Method and Device for Gravity Flow Chromatography

• US 20100170852A1
• Filed: 03/22/2010
• Published: 07/08/2010
• Est. Priority Date: 12/03/2004
• Status: Abandoned Application

• Current Assignee: Chris Suh, Douglas T. Gjerde, Hoang Lee
• Original Assignee: Chris Suh, Douglas T. Gjerde, Hoang Lee
• Inventors: Gjerde, Douglas T., Hoang, Lee, Suh, Chris
• Application Number:
  US12/729,103
• Publication Number:
  US 20100170852A1
• Time in Patent Office: Days
• US Class Current: 210/656
• CPC Class Codes: B01D 15/34 Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
• CPC Class Codes: B01D 15/3804 Affinity chromatography
• CPC Class Codes: B01J 20/286 Phases chemically bonded to a substrate, e.g. to silica or to polymers
• CPC Class Codes: B01J 20/287 Non-polar phases; Reversed phases
• CPC Class Codes: B01J 20/3244 Non-macromolecular compounds
• CPC Class Codes: B01J 2220/54 Sorbents specially adapted for analytical or investigative chromatography
• CPC Class Codes: B01J 2220/64 In a syringe, pipette, e.g. tip or in a tube, e.g. test-tube or u-shape tube in columns B01J2220/58
• CPC Class Codes: C07K 1/16 by chromatography
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: G01N 1/405 by adsorption or absorption
• CPC Class Codes: G01N 2030/062 extracting sample from raw material
• CPC Class Codes: G01N 2035/1053 for separating part of the liquid, e.g. filters, extraction phase
• CPC Class Codes: G01N 30/02 Column chromatography
• CPC Class Codes: G01N 30/466 with separation columns in parallel
• CPC Class Codes: G01N 30/603 retaining the stationary phase, e.g. Frits
• CPC Class Codes: G01N 30/6043 in parallel
• CPC Class Codes: G01N 30/6065 with varying cross section
• CPC Class Codes: G01N 30/6091 Cartridges

Abstract

The invention provides gravity chromatographic columns for the purification of a material (e.g., a biological macromolecule, such as a peptide, protein or nucleic acid) from a sample solution, as well as methods for making and using such columns. The columns typically include a bed of media positioned above a bottom frit or between a bottom and top frit. In some embodiments, the columns employ modified pipette tips as column bodies. In some embodiments, the columns employ modified plates or racks as column bodies. In some embodiments, the invention provides methods and devices for gel filtration, desalting, buffer exchange, ion exchange, ion-pairing, normal phase and reverse phase chromatography. In some embodiments, the invention provides multiplexing gravity flow chromatography on a liquid handling robotic system.

Claims

1. A method for purifying a material from a sample solution using gravity flow chromatography comprising the steps of:

a. providing at least one chromatography column, wherein each column is comprised of

i) a column body having an open upper end, an open lower end, and an open channel between the upper and lower end of the column body,

ii) a bottom frit extending across the open channel,

iii) a packed bed of chromatography medium positioned above the bottom frit, wherein the diameter of each column is within the range of about 12 to about 100 mm²;

b. introducing a sample solution into each column;

c. allowing the sample solution to pass through the column by gravity flow until the flow pauses;

d. introducing an elution liquid into each column;

e. allowing the elution liquid to pass through the column by gravity flow until the flow pauses;

f optionally, repeating steps (d) and (e) at least once;

g. collecting the purified material;

h. optionally, repeating steps (d), (e) and (g).

2. The method of claim 1, wherein the method is automated and steps (b) and (d) are performed by a liquid handler.

3. The method of claim 1, wherein the method is manual and steps (b) and (d) are performed with a pipette.

4. The method of claim 1, wherein the column body is comprised of a modified pipette tip.

5. The method of claim 1, wherein the column body is further comprised of a top frit positioned above the packed bed of chromatography medium.

6. The method of claim 1, wherein prior to step (g), a collection plate is provided and step (g) is performed by touching the open lower end of the columns to the walls of the collection plate wells.

7. The method of claim 1, where in the volume of purified material is in the range of 5 μ

l to 600 μ

l.

8. The method of claim 7, where in the volume of purified material is in the range of 20 μ

l to 90 μ

l.

9. The method of claim 1, wherein the method is performed on a plurality of columns the volume of purified material obtained from the columns has a coefficient of variation of less than 20.

10. The method of claim 1, wherein the distance between the centers of the columns is in the range of about 4.5 mm to about 9.0 mm.

11. The method of claim 10 wherein each column is integrated into a well of a deep-well plate.

12. The method of claim 10, wherein the method is automated and steps (b) and (d) are performed by a liquid handler.

13. The method of claim 10, wherein the method is manual and steps (b) and (d) are performed with a pipette.

14. The method of claim 10, wherein the column body is comprised of a modified pipette tip.

15. The method of claim 10, wherein the column body is further comprised of a top frit positioned above the packed bed of chromatography medium.

16. The method of claim 10, wherein prior to step (g), a collection plate is provided and step (g) is performed by touching the open lower end of the columns to the walls of the collection plate wells or vials.

17. The method of claim 10, where in the volume of purified material is in the range of 5 μ

l to 600 μ

l.

18. The method of claim 17, where in the volume of purified material is in the range of 20 μ

l to 90 μ

l.

19. A plurality of chromatography columns, wherein each column is comprised of

a. a column body having an open upper end, an open lower end, and an open channel between the upper and lower end of the column body;

b. a bottom frit extending across the open channel;

c. a packed bed of medium positioned above the bottom frit; and

d. a top frit extending across the open channel,

wherein the distance between the centers of the columns is within the range of about 4.5 to about 9.0 mm.