U.S. patent application number 12/329319 was filed with the patent office on 2009-03-26 for low dead volume extraction column device.
Invention is credited to Douglas T. Gjerde, Christopher T. Hanna.
Application Number | 20090081084 12/329319 |
Document ID | / |
Family ID | 40471856 |
Filed Date | 2009-03-26 |
United States Patent
Application |
20090081084 |
Kind Code |
A1 |
Gjerde; Douglas T. ; et
al. |
March 26, 2009 |
Low Dead Volume Extraction Column Device
Abstract
The invention provides extraction columns for the purification
of an analyte (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 invention is characterized
by the use of low dead volume columns, which is achieved in part by
the use of low pore volume frits (e.g., membrane screens) to
contain a bed of extraction media in the column. Low dead volume
facilitates the elution of the captured analyte into a very small
volume of desorption solution, allowing for the preparation of low
volume samples containing relatively high concentrations of
analyte.
Inventors: |
Gjerde; Douglas T.;
(Saratoga, CA) ; Hanna; Christopher T.; (San
Francisco, CA) |
Correspondence
Address: |
Douglas T. Gjerde
12295 Woodside Drive
Saratoga
CA
95070
US
|
Family ID: |
40471856 |
Appl. No.: |
12/329319 |
Filed: |
December 5, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10620155 |
Jul 14, 2003 |
7482169 |
|
|
12329319 |
|
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Current U.S.
Class: |
422/400 |
Current CPC
Class: |
B01J 2220/64 20130101;
G01N 2030/009 20130101; Y10T 436/255 20150115; B01D 15/327
20130101; B01L 3/0275 20130101; C07K 16/26 20130101; B01D 15/325
20130101; B01L 2200/0631 20130101; B01D 15/3804 20130101; B01J
20/285 20130101; B01J 20/291 20130101; B82Y 30/00 20130101 |
Class at
Publication: |
422/100 ;
422/101 |
International
Class: |
G01N 1/18 20060101
G01N001/18; B01D 15/08 20060101 B01D015/08 |
Claims
1. A pipette tip extraction column comprising: i) a column body
having an open upper end for attachment to a pump, an open lower
end for passing fluid into and out of the column body, and an open
channel between the upper and lower end of the column body, wherein
the column body comprises a pipette tip; ii) a bottom frit bonded
to and extending across the open channel, the bottom frit having a
pore volume, and wherein the bottom frit is located at or near the
open lower end of the column body; iii) a top frit bonded to and
extending across the open channel between the bottom frit and the
open upper end of the column body, wherein the top frit and bottom
frit are flexible, and wherein the top frit, bottom frit, and
channel surface define an extraction media chamber; and iv) a bed
of gel resin extraction media positioned inside the extraction
media chamber.
2. The pipette tip extraction column of claim 1, wherein the top
frit or the bottom frit is a membrane screen.
3. The pipette tip extraction column of claim 1, wherein the gel
resin is selected from the group consisting of agarose and
sepharose.
4. The pipette tip extraction column of claim 1, wherein the gel
resin extraction media comprises an affinity binding group having
an affinity for a biological molecule of interest.
5. The pipette tip extraction column of claim 4, wherein the
affinity binding group is selected from the group consisting of
Protein A, Protein G, Protein L and an immobilized metal.
6. The pipette tip extraction column of claim 1, wherein the upper
end of the column body is attached to a pump for aspirating fluid
through the lower end of the column body.
7. The pipette tip extraction column of claim 6, wherein the pump
is a pipettor or a syringe.
8. A pipette tip extraction column comprising: i) a column body
having an open upper end for attachment to a pump, an open lower
end for passing fluid into and out of the column body, and an open
channel between the upper and lower end of the column body, wherein
the column body comprises a pipette tip; ii) a bottom frit bonded
to and extending across the open channel, the bottom frit having a
pore volume, and wherein the bottom frit is located at or near the
open lower end of the column body; iii) a top frit bonded to and
extending across the open channel between the bottom frit and the
open upper end of the column body, wherein the top frit and bottom
frit are flexible membrane screens, and wherein the top frit and
the bottom frit are bonded to the column body by gluing or welding,
and wherein the top frit, bottom frit, and channel surface define
an extraction media chamber; and iv) a bed of gel resin extraction
media positioned inside the extraction media chamber.
9. The pipette tip extraction column of claim 8, wherein the gel
resin is selected from the group consisting of agarose and
sepharose.
10. The pipette tip extraction column of claim 8, wherein the gel
resin extraction media comprises an affinity binding group having
an affinity for a biological molecule of interest.
11. The pipette tip extraction column of claim 10, wherein the
affinity binding group is selected from the group consisting of
Protein A, Protein G, Protein L and an immobilized metal.
12. The pipette tip extraction column of claim 8, wherein the upper
end of the column body is attached to a pump for aspirating fluid
through the lower end of the column body.
13. The pipette tip extraction column of claim 12, wherein the pump
is a pipettor or a syringe.
14. A pipette tip extraction column comprising: i) a column body
having an open upper end for attachment to a pump, an open lower
end for passing fluid into and out of the column body, and an open
channel between the upper and lower end of the column body, wherein
the column body comprises a pipette tip; ii) a bottom frit
extending across the open channel, the bottom frit having a pore
volume, and wherein the bottom frit is located at or near the open
lower end of the column body; iii) a top frit extending across the
open channel between the bottom frit and the open upper end of the
column body, wherein the top frit and bottom frit are attached to
the column body by a means selected from the group consisting of an
annular pip, gluing, and welding, and wherein the top frit, bottom
frit, and channel surface define an extraction media chamber; and
iv) a bed of gel resin extraction media positioned inside the
extraction media chamber.
15. The pipette tip extraction column of claim 14, wherein the top
frit or the bottom frit is a membrane screen.
16. The pipette tip extraction column of claim 14, wherein the gel
resin is selected from the group consisting of agarose and
sepharose.
17. The pipette tip extraction column of claim 14, wherein the gel
resin extraction media comprises an affinity binding group having
an affinity for a biological molecule of interest.
18. The pipette tip extraction column of claim 17, wherein the
affinity binding group is selected from the group consisting of
Protein A, Protein G, Protein L and an immobilized metal.
19. The pipette tip extraction column of claim 14, wherein the
upper end of the column body is attached to a pump for aspirating
fluid through the lower end of the column body.
20. The pipette tip extraction column of claim 19, wherein the pump
is a pipettor or a syringe.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of U.S. patent
application Ser. No. 10/620,155 filed Jul. 14, 2003 which is
incorporated by reference herein in its entirety for all
purposes.
FIELD OF THE INVENTION
[0002] This invention relates to a device and method for the
capture of analytes by solid phase extraction with a column device
and collection of the analytes into a controlled volume of solvent.
The analytes can include biomolecules, particularly biological
macromolecules such as proteins and peptides. The device and method
of this invention are particularly useful in proteomics for sample
preparation and analysis with analytical technologies employing
biochips, mass spectrometry and other instrumentation.
BACKGROUND OF THE INVENTION
[0003] Proteomics can be defined as the comprehensive study of
proteins and their functional aspects. Proteins perform the work of
the cell. Single proteins can have many forms. The function of a
protein depends on the form, interactions, and complexes of the
protein. A deeper understanding of the biological functions of
proteins is needed so that drugs can be developed.
[0004] Protein sample processing is a complex problem within
proteomics. Proteins can function individually or as complexes
(groups of proteins bound as a complex). Proteins cannot be
amplified, as DNA is amplified with polymerase chain reaction (PCR)
methods. Proteins must be enriched and purified before they can be
analyzed. Protein processing methods and systems must be flexible;
more than a million possible proteins are expressed. For analysis
it is necessary to separate and concentrate the proteins of
interest from many thousands of other proteins, while selectively
removing other materials that will interfere with the protein
analytical process including cellular material such as other
proteins, sugars, carbohydrates, lipids, DNA, RNA and salts.
Reproducible recovery is needed and in most cases protein function
must be retained during processing. Structural differences between
forms must be preserved and final processing of samples must be
easily integrated into many different detection schemes, for
example mass spectrometry, protein chips, and the like.
[0005] Solid phase extraction is one of the primary tools for
preparing protein samples prior to analysis. The method purifies
proteins according to their identity, class type or structure, or
function to prepare them for analysis by mass spectrometry or other
analytical methods.
[0006] The process of solid phase extraction uses an extraction
phase in the form of a column or bed, and the sample may be either
loaded onto the column or added to a bulk solution to extraction
beads. The extraction phase retains the sample protein, the
extraction phase is washed to remove contaminants, and then the
sample protein is removed with the extraction or recovery
solvent.
[0007] Extraction columns are used to prepare the protein samples
for analysis. Often very low amounts of proteins are expressed in a
sample, and sample preparation procedures are needed to isolate and
recover the protein before analysis.
[0008] The solid phase extraction of biomolecules such as nucleic
acids and proteins is commonly performed by columns packed with a
variety of extraction phases.
[0009] The need for biomolecule extraction for proteins is
increasing rapidly. Large numbers of samples need to be analyzed by
a variety of techniques to determine the function of proteins.
Typical sample volume is 0.5 to 5 mL or more on a typical column
bed volume of 1 to 5 mL, requiring a typical desorption solvent
volume of 2 to 10 mL.
[0010] There are a number of companies that have developed products
whose principle aim is the purification of certain proteins or
protein classes by solid phase extraction. The intent of these
products is the simplification of proteomic analyses by providing a
sample of only those proteins in which the investigator is
interested. These products are often packaged for a single use and
disposal. Packed-bed columns operate at relatively low pressures,
thus making them simple to operate in a highly parallel and
automated manner. Due to the very nature of a conventional
packed-bed approach, it is limited with respect to reliable
quantification and/or enrichment of sample. A packed-bed approach
is extremely difficult to apply in a manner that is both
cost-effective and reliable. It cannot be effectively applied to a
microscale process level.
