fig. 2C and 2D, the lower portion of the receiving plate may include a plurality of reinforcing ribs 180.

An illustrative embodiment of the intermediate filter plate 200 is shown in more detail in FIGS. 1B, 1C, and 3A-3E. The illustrated intermediate filter plate includes two pairs of opposing sidewalls 210 and 220 and a plurality of wells 201 for receiving a fluid sample containing cells that passes through the wells 101 of the upper receiving plate. The hole 201 has: a sidewall 202; having an inner surface 202A; tip end 230A (tapered for those ends that fit in groove 109', as shown in fig. 1C); a bottom end 230B; a bottom wall 203 having a rib structure 215 projecting upwardly therefrom, the rib structure having a top surface 215A spaced from the inner surface of the side wall (see fig. 1C and 3E); a filter plate fluid flow port 205 in communication with the spout 207; and a downwardly projecting sleeve 204 surrounding and spaced from the spout, the sleeve having an inner surface 204A and an outer surface 204B. Fig. 3E shows the bottom wall sloping downwardly towards the fluid flow port. The space between the outer surfaces of the downwardly projecting sleeves of adjacent intermediate filter plate apertures forms a recess 209 having a lower opening for receiving the top end portion 330A of the corresponding aperture 301 in the lower collection plate 300 (see fig. 1B).

The illustrated rib structure 215 has an annular upwardly projecting outer rib adjacent the sidewall, and a more centrally located plurality of radially disposed upwardly projecting ribs. Preferably, as shown, the upper surface of the rib is non-planar, e.g. rounded. A plurality of radially arranged upwardly projecting ribs may provide a drainage grid for the apertures.

As shown in fig. 3C, the lower portion of the middle filter plate may include a plurality of reinforcing ribs 280.

As shown in fig. 1B and 1C, the pores 201 of the intermediate filter plate 200 include filters 250, each of which includes an upper layer having a depth filter 251, an intermediate layer having a microporous membrane 252, and a bottom layer having a microporous membrane 253. Each layer has an upstream surface and a downstream surface, with the upstream surface of the upper layer comprising the upstream surface 251A of the filter and the downstream surface of the bottom layer comprising the downstream surface 253A of the filter.

As shown in fig. 1C, the sleeve end 104C of the upper receiving plate 100 compresses the upstream surface of the filter in the bore of the intermediate filter plate and the upper surface 215A of the rib structure 215 (particularly the upper surface of the annular outer rib near the sidewall) compresses the downstream surface of the filter, thereby sealing the filter in the bore without allowing bypass.

The filter layer may have any suitable pore structure, such as pore size (e.g., as evidenced by bubble point, or as evidenced by KL, as described, for example, in U.S. patent 4,340,479, or as evidenced by capillary condensate flow orifice method), pore grade, pore size (e.g., as characterized by the modified OSU F2 test, as described, for example, in U.S. patent 4,925,572), or a removal rate that reduces or allows passage of one or more target species as a fluid passes through the layer.

Typically, the average pore size of the depth filter layer is in the range of about 1 micron to about 20 microns, preferably in the range of about 6 microns to about 15 microns. Typically, the average pore size of the membrane of the intermediate layer is in the range of about 0.4 microns to about 0.8 microns, and in some embodiments, it has an average pore size of about 0.65 microns. The membrane of the bottom layer is preferably nominally used to provide sterile filtration, e.g., having an average pore size nominally about 0.2 microns.

A variety of suitable filter layers are commercially available.

The filter layer may have any desired critical wetting surface tension (CWST, as defined, for example, in U.S. patent 4,925,572). CWST may be selected as known in the art, for example, as additionally disclosed in U.S. Pat. nos. 5,152,905, 5,443,743, 5,472,621, and 6,074,869.

An illustrative embodiment of the lower collection plate 300 is shown in greater detail in FIGS. 1B and 4A-4C. The illustrated lower collection plate includes two pairs of opposing sidewalls 310 and 320 and a plurality of apertures 301 for receiving filtered fluid sample passing through the apertures 201 of the middle filter plate. The aperture 301 has a side wall 302 (having an inner surface 302A), a top end 330A, a bottom end 330B, and a bottom wall 303. As shown in fig. 1B, the top end 330A of the hole in the lower collection plate 300 can be received in the groove 209 of the downwardly protruding sleeve 204 surrounding the corresponding hole 201 in the intermediate filter plate 200.

The plate may be made of any suitable rigid impermeable material, including any impermeable thermoplastic material compatible with the fluid to be treated. For example, the plates may be made of metal such as stainless steel or of a polymer. In a preferred embodiment, the plate is made of a polymer, such as acrylic, polypropylene, polystyrene or polycarbonate resin.

The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

Examples of the invention

This example demonstrates the isolation of a protein from a fluid sample containing cells of a mammal (Chinese hamster ovary; (CHO)). The sample was a 5ml CHO cell culture with a density >2500 ten thousand cells/ml, and gamma globulin (containing IgG) was added to evaluate more protein (about 10mg total protein, about 3mg IgG).

The assembly of the porous device is generally as shown. The filter comprises three layers. The most upstream layer was a depth filter sheet (SEITZ K Cellulose 700P; Parl, N.Y. Washington, N.Y.), the middle layer was a 0.65 μm asymmetric polyethersulfone membrane and the bottom layer was a 0.2 μm symmetric polyethersulfone membrane (the middle layer in combination with the bottom layer are commercially available as SUPOR EKV filters; Parl, N.Y. Washington, N.Y.).

One set of samples was filtered using a vacuum manifold apparatus (a multi-well plate vacuum manifold from parr) and the other set of cells was filtered using centrifugation (Eppendorf 5810 centrifuge).

The results are as follows:

the results using vacuum and centrifugal filtration are generally similar.

This example shows that the device removes cells that affect absorbance and turbidity and provides for recovery of proteins.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms "a" and "an" and "the" and "at least one" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both aspects: singular and plural, unless otherwise indicated herein or clearly contradicted by context. The term "at least one" followed by a list of one or more items (e.g., "at least one of a and B") should be understood to refer to one item selected from the listed items (a or B), or any combination of two or more of the listed items (a and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "with" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.