[0011] Moreover, packed columns have extensive carry-over from
sample to sample, are expensive to manufacture, and may be
difficult to multiplex (extract multiple samples simultaneously).
Proteins may be irreversibly adsorbed to the extraction phase or
may be trapped by frits and other "dead zones" within the column
making recovery of the proteins incomplete.
[0012] Other drawbacks include losses of materials due to unswept
volumes leading to low recoveries and irreproducibility of results;
dilution of materials due to large elution volumes applied in an
attempt to minimize these selfsame unswept volumes; depending on
implementation, requirements often to adhere to a flow
"directionality" introducing limitations on full integration of
sample processing; manufacturing difficulties and costs for micro-
or nanoscale volume systems; and porosity of construction materials
used in commercially available systems that cause severe loss of
biomaterials.
[0013] Spin columns and pipette tip columns are disposable column
technologies commonly used for processing samples. At present, most
of these columns contain filters or frits. Conventional frits,
porous discs used to contain the column beds, have significant dead
volume. This leads to significant sample loss when very small
sample volumes are separated.
[0014] One conventional method for making sample preparation
devices involves first inserting a precut porous plug obtained
from, for example, a fibrous glass or cellulose sheet, into the tip
of a pipette. This is followed by the addition of loose particles
and a second porous plug. The plugs serve to retain the particles
in place in the pipette tip. However, the plugs also entrap excess
liquid thereby creating dead space or volume (i.e., space not
occupied by media or polymer that can lead to poor sample recovery,
contamination such as by sample carry-over, etc.).
[0015] Current available methods are not well suited for the
separation and recovery of very small volumes in the low microliter
range.
[0016] Also, since the volume of the filter is often as large as
the volume of the micro volume sample itself, the extraction or
separation process or chromatography process is adversely affected
due to the large volume of filter material through which the sample
must pass.
[0017] In addition, the adsorption of biomolecules can be a
problem. Since the concentration of biomolecules in micro volume
samples is so small, the adsorption of biomolecules on the filter
can result in significant loss of the total sample mass. The filter
material may also absorb proteins or biomolecules from the sample,
resulting in lower than desirable sample recovery. Also, the filter
material may behave differently in different elution media,
subsequently interfering with both the quality of the separation
process and the volume of the sample retained.
[0018] Collecting samples in the 1 to 20 .mu.L range is a critical
need. At such low volumes, efficient sample handling is crucial to
avoid loss. Conventional methods and devices for sample preparation
are not practical for handling the "microseparation" of such small
sample volumes.
[0019] Ultrafiltration can only effectively concentrate and desalt,
and thus the application of adsorption technology at this scale
could offer an entirely new approach to micro-mass sample
preparation.
[0020] However, these procedures cannot be used with extremely
small liquid delivery devices such as conventional pipette tips, as
there is no practical way to load either the plug or the particles
to obtain a micro-adsorptive device that contains 20 milligrams or
less of adsorbent, the amount suitable for use with the
aforementioned extremely small sample loads.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIG. 1 depicts an embodiment of the invention where the
extraction column body is constructed from a tapered pipette
tip.
[0022] FIG. 2 is an enlarged view of the extraction column of FIG.
1.
[0023] FIG. 3 depicts an embodiment of the invention where the
extraction column is constructed from two cylindrical members.
[0024] FIG. 4 depicts a syringe pump embodiment of the invention
with a cylindrical bed of solid phase media in the tip.
[0025] FIG. 5 is an enlarged view of the extraction column element
of the syringe pump embodiment of FIG. 4.
[0026] FIGS. 6-10 show successive stages in the construction of the
embodiment depicted in FIGS. 1 and 2.
[0027] FIG. 11 depicts an embodiment of the invention with a
straight connection configuration as described in Example 8.
[0028] FIG. 12 depicts an embodiment of the invention with an end
cap and retainer ring configuration as described in Example 9.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0029] This invention is used for the capture of analytes by solid
phase extraction with a column device and collection of the
analytes into a controlled volume of solvent. This invention is
useful for analytes including biomolecules and is compatible with
requirements for sample preparation and analysis by analytical
technology--especially biochips and mass spectrometry.
[0030] The invention is characterized by the use of extraction
columns having low dead volumes. This is achieved in part by the
use of a low volume frit or frits to contain a bed of extraction
media in an extraction media chamber positioned in the column. Low
dead volume facilitates the elution of the captured analyte into a
very small volume of desorption solution, allowing for the
preparation of low volume samples containing relatively high
concentrations of analyte. Low volume, high concentration solutions
particularly useful with regard to protein preparations for
analysis by techniques such as mass spectrometery and protein
chips.
I. Terminology
[0031] Before describing the present invention in detail, it is to
be understood that this invention is not limited to specific
embodiments described herein. It is also to be understood that the
terminology used herein for the purpose of describing particular
embodiments is not intended to be limiting. As used in this
specification and the appended claims, the singular forms "a", "an"
and "the" include plural referents unless the context clearly
dictates otherwise. Thus, for example, reference to polymer bearing
a protected carbonyl would include a polymer bearing two or more
protected carbonyls, and the like.
[0032] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, specific examples of appropriate materials and methods
are described herein.
[0033] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions set out below.
[0034] The term "bed volume" as used herein is defined as the
volume of a bed of extraction media in an extraction column.
Depending on how densely the bed is packed, the volume of the
extraction media in the column bed is typically about half to one
third of the total bed volume; well packed beds have less space
between the beads and hence generally have lower interstital
volumes.
[0035] The term "interstitial volume" of the bed refers to the
volume of the bed of extraction media that is accessible to
solvent, e.g., aqueous sample solutions, wash solutions and
desorption solvents. For example, in the case where the extraction
media is a chromatography bead (e.g., agarose or sepharose), the
interstitial volume of the bed constitutes the solvent accessible
volume between the beads, as well as any solvent accessible
internal regions of the bead, e.g., solvent accessible pores. The
interstitial volume of the bed represents the minimum volume of
liquid required to saturate the column bed.
[0036] The term "dead volume" as used herein with respect to a
column is defined as the interstitial volume of the extraction bed,
tubes, membrane or frits, and passageways in a column. In the
device of this invention with gel-type extraction media and the
pore volume of the frits. Since the bottom frit of the column
directly contacts the sample, wash, and elution liquids, minimal
tubing or passageway dead volume is present in this device.
[0037] The term "elution volume" as used herein is defined as the
volume of desorption or elution liquid into which the analytes are
desorbed and collected. The terms "desorption solvent," elution
liquid" and the like are used interchangeably herein.
[0038] The term "enrichment factor" as used herein is defined as
the ratio of the sample volume divided by the elution volume,
assuming that there is no contribution of liquid coming from the
dead volume. To the extent that the dead volume either dilutes the
analytes or prevents complete adsorption, the enrichment factor is
reduced.
[0039] The terms "extraction column" and "extraction tip" as used
herein are defined as a column device used in combination with a
pump, the column device containing a bed of solid phase extraction
material, i.e., extraction media.
[0040] The term "frit" as used herein are defined as porous
material for holding the extraction media in place in a column. An
extraction media chamber is typically defined by a top and bottom
frit positioned in an extraction column. In preferred embodiments
of the invention the frit is thin, and has a low pore volume, e.g.,
a membrane screen.
[0041] The term "gel-type packing material" as used herein is
defined as non-porous or micro-porous beads such as agarose or
sepharose beads, the beads containing a functional group or having
a surface that binds selectively with the analyte of interest.
[0042] The term "lower column body" as used herein is defined as
the column bed and bottom membrane screen of a column.
[0043] The term "membrane screen" as used herein is defined as a
woven or non-woven fabric or screen for holding the column packing
in place in the column bed, the membranes having a low dead volume.
The membranes are of sufficient strength to withstand packing and
use of the column bed and of sufficient porosity to allow passage
of liquids through the column bed. The membrane is thin enough so
that it can be sealed around the perimeter or circumference of the
membrane screen so that the liquids flow through the screen.
[0044] The term "sample volume", as used herein is defined as the
volume of the liquid of the original sample solution from which the
analytes are separated or purified.
[0045] The term "upper column body", as used herein is defined as
the chamber and top membrane screen of a column.
[0046] The term "biomolecule" as used herein refers to biomolecule
derived from a biological system. The term includes biological
macromolecules, such as a proteins, peptides, and nucleic
acids.
[0047] The term "protein chip" is defined as a small plate or
surface upon which an array of separated, discrete protein samples
are to be deposited or have been deposited. These protein samples
are typically small and are sometimes referred to as "dots." In
general, a chip bearing an array of discrete proteins is designed
to be contacted with a sample having one or more biomolecules which
may or may not have the capability of binding to the surface of one
or more of the dots, and the occurrence or absence of such binding
on each dot is subsequently determined. A reference that describes
the general types and functions of protein chips is Gavin MacBeath,
Nature Genetics Supplement, 32:526 (2002).
II. Low Dead Volume Extraction Columns
[0048] Column Body
[0049] The column body is a tube having two open ends connected by
an open channel. The tube can be in any shape, including but not
limited to cylindrical or frustoconical, and of any dimensions
consistent with the function of the column as described herein. In
certain embodiments of the invention the column body takes the form
of a pipette tip, a syringe, a luer adapter or similar tubular
bodies.
[0050] One of the open ends of the column, sometimes referred to
herein as the open upper end of the column, is adapted for
attachment to a pump. In some embodiments of the invention the
upper open end is operatively attached to a pump, whereby the pump
can be used for aspirating a fluid into the extraction column
through the other open end of the column, and optionally for
discharging fluid out through the open lower end of the column.
Thus, it is a feature of the present invention that fluid enters
and exits the extraction column through the same open end of the
column. This is in contradistinction with the operation of some
extraction columns, where fluid enters the column through one open
end and exits through the other end after traveling through an
extraction media, i.e., similar to conventional column
chromatography. The fluid can be a liquid, such as a sample
solution, wash solution or desorption solvent.
[0051] The column body can be can be composed of any material that
is sufficiently non-porous that it can retain fluid and that is
compatible with the solutions, media, pumps and analytes used. A
material should be employed that does not substantially react with
substances it will contact during use of the extraction column,
e.g., the sample solutions, the analyte of interest, the extraction
media and desorption solvent. A wide range of suitable materials
are available and known to one of skill in the art, and the choice
is one of design. Various plastics make ideal column body
materials, but other materials such as glass, ceramics or metals
could be used in some embodiments of the invention. Some examples
of materials include polysulfone, polypropylene, polyethylene,
polyethyleneterephthalate, polyethersulfone,
polytetrafluoroethylene, cellulose acetate, cellulose acetate
butyrate, acrylonitrile PVC copolymer, polystyrene,
polystyrene/acrylonitrile copolymer, polyvinylidene fluoride,
glass, metal, silica, and combinations of the above listed
materials.
[0052] Some specific examples of suitable column bodies are
provided in the Examples.
[0053] Extraction Media
[0054] The extraction media used in the column is preferably a form
of water-insoluble particle (e.g., a porous or non-porous bead)
that has an affinity for an analyte of interest. Typically the
analyte of interest is a protein, peptide or nucleic acid. The
extraction processes can be affinity, reverse phase, normal phase,
ion exchange, hydrophobic interaction chromatography, or
hydrophilic interaction chromatography agents.
[0055] The bed volume of the extraction media used in the
extraction columns of the invention is typically small, preferably
in the range of 0.5-100 .mu.L, more preferably in the range of 1-50
.mu.L, and still more preferably in the range of 2-25 .mu.L. The
low bed volume results in a low interstitial volume of the bed,
contributing to the low dead volume of the column, thereby
facilitating the recovery of the analyte in a small volume of
desorption solvent.
[0056] The low bed volumes employed in certain embodiments allow
for the use of relatively small amounts of extraction media, e.g.,
soft, gel-type beads. For example, some embodiments of the
invention employ a bed of extraction media having a dry weight of
less than 10 mg (e.g., in the range of 0.1-10 mg, 0.5-10 mg, 1-10
mg or 2-10 mg), less than 2 ms (e.g., in the range of 0.1-2 mg,
0.5-2 mg or 1-2 mg), or less than 1 mg (e.g., in the range of 0.1-1
mg or 0.5-1 mg).
[0057] Many of the extraction media types suitable for use in the
invention are selected from a variety of classes of chromatography
media. It has been found that many of these chromatography media
types and the associated chemistries are suited for use as solid
phase extraction media in the devices methods of this
invention.
[0058] Thus, examples of suitable extraction media include
agarose-based materials, sepharose-based materials,
polystyrene/divinylbenzene copolymers, poly methylmethacrylate,
protein G beads (e.g., for IgG protein purification), MEP
Hypercel.TM. beads (e.g., for IgG protein purification), affinity
phase beads (e.g., for protein purification), ion exchange phase
beads (e.g., for protein purification), hydrophobic interaction
beads (e.g., for protein purification), reverse phase beads (e.g.,
for nucleic acid or protein purification), and beads having an
affinity for molecules analyzed by label-free detection. Silica
beads are also suitable.
[0059] Soft gel-type beads, such as agarose and sepharose based
beads, are found to work surprisingly well in columns and methods
of this invention. In conventional chromatography fast flow rates
can result in bead compression, which results in increased back
pressure and adversely impacts the ability to use these gels with
faster flow rates. In the present invention relatively small bed
volumes are used, and it appears that this allows for the use of
high flow rates with a minimal amount of bead compression and the
problem attendant with such compression.
[0060] Affinity extractions use a technique in which a biospecific
adsorbent is prepared by coupling a specific ligand (such as an
enzyme, antigen, or hormone) for the analyte, (e.g., macromolecule)
of interest to a solid support. This immobilized ligand will
interact selectively with molecules that can bind to it. Molecules
that will not bind elute unretained. The interaction is selective
and reversible. The references listed below show examples of the
types of affinity groups that can be employed in the practice of
this invention are hereby incorporated by reference herein in their
entireties. Antibody Purification Handbook, Amersham Biosciences,
Edition AB, 18-1037-46 (2002); Protein Purification Handbook,
Amersham Biosciences, Edition AC, 18-1132-29 (2001); Affinity
Chromatography Principles and Methods, Amersham Pharmacia Biotech,
Edition AC, 18-1022-29 (2001); The Recombinant Protein Handbook,
Amersham Pharmacia Biotech, Edition AB, 18-1142-75 (2002); and
Protein Purification: Principles, High Resolution Methods, and
Applications, Jan-Christen Janson (Editor), Lars G. Ryden (Editor),
Wiley, John & Sons, Incorporated (1989).
[0061] Examples of suitable affinity binding agents are summarized
in Table I, wherein the affinity agents are from one or more of the
following interaction categories: [0062] 1. Chelating metal--ligand
interaction [0063] 2. Protein--Protein interaction [0064] 3.
Organic molecule or moiety--Protein interaction [0065] 4.
Sugar--Protein interaction [0066] 5. Nucleic acid--Protein
interaction [0067] 6. Nucleic acid--nucleic acid interaction
TABLE-US-00001 [0067] TABLE I Examples of Affinity molecule or
moiety fixed at Interaction surface Captured biomolecule Category
Ni-NTA His-tagged protein 1 Ni-NTA His-tagged protein within a 1, 2
multi-protein complex Fe-IDA Phosphopeptides, 1 phosphoproteins
Fe-IDA Phosphopeptides or 1, 2 phosphoproteins within a
multi-protein complex Antibody or other Proteins Protein antigen 2
Antibody or other Proteins Small molecule-tagged 3 protein Antibody
or other Proteins Small molecule-tagged 2, 3 protein within a
multi- protein complex Antibody or other Proteins Protein antigen
within a 2 multi-protein complex Antibody or other Proteins
Epitope-tagged protein 2 Antibody or other Proteins Epitope-tagged
protein 2 within a multi-protein complex Protein A, Protein G or
Antibody 2 Protein L Protein A, Protein G or Antibody 2 Protein L
ATP or ATP analogs; 5'- Kinases, phosphatases 3 AMP (proteins that
requires ATP for proper function) ATP or ATP analogs; 5'- Kinase,
phosphatases 2, 3 AMP within multi-protein complexes Cibacron 3G
Albumin 3 Heparin DNA-binding protein 4 Heparin DNA-binding
proteins 2, 4 within a multi-protein complex Lectin Glycopeptide or
4 glycoprotein Lectin Glycopeptide or 2, 4 glycoprotein within a
multi-protein complex ssDNA or dsDNA DNA-binding protein 5 ssDNA or
dsDNA DNA-binding protein 2, 5 within a multi-protein complex ssDNA
Complementary ssDNA 6 ssDNA Complementary RNA 6 Streptavidin/Avidin
Biotinylated peptides 3 (ICAT) Streptavidin/Avidin Biotinylated
engineered tag 3 fused to a protein (see avidity.com)
Streptavidin/Avidin Biotinylated protein 3 Streptavidin/Avidin
Biotinylated protein within 2, 3 a multi-protein complex
Streptavidin/Avidin Biotinylated engineered tag 2, 3 fused to a
protein within a multi-protein complex Streptavidin/Avidin
Biotinylated nucleic acid 3 Streptavidin/Avidin Biotinylated
nucleic acid 2, 3 bound to a protein or multi- protein complex
Streptavidin/Avidin Biotinylated nucleic acid 3, 6 bound to a
complementary nucleic acid
[0068] In one aspect of the invention an extraction media is used
that contains a surface functionality that has an affinity for a
protein fusion tag used for the purification of recombinant
proteins. A wide variety of fusion tags and corresponding affinity
groups are available and can be used in the practice of the
invention.
[0069] One of the most common fusion tags is the so-called "His"
tag, which is comprised of a series of consecutive histidine
residues, e.g., two, four or six consecutive histidine residues.
There are a number of metal-chelate groups that can be attached to
the surface of an extraction media for purification of "His-tagged
proteins, including metal-IDA (IDA: iminodiacetate), metal-NTA
(NTA: nitrilotriacetate), and metal-CMA (CMA: carboxymethylated
aspartate), where the metal is typically selected from nickel,
copper, iron, zinc and cobalt. The trapped fusion protein is eluted
by disrupting the histidine-metal coordination by some suitable
salt such as imidazole or ethylene diamine tetra acetic acid
(EDTA).
[0070] There are other affinity groups available for purifying
recombinant proteins through their fusion tags, and these groups
can be attached to an extraction media for use in the invention.
Antibodies can be used for purification through any peptide
sequence (a common one is the FLAG tag); avidin (monomeric or
multimeric) can be used for purifying a peptide sequence that is
selectively biotinylated within the expression system; calmodulin
charged with calcium can be used for purifying a peptide sequence
that is often referred to as a "calmodulin binding peptide" (or,
CBP), where elution is performed by removing the calcium with
ethylene glycol tetra acetic acid (EGTA); glutathione can be used
for purifying a fusion protein that carries the glutathione
S-transferase protein (GST), where the GST is often cleaved off
with a specific protease; amylose can be used for purifying a
fusion protein that carries the maltose binding protein (MBP),
where the MBP is often cleaved off with a specific protease;
cellulose can be used for purifying a fusion protein that carries a
peptide that is referred to as the cellulose-binding domain tag,
followed by elution with ethylene glycol; S-protein (derived from
ribonuclease A) can be used for purifying a fusion protein that
carries a peptide with specific affinity for S-protein, where the
peptide can be cleaved off with a specific protease.
[0071] It is also possible to create an affinity surface that has
the bis-arsenical fluorescein dye FlAsH. For example, a FlAsH dye
can be used for purifying a fusion protein that carries the peptide
sequence tag CCxxCC (where xx is any amino acid, such as RE). The
protein is then eluted with 1,4-dithiothreitol, or DTT.
[0072] In one aspect the invention is used for purification of
antibodies. Antibodies are frequently purified on the basis of
highly conserved structural characteristics. For example, it is
possible to pack columns with extraction media containing Protein
A, Protein G, or Protein A/G fusions to purify IgG antibodies
through their Fc region (with lower affinity for the Fab antibody
fragment region in the case of Protein G). These are often eluted
by using low pH 2.5. It is also possible to purify IgG antibodies
through their Fab antibody fragment region, provided their light
chain is a kappa light chain. This is achieved by using a surface
of Protein L.
[0073] In one aspect the extraction media comprises small molecule
ligands that are capable of achieving separations on the basis of
hydrophobic charge interactions. Ligands such as
4-mercapto-ethyl-pyridine and 2-mercaptopyridine are capable of
trapping antibodies such as IgGs, which are eluted by changes to
low pH much milder than in the case of Protein A or Protein G. For
example, elution is accomplished with 4-mercapto-ethyl-pyridine at
pH 4 (as opposed to pH 2.5 for the Protein A and Protein G).
[0074] In addition, other antibodies can be used for purification
of antibodies. For example, it is possible to use an extraction
media comprising an immobilized antibody for the purification of
IgE (with an anti-IgE surface), the purification of IgM (with an
anti-IgM surface), the purification of IgA (with an anti-IgA
surface), the purification of IgD (with an anti-IgD surface), as
well as the purification of IgG (with an anti-IgG surface).
[0075] Extraction columns of the invention can be used for
purification of phosphopeptides and phosphoproteins by the
inclusion of an appropriate affinity group on the extraction media.
One alternative is to exploit the natural interaction between
phosphate groups and metal ions. Therefore, phosphopeptides and
phosphoproteins can be purified on metal-chelate surfaces made from
IDA, NTA, or CMA.
[0076] It is also possible to purify these phosphopeptides and
phosphoproteins with immobilized antibodies. For example, it is
possible to use antibodies on the packing material that are
specific to phosphotyrosine residues, as well as phosphoserine and
phosphothreonine residues. It is also possible to use antibodies
that are bind to specific phosphorylated sites within a protein,
such as specifically-binding phosphorylated tyrosine within a
specific kinase. These antibodies are often referred to as
phosphorylation site-specific antibodies (PSSAs). Once adsorbed the
trapped phosphoprotein and phosphopeptides can be eluted at low
pH.
[0077] Yet another approach to the purification of phosphopeptides
and phosphoproteins involves the derivitization of the phosphate
group such that biotin is attached to it. This biotinylated
phosphoprotein or phosphopeptide can be purified using an
avidin-derivatized extraction media, wherein the avidin can be
monomeric or multimeric.
[0078] In some embodiments of the invention an extraction column is
used for the purification of protein complexes. One embodiment
involves the use of a recombinant "bait" protein that will form
complexes with its natural interaction partners. These multiprotein
complexes are then purified through a fusion tag that is attached
to the "bait." These tagged "bait" proteins can be purified through
groups incorporated into the extraction media such as metal-chelate
groups, antibodies, calmodulin, or any of the other surface groups
described above for the purification of recombinant proteins.
[0079] It is also possible to purify "native" (i.e.
non-recombinant) protein complexes without having to purify through
a fusion tag. This is achieved by immobilizing an antibody for one
of the proteins within the multiprotein complex. This process is
often referred to as "co-immunoprecipitation." The multiprotein
complexes can be eluted with low pH.
[0080] Extraction columns of the invention can be used to purify
entire classes of proteins on the basis of highly conserved motifs
within their structure, whereby an affinity ligand attached to the
packing reversibly binds to the conserved motif. For example, it is
possible to immobilize particular nucleotides on the extraction
media. Examples include, but are not limited to, adenosine
5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine
5'-monophosphate (AMP), nicotinamide adenine dinucleotide (NAD), or
nicotinamide adenine dinucleotide phosphate (NADP). These
nucleotides can be used for the purification of enzymes that are
dependent upon these nucleotides such as kinases, phosphatases,
heat shock proteins and dehydrogenases, to name a few.
[0081] There are other affinity groups that can be incorporated
into the extraction media for purification of protein classes.
Lectins can be used for the purification of glycoproteins.
Concanavilin A (Con A) and lentil lectin can be used for the
purification of glycoproteins and membrane proteins, and wheat germ
lectin can be used for the purification of glycoproteins and cells
(especially T-cell lymphocytes). Though it is not a lectin, the
small molecule phenylboronic acid can also be incorporated into the
extraction media and used for purification of glycoproteins.
[0082] It is also possible to incorporate heparin into the
extraction media, which is useful for the purification of
DNA-binding proteins (e.g. RNA polymerase I, II and III, DNA
polymerase, DNA ligase). In addition, immobilized heparin can be
used for purification of various coagulation proteins (e.g.
antithrombin III, Factor VII, Factor IX, Factor XI, Factor XII and
XIIa, thrombin), other plasma proteins (e.g. properdin, BetaIH,
Fibronectin, Lipses), lipoproteins (e.g. VLDL, LDL, VLDL
apoprotein, HOLP, to name a few), and other proteins (platelet
factor 4, hepatitis B surface antigen, hyaluronidase). These types
of proteins are often blood and/or plasma borne. Since there are
many efforts afoot to rapidly profile the levels of these types of
proteins by technologies such as protein chips, the performance of
these chips will be enhanced by performing an initial purification
and enrichment of the targets prior to protein chip analysis.
[0083] It is also possible to use extraction media with protein
interaction domains for purification of those proteins that are
meant to interact with that domain. One interaction domain that can
be used is the Src-homology 2 (SH2) domain that binds to specific
phophotyrosine-containing peptide motifs within various proteins.
The SH2 domain has previously been immobilized on a resin and used
as an affinity reagent for performing affinity chromatography/mass
spectrometry experiments for investigating in vitro phosphorylation
of epidermal growth factor receptor (EGFR) (see Christian Lombardo,
et al., Biochemistry, 34:16456 (1995)). Other than the SH2 domain,
other protein interaction domains can be used for the purposes of
purifying those proteins that possess their recognition domains.
Many of these protein interaction domains have been described (see
Tony Pawson, Protein Interaction Domains, Cell Signaling Technology
Catalog, 264-279 (2002)) for additional examples of these protein
interaction domains).
[0084] Benzamidine is another example of a class-specific affinity
ligand, which can be incorporated into the extraction media for
purification of serine proteases. The dye ligand Procion Red HE-3B
can be used for the purification of dehydrogenases, reductases and
interferon, to name a few.
[0085] Reversed-phase chromatography media can also function as an
extraction media in certain embodiments of the invention. In
reversed-phase chromatography, an aqueous/organic solvent mixture
is commonly used as the mobile phase, and a high-surface-area
nonpolar solid is employed as the stationary phase. The latter can
be an alkyl-bonded silica packing, e.g., with C.sub.8 or C.sub.18
groups covering the silica surface. The basis of solute retention
in reversed-phase chromatography is still somewhat controversial;
some workers favor an adsorption, while others believe that the
solute partitions into the nonpolar stationary phase. Probably both
processes are important for many samples. Competition between
solute and mobile-phase molecules exists for a place on the
stationary-phase surface. That is, an adsorbed molecule will
displace some number of previously adsorbed molecules
(Chromatography, 5.sup.th edition, PART A: FUNDAMENTALS AND
TECHNIQUES, editor: E. Heftmann, Elsevier Science Publishing
Company, New York, pp A25 (1992)). The near universal application
of reversed-phase chromatography stems from the fact that virtually
all organic molecules have hydrophobic regions in their structure
and are capable of interacting with the stationary phase. Since the
mobile phase is polar and generally contains water, the method is
ideally suited to the separation of polar molecules which are
either insoluble in organic solvents or bind too strongly to
inorganic oxide adsorbents for normal elution. Reversed-phase
chromatography employing acidic, low ionic strength eluents has
become a widely established technique for the purification and
structural elucidation of proteins. However, the structure of
biopolymers is very sensitive to mobile phase composition, pH and
the presence of complexing species which can result in anomalous
retention and even denaturing of proteins. A general characteristic
of reversed-phase systems is that a decrease in polarity of the
mobile phase, that is increasing the volume fraction of organic
solvent in an aqueous organic mobile phase, leads to a decrease in
retention; a reversal of the general trends observed in
liquid-solid chromatography or normal phase chromatography. It is
also generally observed for reversed-phase chromatography that for
members of a homologous or oligomous series, the logarithm of the
solute capacity factor is a linear function of the number of
methylene groups or repeat units of the oligomeric structure
(ADVANCED CHROMATOGRAPHIC AND ELECTROMIGRATION METHODS IN
BIOSCIENCES, editor: Z. Deyl, Elsevier Science BV, Amsterdam, The
Netherlands, pp 528 (1998); CHROMATOGRAPHY TODAY, Colin F. Poole
and Salwa K. Poole, and Elsevier Science Publishing Company, New
York, pp 394 (1991)).
[0086] The references listed below show different types of surfaces
used for reverse phase separations and are hereby incorporated by
reference herein in their entireties: CHROMATOGRAPHY, 5.sup.th
edition, Part A: Fundamentals and Techniques, editor: E. Heftmann,
Elsevier Science Publishing Company, New York, pp A25 (1992);
ADVANCED CHROMATOGRAPHIC AND ELECTROMIGRATION METHODS IN
BIOSCIENCES, editor: Z. Deyl, Elsevier Science BV, Amsterdam, The
Netherlands, pp 528 (1998); CHROMATOGRAPHY TODAY, Colin F. Poole
and Salwa K. Poole, and Elsevier Science Publishing Company, New
York, pp 394 (1991).
[0087] Ion-pair chromatography media can also function as an
extraction media in certain embodiments of the invention. In
ion-pair chromatography, the column packing is usually the same as
in reversed-phase chromatography; e.g., a C.sub.8 or C.sub.18
silica. The mobile phase is likewise similar to that used in
reverse phase chromatography: an aqueous/organic solvent mixture
containing a buffer plus a so-called ion-pair reagent. The ion-pair
reagent will be positively charged for the retention and separation
of sample anions and negatively charged for the retention of sample
cations. Typical examples of ion-pair reagents are hexane sulfonate
and tetrabutylammonium. The basis of retention in ion-pair
chromatography is still controversial, two different processes
being possible: (a) adsorption of ion pairs or (b) formation of an
in situ ion exchanger. Although these two processes appear somewhat
different, they lead to quite similar predictions of retention as a
function of experimental conditions. Retention in ion-pair
chromatography can be continuously varied from a reversed-phase
process to an ion-exchange process. This capability provides a
number of practical advantages. For example, variation of the
mobile phase composition allows a considerable control over the
retention of individual sample ions. This can be used to separate
particularly difficult samples, e.g., mixtures of anionic,
cationic, and/or neutral molecules (CHROMATOGRAPHY, 5.sup.th
Edition, Part A: Fundamentals And Techniques, editor: E. Heftmann,
Elsevier Science Publishing Company, New York, pp A28 (1992)).
[0088] The references listed below show different types of groups
used for ion-pair chromatography and are hereby incorporated by
reference herein in their entireties: Reference: CHROMATOGRAPHY,
5.sup.th Edition, Part A: Fundamentals and Techniques, editor: E.
Heftmann, Elsevier Science Publishing Company, New York, pp A28
(1992); and CHROMATOGRAPHY TODAY, Colin F. Poole and Salwa K.
Poole, Elsevier Science Publishing Company, New York, pp 411
(1991).
[0089] Normal phase chromatography media can also function as an
extraction media in certain embodiments of the invention. In normal
phase chromatography, the stationary phase is a high-surface-area
polar adsorbent, e.g., silica or a bonded silica with polar surface
groups. The mobile phase (a mixture of organic solvents) is less
polar than the stationary phase. Consequently, more polar solutes
are preferentially retained; there is often little difference in
the retention of different homologues or a particular compound
class. This has led to the use of normal phase chromatography for
so-called compound-class (group-type) separations, where, e.g.,
alcohols are separated as a group from monoesters and other
compound classes. The basis of normal phase chromatography
retention is an adsorption/displacement process. Another feature of
normal phase chromatography retention is the so-called localization
of adsorbed solute and mobile-phase molecules on the
stationary-phase surface. Localization refers to the formation of
discrete bonds (by dipole/dipole or hydrogen-bonding interactions)
between polar sites on the adsorbent and polar substituents in the
solute molecule. Localization, in turn, confers a high degree of
specificity to the interaction of solute isomers with the adsorbent
surface, leading to typically better separations of isomers by
normal phase chromatography than by other chromatographic methods
(CHROMATOGRAPHY, 5.sup.th edition, Part A: Fundamentals and
Techniques, editor: E. Heftmann, Elsevier Science Publishing
Company, New York, pp A27 (1992)).
[0090] The references listed below show different types of affinity
groups used for normal phase chromatography and are hereby
incorporated by reference herein in their entireties:
CHROMATOGRAPHY, 5.sup.th edition, Part A: Fundamentals and
Techniques, editor: E. Heftmann, Elsevier Science Publishing
Company, New York, pp A27 (1992); and CHROMATOGRAPHY TODAY, Colin
F. Poole and Salwa K. Poole, Elsevier Science Publishing Company,
New York, pp 375 (1991).
[0091] Ion Exchange chromatography media can also function as an
extraction media in certain embodiments of the invention. Ion
Exchange (IEX) is a mode of chromatography in which ionic
substances are separated on cationic or anionic sites of the
packing. The surface in ion exchange is usually an organic matrix
which is substituted with ionic groups, e.g., sulfonate or
trimethylammonium. The mobile phase typically consists of water
plus buffer and/or salt. The retention of a solute ion occurs via
ion exchange with a mobile phase ion or similar (positive or
negative) charge. Ion exchange chromatography is often applied to
the separation of acidic or basic samples, whose charge varies with
pH. In the simple case of solute molecules bearing a single acidic
or basic group, the solute will be present as some mixture of
charged and neutral species. The fraction of solute molecules that
are ionized then determines retention. In the case of ion exchange,
the retention of the uncharged species can be ignored
(CHROMATOGRAPHY, 5.sup.th Edition, Part A: Fundamentals and
Techniques, editor: E. Heftmann, Elsevier Science Publishing
Company, New York, pp A28 (1992)). Ion exchange chromatography is
one of the oldest and most traditional techniques for separating
complex mixtures of proteins. The references listed below show
different types of groups and surfaces used for ion exchange
chromatography and are hereby incorporated by reference herein in
their entireties; CHROMATOGRAPHY, 5.sup.th Edition, Part A:
Fundamentals and Techniques, editor: E. Heftmann, Elsevier Science
Publishing Company, New York, pp A28 (1992); CHROMATOGRAPHY TODAY,
Colin F. Poole and Salwa K. Poole, Elsevier Science Publishing
Company, New York, pp 422 (1991); and ADVANCED CHROMATOGRAPHIC AND
ELECTROMIGRATION METHODS IN BIOSCIENCES, editor: Z. Deyl, Elsevier
Science BV, Amsterdam, The Netherlands, pp 540 (1998).
[0092] Hydrophobic Interaction Chromatography media can also
function as an extraction media in certain embodiments of the
invention. Hydrophobic Interaction Chromatography is widely used
for the separation and purification of proteins. During separation,
proteins are induced to bind to a weakly hydrophobic stationary
phase using a buffered mobile phase of high ionic strength and then
selectively desorbed during a decreasing salt concentration
gradient. Proteins are usually separated in hydrophobic interaction
chromatography according to their degree of hydrophobicity, much as
in reversed-phase chromatography, but because of the gentler nature
of the separation mechanism, there is a greater probability that
they will elute with their conformational structure (biological
activity) intact. In reversed-phase chromatography, proteins unfold
on the bonded phase surface as a consequence of the high
interfacial tension existing between the mobile and the bonded
stationary phases. These conditions are minimized in hydrophobic
interaction chromatography by using stationary phases of lower
hydrophobicity together with totally aqueous mobile phases, in
general, since solvent strength is controlled by varying ionic
strength rather than by increasing the volume fraction of an
organic modifier. Retention and selectivity in hydrophobic
interaction chromatography depend substantially on the type of
stationary phase. Retention increases for more hydrophobic ligands
and with it the possibility of denaturing certain proteins. Some
proteins are only satisfactorily handled on hydrophilic stationary
phases. The ligand density and structure as well as the
hydrophobicity of the stationary phase are the primary stationary
phase variables that should be optimized for the separation of
individual proteins. Mobile phase parameters that have to be
optimized are the salt concentration, salt type, slope of the salt
gradient, pH, addition of surfactant or organic modifier and
temperature. In the absence of specific binding of the salt to the
protein molecule and at relatively high salt concentration in the
mobile phase, retention increases linearly with the salt molality
and at constant salt concentration with the molal surface tension
increment of the salt used in the aqueous mobile phase.
[0093] The reference listed below shows different types of groups
and surfaces used for hydrophobic interactions and is hereby
incorporated by reference herein in its entirety: CHROMATOGRAPHY
TODAY, Colin F. Poole and Salwa K. Poole, Elsevier Science
Publishing Company, New York, 402 (1991).
[0094] Frits
[0095] Columns of the invention employ frits having a low pore
volume, which contributes to the low dead volume of the columns.
The frits of the invention are porous, since it is necessary for
fluid to be able to pass through the frit. The frit should have
sufficient structural strength so that frit integrity can contain
the extraction media in the column. It is desirable that the frit
have little or no affinity for chemicals with which it will come
into contact during the extraction process, particularly the
analyte of interest. In many embodiments of the invention the
analyte of interest is a biomolecule, particularly a biological
macromolecule. Thus in many embodiments of the invention it
desirable to use a frit that has a minimal tendency to bind or
otherwise interact with biological macromolecules, particularly
proteins, peptides and nucleic acids.
[0096] Frits of various pores sizes and pore densities may be used
provided the free flow of liquid is possible and the beads are held
in place within the extraction media bed.
[0097] One frit, i.e., a lower frit, is bonded to and extends
across the open channel of the column body. A second frit is bonded
to and extends across the open channel between the bottom frit and
the open upper end of the column body.
[0098] The top frit, bottom frit and channel surface define an
extraction media chamber wherein a bed of extraction media is
positioned. The frits should be securely attached to the column
body and extend across the opening and/or open end so as to
completely occlude the channel, thereby substantially confining the
bed of extraction media inside the extraction media chamber. In
certain embodiments of the invention the bed of extraction media
occupies at least 80% of the volume of the extraction media
chamber, more preferably 90%, 95%, 99%, or substantially 100% of
the volume. In some embodiments the invention the space between the
extraction media bed and the upper and lower frits is negligible,
i.e., the frits are in substantial contact with upper and lower
surfaces of the extraction media bed, holding a well-packed bed of
extraction media securely in place.
[0099] In some embodiments of the invention the bottom frit is
located at the open lower end of the column body. This
configuration is shown in the figures and exemplified in the
Examples, but is not required, i.e., in some embodiments the bottom
frit is located at some distance up the column body from the open
lower end. However, in view of the importance of minimizing dead
volume in the column it is desirable that the lower frit and
extraction media chamber be located at or near the lower end. In
some cases this can mean that the bottom frit is attached to the
face of the open lower end, as shown in FIGS. 1-10. However, in
some cases there can be some portion of the lower end extending
beyond the bottom frit, as exemplified by the embodiment depicted
in FIG. 11. For the purposes of this invention, so long as the
length as this extension is such that it does not substantially
introduce dead volume into the extraction column or otherwise
adversely impact the function of the column, the bottom frit is
considered to be located at the lower end of the column body. In
some embodiments of the invention the volume defined by the bottom
frit, channel surface, and the face of the open lower end (i.e.,
the channel volume below the bottom frit) is less than the volume
of the extraction media chamber, or less than 10% of the volume of
the extraction media chamber, or less than 1% of the volume of the
extraction media chamber.
[0100] The frits used in the invention are characterized by having
a low pore volume. Some embodiments of the invention employ frits
having pore volumes of less than 1 microliter (e.g., in the range
of 0.015-1 microliter, 0.03-1 microliter, 0.1-1 microliter or 0.5-1
microliter), preferably less than 0.5 microliter (e.g., in the
range of 0.015-0.5 microliter, 0.03-0.5 microliter or 0.1-0.5
microliter), less than 0.1 microliter (e.g., in the range of
0.015-0.1 microliter or 0.03-0.1 microliter) or less than 0.03
microliters (e.g., in the range of 0.015-0.03 microliter).
[0101] Frits of the invention preferably have pore openings or mesh
openings of a size in the range of about 5-100 .mu.m, more
preferably 10-100 .mu.m, and still more preferably 15-50 .mu.m,
e.g, about 43 .mu.m. The performance of the column is typically
enhanced by the use of frits having pore or mesh openings
sufficiently large so as to minimize the resistance to flow. The
use of membrane screens as described herein typically provide this
low resistance to flow and hence better flow rates, reduced back
pressure and minimal distortion of the bed of extraction media. The
pore or mesh openings of course should not be so large that they
are unable to adequately contain the extraction media in the
chamber.
[0102] The frits used in the practice of the invention are
characterized by having a low pore volume relative to the
interstitial volume of the bed of extraction media contained by the
frit. Thus, in certain embodiments of the invention the frit pore
volume is equal to 10% or less of the interstitial volume of the
bed of extraction media (e.g., in the range 0.1-10%, 0.25-10%,
1-10% or 5-10% of the interstitial volume), more preferably 5% or
less of the interstitial volume of the bed of extraction media
(e.g., in the range 0.1-5%, 0.25-5% or 1-5% of the interstitial
volume), and still more preferably 1% or less of the interstitial
volume of the bed of extraction media (e.g., in the range 0.1-1% or
0.25-0% of the interstitial volume).
[0103] The pore density will allow flow of the liquid through the
membrane and is preferably 10% and higher to increase the flow rate
that is possible and to reduce the time needed to process the
sample.
[0104] Some embodiments of the invention employ a thin frit,
preferably less than 350 .mu.m in thickness (e.g., in the range of
20-350 .mu.m, 40-350 .mu.m, or 50-350 .mu.m), more preferably less
than 200 .mu.m in thickness (e.g., in the range of 20-200 .mu.m,
40-200 .mu.m, or 50-200 .mu.m), more preferably less than 100 .mu.m
in thickness (e.g., in the range of 20-100 .mu.m, 40-100 .mu.m, or
50-100 .mu.m), and most preferably less than 75 .mu.m in thickness
(e.g., in the range of 20-75 .mu.m, 40-75 .mu.Mm, or 50-75
.mu.m).
[0105] Some embodiments of the invention employ a membrane screen
as the frit. The membrane screen should be strong enough to not
only contain the extraction media in the column bed, but also to
avoid becoming detached or punctured during the actual packing of
the media into the column bed. Membranes can be fragile, and in
some embodiments must be contained in a framework to maintain their
integrity during use. However, it is desirable to use a membrane of
sufficient strength such that it can be used without reliance on
such a framework. The membrane screen should also be flexible so
that it can conform to the column bed. This flexibility is
advantageous ins the packing process as it allows the membrane
screen to conform to the bed of extraction media, resulting in a
reduction in dead volume.
[0106] Preferably the membrane is a woven or non-woven mesh of
fibers that may be a mesh weave, a random orientated mat of fibers
i.e. a " " polymer paper," a spun bonded mesh, an etched or "pore
drilled" paper or membrane such as nuclear track etched membrane or
an electrolytic mesh (see, e.g., U.S. Pat. No. 5,556,598). The
membrane may be polymer, glass, or metal provided the membrane is
low dead volume, allows movement of the various sample and
processing liquids through the column bed, may be attached to the
column body, is strong enough to withstand the bed packing process,
is strong enough to hold the column bed of beads, and does not
interfere with the extraction process i.e. does not adsorb or
denature the sample molecules.
[0107] The frit can be attached to the column body by any means
which results in a stable attachment. For example, the screen can
be bonded to the column body through welding or gluing. Gluing can
be done with any suitable glue, e.g., silicone, cyanoacrylate glue,
epoxy glue, and the like. The glue or weld joint must have the
strength required to withstand the process of packing the bed of
extraction media and to contain the extraction media with the
chamber. For glue joints, a glue should be selected employed that
does not adsorb or denature the sample molecules.
[0108] Alternatively, a frit can be attached by means of an annular
pip, as described in U.S. Pat. No. 5,833,927. This mode of
attachment is particularly suited to embodiment where the frit is a
membrane screen.
[0109] The frits of the invention, e.g., a membrane screen, can be
made from any material that has the required physical properties as
described herein. Examples of suitable materials include nylon,
polyester, polyamide, polycarbonate, cellulose, polyethylene,
nitrocellulose, cellulose acetate, polyvinylidine difluoride,
polytetrafluoroethylene (PTFE), polypropylene and glass. A specific
example of a membrane screen is the 43 .mu.m pore size
Spectra/Mesh.RTM. polyester mesh material which is available from
Spectrum Labs (Ranch Dominguez, Calif., PN 145837).
[0110] Extraction Column Assembly
[0111] The extraction columns of the invention can be constructed
by a variety of methods using the teaching supplied herein. In some
embodiments the extraction column can be constructed by the
engagement (i.e., attachment) of upper and lower tubular members
that combine to form the extraction column. Examples of this mode
of column construction are described in the Examples and depicted
in the figures.
[0112] For example, an embodiment of the invention wherein in the
two tubular members are sections of pipette tips is depicted in
FIG. 1 (FIG. 2 is an enlarged view of the open lower end and
extraction media chamber of the column). This embodiment is
constructed from a frustoconical upper tubular member 2 and a
frustoconical lower tubular member 3 engaged therewith. The
engaging end 4 of the upper tubular member has a tapered bore that
matches the tapered external surfaced of the engaging end 6 of the
lower tubular member, the engaging end of the lower tubular member
receiving the engaging end of the upper tubular member in a
telescoping relation. The tapered bore engages the tapered external
surface snugly so as to form a good seal in the assembled
column.
[0113] A membrane screen 10 is bonded to and extends across the tip
of the engaging end of the upper tubular member and constitutes the
upper frit of the extraction column. Another membrane screen 14 is
bonded to and extends across the tip of the lower tubular member
and constitutes the lower frit of the extraction column. The
extraction media chamber 16 is defined by the membrane screens 10
and 14 and the channel surface 18, and is packed with extraction
media 20.
[0114] The pore volume of the membrane screens 10 and 14 is low to
minimize the dead volume of the column. The sample and desorption
solution can pass directly from the vial or reservoir into the bed
of extraction media. The low dead volume permits desorption of the
analyte into the smallest possible desorption volume, thereby
maximizing analyte concentration.
[0115] The volume of the extraction media chamber 16 is variable
and can be adjusted by changing the depth to which the upper
tubular member engaging end extends into the lower tubular member,
as determined by the relative dimensions of the tapered bore and
tapered external surface.
[0116] The sealing of the upper tubular member to the lower tubular
in this embodiment is achieved by the friction of a press fit, but
could alternatively be achieved by welding, gluing or similar
sealing methods.
[0117] FIG. 3 depicts an embodiment of the invention comprising an
upper and lower tubular member engaged in a telescoping relation
that does not rely on a tapered fit. Instead, in this embodiment
the engaging ends 34 and 35 are cylindrical, with the outside
diameter of 34 matching the inside diameter of 35, so that he
concentric engaging end form a snug fit. The engaging ends are
sealed through a press fit, welding, gluing or similar sealing
methods. The volume of the extraction bed can be varied by changing
how far the length of the engaging end 34 extends into engaging end
35. Note that the diameter of the upper tubular member 30 is
variable, in this case it is wider at the upper open end 31 and
tapers down to the narrower engaging end 34. This design allows for
a larger volume in the column channel above the extraction media,
thereby facilitating the processing of larger sample volumes and
wash volumes. The size and shape of the upper open end can be
adapted to conform to a pump used in connection with the column.
For example, upper open end 31 can be tapered outward to form a
better friction fit with a pump such as a pipettor or syringe.
[0118] A membrane screen 40 is bonded to and extends across the tip
38 of engaging end 34 and constitutes the upper frit of the
extraction column. Another membrane screen 44 is bonded to and
extends across the tip 42 of the lower tubular member 36 and
constitutes the lower frit of the extraction column. The extraction
media chamber 46 is defined by the membrane screens 40 and 44 and
the open interior channel of lower tubular member 36, and is packed
with extraction media 48.
[0119] FIG. 4 is a syringe pump embodiment of the invention with a
cylindrical bed of extraction media in the tip, and FIG. 5 is an
enlargement of the top of the syringe pump embodiment of FIG. 4.
These figures show a low dead volume column based on using a
disposable syringe and column body. Instead of a pipettor, a
disposable syringe is used to pump and contain the sample.
[0120] The upper portion of this embodiment constitutes a syringe
pump with a barrel 50 into which a plunger 52 is positioned for
movement along the central axis of the barrel. A manual actuator
tab 54 is secured to the top of the plunger 52. A concentric
sealing ring 56 is secured to the lower end of the plunger 52. The
outer surface 58 of the concentric sealing ring 56 forms a sealing
engagement with the inner surface 60 of the barrel 50 so that
movement of the plunger 52 and sealing ring 56 up or down in the
barrel moves liquid up or down the barrel.
[0121] The lower end of the barrel 50 is connected to an inner
cylinder 62 having a projection 64 for engaging a Luer adapter. The
bottom edge 66 of the inner cylinder 62 has a membrane screen 68
secured thereto. The inner cylinder 62 slides in an outer sleeve 70
with a conventional Luer adaptor 72 at its upper end. The lower
segment 74 of the outer sleeve 70 has a diameter smaller than the
upper portion 76, outer sleeve 70 forming a ledge 78 positioned for
abutment with the lower end 66 and membrane screen 68. A membrane
screen 80 is secured to the lower end 82 of the lower segment 74.
The extraction media chamber 84 is defined by the upper and lower
membrane screens 68 and 80 and the inner channel surface of the
lower segment 74. The extraction beads 86 are positioned in the
extraction media chamber 84. The volume of extraction media chamber
84 can be adjusted by changing the length of the lower segment
74.
[0122] Other embodiments of the invention exemplifying different
methods of construction are also described in the examples.
[0123] Pump
[0124] In using the extraction columns of the invention a pump is
attached to the upper open end of the column and used to aspirated
and discharge the sample from the column. The pump can take any of
a variety of forms, so long as it is capable of generating a
negative internal column pressure to aspirate a fluid into the
column channel through the open lower end. In some embodiments of
the invention the pump is also able to generate a positive internal
column pressure to discharge fluid out of the open lower end.
Alternatively, other methods can be used for discharging solution
from the column, e.g., centrifugation.
[0125] The pump should be sufficiently strong so as to be able to
draw a desired sample solution, wash solution and/or desorption
solvent through the bed of extraction media.
[0126] In some embodiments of the invention the pump is capable of
controlling the volume of fluid aspirated and/or discharged from
the column, e.g., a pipettor. This allows for the metered intake
and outtake of solvents, which facilitates more precise elution
volumes to maximize sample recovery and concentration.
[0127] Non-limiting examples of suitable pumps include a pipettor,
syringe, peristaltic pump, electrokinetic pump, or an induction
based fluidics pump.
III. Methods for Using the Extraction Columns
[0128] Extraction columns of the invention should be stored under
conditions that preserve the integrity of the extraction media. For
example, columns containing agarose- or sepharose-based extraction
media should be stored under cold conditions (e.g., 4 degrees
Celsius) and in the presence of 0.01 percent sodium azide or 20
percent ethanol.
[0129] The sample solution can be any solution containing an
analyte of interest. The invention is particularly useful for
extraction and purification of biological molecules, hence the
sample solution is often of biological origin, e.g., a cell lysate.
In one embodiment of the invention the sample solution is a
hybridoma cell culture supernatant.
[0130] Prior to extraction, a conditioning step may be employed. If
analyte extraction is incomplete in a single pass, the sample
solution can be passed back and forth through the media several
times. An optional wash step between the extraction and desorption
steps can also improve the purity of the final product. Typically
water or a buffer is used for the wash solution. The wash solution
is preferably one that will remove unwanted contaminants with a
minimal desorption of the analyte of interest.
[0131] The volume of desorption solvent used can be very small,
approximating the interstitial volume of the bed of extraction
media. In certain embodiments of the invention the amount of
desorption solvent used is less than 10-fold greater than the
interstitial volume of the bed of extraction media, more preferably
less than 5-fold greater than the interstitial volume of the bed of
extraction media, still more preferably less than 3-fold greater
than the interstitial volume of the bed of extraction media, still
more preferably less than 2-fold greater than the interstitial
volume of the bed of extraction media, and most preferably is equal
to or less than the interstitial volume of the bed of extraction
media.
[0132] The desorption solvent will vary depending upon the nature
of the analyte and extraction media. For example, where the analyte
is a His-tagged protein and the extraction media an IMAC resin, the
desorption solution will contain imidazole or the like to release
the protein from the resin. In some cases desorption is achieved by
a change in pH or ionic strength, e.g., by using low pH or high
ionic strength desorption solution. A suitable desorption solution
can be arrived at using available knowledge by one of skill in the
art.
[0133] In one embodiment, the extraction column may be used for
multidimensional stepwise solid phase extraction of isotope-coded
affinity tagged (ICAT) peptides. The fractions are collected on the
basis of increasing ionic strength or pH, and can be processed in
the affinity separation dimension described below, but with
suitable adjustments being made for larger sample volumes being
introduced into the affinity capillary and/or possible differences
in pH. In certain instances the fractions collected from the avidin
affinity column may be processed further for cleavage of the
affinity tag from the isotope-coding region, prior to separation in
the reversed-phase separation dimension described below.
[0134] The cleavage can be performed directly upon the collected
fraction by photocleavage as described in Huilin Zhou, et al.,
Nature Biotech., 19:512 (2002), or acid cleavage with
TFA-triethylsilane as described in Brian Williamson, et al.,
Proceedings of the 50.sup.th ASMS Conference on Mass Spectrometry
and Allied Topics, Orlando, Fla., Jun. 2-6, 2002, Orlando, Fla.,
Poster # WPA023, or by evaporating the collected fraction to
dryness by standard means and adding TFA-triethylsilane reagent to
achieve acid cleavage as described in Williamson, et al, 50.sup.th
ASMS Conference Proceedings, Jun. 2-6, 2002, Orlando, Fla., Poster
# WPA023 (2002).
[0135] In instances where the peptide mixture generated by the
release, labeling and proteolysis is not excessively complex, it
may be possible to bypass the ion-exchange separation dimension and
proceed directly to the affinity separation dimension. An example
of bypassing the ion-exchange separation dimension is given in LC
Packings/Dionex' Application Note, "2D Analysis of Isotope Coded
Affinity Tag (ICAT) Labeled Proteins," Application Note UltiMate
Capillary and Nano LC System, Proteomics #09. However, if this
strategy is applied it is advised that some suitable means be
applied for removal of the unincorporated ICAT tags prior to
introducing the sample to the monomeric avidin column, which would
otherwise be removed in the ion-exchange separation dimension.
[0136] In certain instances it may be possible to bypass the
ion-exchange separation and affinity separation dimensions and
proceed directly from the sample protein release, lysis and
labeling step (i.e. the first step described at the beginning of
this example) to the reversed-phase separation dimension, such as
when solid-phase isotope-coded tagging reagents are being utilized
as described in Huilin Zhou, et al., Nature Biotech., 19:512
(2002); in this case the cleavage of the isotope-coded peptide from
the solid-phase support can be achieved by photocleavage as
described in Huilin Zhou, et al., Nature Biotech., 19:512 (2002) or
by acid cleavage as described in Brian Williamson, et al.,
Proceedings of the 50.sup.th ASMS Conference on Mass Spectrometry
and Allied Topics, Orlando, Fla., Jun. 2-6, 2002, Orlando, Fla.,
Poster # WPA023.
[0137] The device, apparatus and method of this invention can be
used to prepare materials for protein chips, DNA chips or other
biochips.
[0138] Protein chips dynamics can be represented by the following
equation:
A+B=AB
[0139] AB is capable of generating an analytical signal, where A is
the chip-bound moiety and B is its cognate binder introduced to the
chip. An assumption of specific interactions is always assumed.
Binding events other than "AB" can have the appearance of AB, the
variance being caused by non-A (i.e. contaminating) moieties having
some affinity for B, non-B (i.e. contaminating) moieties having
some affinity for A, or a combinations of the two; any of these
events will have the appearance of a true AB event. This
characteristic will define the success or failure of a particular
protein chip experiment, and is the most trivialized or ignored
aspects of the technology.
[0140] For some non-protein chips (specifically DNA chips), the A
groups do not require purification or enrichment since they are
synthesized in place, or are amplified via PCR and spotted. With
the exception of very short peptides, the structural complexity of
proteins will not allow for on-chip synthesis of A. Therefore,
preparation of A materials for use within protein chips will place
a premium on the purity of the material. In addition, the A
materials will often need to be highly enriched so as to provide
maximum opportunity for AB to occur.
[0141] Protein chips are characterized by having small volumes of
"A" applied to the surface. The volumes are often on the order of
10 mL or less for each spot. Since many proteins are difficult
and/or expensive to prepare, the ability to purify and enrich at
scales on par with the spots would significantly reduce waste. It
would also allow for "just-in-time" purification, so that the chip
is prepared just as the protein is being purified.
[0142] Different materials are brought to the chip as A, and each
material require purification and/or enrichment. Examples of these
materials are antibodies (i.e. IgG, IgY, etc) as affinity
molecules, general affinity proteins (i.e. scFvs, Fabs, affibodies,
peptides, etc) as affinity molecules, other proteins that are being
screened for general affinity characteristics, and nucleic
acids/(photo) aptamers as affinity molecules, for example.
[0143] Different means of attaching A to chip surfaces, and each
will require purification and enrichment procedures that are
compatible with the attachment chemistry. Examples of attachment
chemistry include direct/passive immobilization to protein chip
substrates, and these can become covalent in cases of native thiols
associating with gold surfaces, as one example. Covalent attachment
is another method of attachment of functional groups at chip
surface, and these can be self-assembled monolayers with and
without additional groups, immobilized hydrogel, and the like.
Non-covalent/affinity attachment to functional groups/ligands at
chip surface is another method of attachment; examples of this
method are ProA or ProG for IgGs, phenyl(di)boronic acid with
salicylhydroxamic acid groups; streptavidin monolayers with
biotinylation of native lysines/cysteines, and the like.
[0144] The samples or analyte to be brought to the chip can be
varied in composition and mode of interaction with A.
[0145] There is more than one way to achieve specific AB
interactions through the manipulation of B. One means is to remove
potentially interfering non-B contaminants by their specific
removal, provided these contaminants are sufficiently well-defined
such as albumin, fibrin, etc.
[0146] Another means is the removal of non-B contaminants by
trapping B (either individually or as a class), removing
contaminants by washing, and releasing B. This simultaneously
allows for enrichment of B, thus enhancing the sensitivity for the
AB event.
[0147] Just as the scale of the chip is very small, there are
opportunities to make the scale of the sample small--therefore
allowing for analysis of very small samples. Since samples are
precious materials, the scale of purification and enrichment would
allow for this to occur. As with chip preparation, this can occur
in a "just-in-time" manner.
[0148] The detection event requires some manner of A interacting
with B, so the central player in the detection event (since it
isn't part of the protein chip itself) is B. The means of detecting
the presence of B (or, B-like substances described above) are
varied and can include label-free detection of B (or B-like
substances) interacting with A such as surface plasmon resonance
imaging as practiced by HTS Biosystems--grating-coupled SPR or
BiaCore--prism or Kretschmann-based SPR, or Micro-cantilever
detection schemes as practiced by Protiveris.
[0149] The detection means can include physical labeling of B (or
B-like substances) interacting with A, followed by spatial imaging
of AB pair (i.e. Cy3/Cy5 differential labeling with standard
fluorescent imaging as practiced by BD Biosciences Clontech,
radioactive ATP labeling of kinase substrates with autoradiography
imaging as practiced by Jerini or other suitable imaging
techniques. In the case of fluorescent tagging, one can achieve
higher sensitivity with fluorescent waveguide imaging as practiced
by ZeptoSens.
[0150] The detection means can also include interaction of AB
complex with a third B-specific affinity partner C, where C is
capable of generating a signal by being fluorescently tagged, or is
tagged with a group that allows a chemical reaction to occur at
that location (such as generation of a fluorescent moiety, direct
generation of light, etc). Detection of this AB-C binding event can
occur via fluorescent imaging as practiced by Zyomyx and SomaLogic,
chemilumine-scence imaging as practiced by HTS Biosystems and
Hypromatrix, fluorescent imaging via waveguide technology, or other
suitable detection means.
[0151] Arrayers are instruments for spotting nucleic acids,
proteins or other reagent onto chips that are used for molecular
biology research or diagnostic work. The arrayers can be used both
in the manufacture of the chips and in the use of the chip. In
manufacturing, an arrayer can be used to transport the chemical
reactants to specific spots on the chip. This may be a multi-step
process as the chemical complex used for detection is built at each
particular spot in the array.
[0152] Each process can require sample preparation. In some cases,
DNA is purified and deposited to a surface on a chip. Then samples
containing complementary DNA or RNA are reacted with the chip.
Before the samples can be reacted, the nucleic acid is purified
away from the other materials (proteins, particulate,
carbohydrates, etc.) found in the samples. In other cases, protein
chips may be manufactured by depositing specific proteins in an
array. Then samples containing proteins can be reacted with various
array sites to measure protein/protein interactions.
[0153] In application of mass spectrometry for the analysis of
biomolecules, the molecules must be transferred from the liquid or
solid phases to gas phase and to vacuum phase. Since most
biomolecules are both large and fragile, the most effective methods
for their transfer to the vacuum phase are matrix-assisted laser
desorption ionization (MALDI) or electrospray ionization (ESI).
[0154] Mass spectrometry provides essentially two methods for
analyzing proteins: bottom up and top down analysis. In bottom up
analysis, the protein is manipulated and broken up in a controlled
manner (usually through an enzymatic digestion process), analyzed,
and then reassembled using the data from the various parts. Top
down analysis works with the whole protein, optionally using an ion
source to break apart the protein and determine the identity of the
protein.
[0155] While both methods may require long mass spectrometer
analysis times, top down approaches usually require the longest
time. Under ideal cases, a static sample is measured and parameters
on the manner in which the source is directed or implemented. The
methods in which the data are analyzed are varied to perform a full
analysis of the protein.
[0156] Many sample introduction methods introduce samples
"on-the-fly." The sample is introduced from an HPLC column as
continuous flow into the nozzle of the electrospray ionization
(ESI) source. In order to introduce samples so that top down
analysis can be implemented, the flow of the sample may be slowed.
The method is called peak parking. In this way, the sample
residence time can be increased by a factor of 10 or greater
increasing the sensitivity of the analysis by a factor of 8 or
greater. However, this method is still inflexible and inadequate
because the analysis must still be performed quickly--often more
quickly than the instrument is capable of performing.
[0157] This is also true for introduction of samples from a solid
phase extraction device. One may introduce the entire sample before
the analysis is completed. It is much better to introduce a
discrete uniform sample into the mass spectrometer. In this way,
the mass spectrometry method and procedure can be adapted to the
sample in the best manner.
[0158] This can be accomplished by using an apparatus where the
desorbed material from an open tube extraction device is deposited
directly into an electrospray nozzle.
[0159] MALDI is commonly interfaced to time of flight (TOF) mass
spectrometers (MALDI-TOF) and ESI is interfaced to quadrupole, ion
trap and TOF mass analyzers. Both MALDI and ESI approaches are
useful for determining the full masses of proteins and peptides in
mixtures, before and after purification and to induce fragmentation
of peptides for ms/ms analysis. Modern mass spectrometry is
accurate enough to be useful for evaluating the correct translation
or chemical synthesis of biomolecules. Any deviation of the
observed mass of the sample from its calculated mass indicates
incorrect synthesis or the presence of post-translational or
chemical modifications. Biomolecules can be purposely fragmented in
the mass spectrometer and the masses of the resulting fragments can
be accurately determined. The patterns of such fragment masses are
useful for ms/ms sequencing of the peptides and their
identification in the data banks.
[0160] Electrospray is performed by mixing the sample with volatile
acid and organic solvent and infusing it through a conductive
needle charged with high voltage. The charged droplets that are
sprayed (or ejected) from the needle end, are directed into the
mass spectrometer, and are dried up by heat and vacuum as they fly
in. After the drops dry, the remaining charged molecules are
directed by electromagnetic lenses into the mass detector and mass
analyzed. Electrospray mass spectrometry can be used to determine
the masses of different molecules, from small peptides to intact
large proteins. Even though the mass-range of the currently
available instruments is only 2000 to 10000 mass unit, most
proteins become multi-charged during the electrospray step and
since the instrument measures the mass to charge ratio (m/z) of the
molecules, most proteins are sufficiently charged to have an m/z
that is within the mass range. To calculate the full mass of the
protein from the different m/z measured, a deconvolution is
performed, returning the full mass of the proteins.
[0161] For MALDI-TOF the proteins are deposited on metal targets,
as co-crystallized with an organic matrix. The samples are dried
and inserted into the mass spectrometer. After vacuum is
established, the matrix crystals absorb the light energy from short
flashes of a high-energy laser. The matrix rapidly sublimes,
carrying with it the biomolecule into the vacuum phase. The sample
and matrix plume enter a strong electromagnetic field that
accelerate the charged molecules into a free flight zone where they
fly until they hit a detector located at its far end. The mass of
the protein can be calculated from its flight time. Accurate
determination of the masses is obtained by the flight time to that
of a standard of known mass. The flight time is proportional to the
log of mass of the protein and the larger proteins fly slower and
reach the detector later.
[0162] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0163] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples, which are provided by way of illustration, and are not
intended to be limiting of the present invention, unless so
specified.
EXAMPLES
[0164] The following preparations and examples are given to enable
those skilled in the art to more clearly understand and practice
the present invention. They should not be construed as limiting the
scope of the invention, but merely as being illustrative and
representative thereof.
Example 1
Preparation of an Extraction Column Body from Pipette Tips
[0165] Two 1000 .mu.L polypropylene pipette tips of the design
shown in FIG. 6 (VWR, Brisbane, Calif., PN 53508-987) were used to
construct one extraction column. In this example, two extraction
columns were constructed: a 10 .mu.L bed volume and 20 .mu.L bed
volume. To construct a column, various components were made by
inserting the tips into several custom aluminum cutting tools and
cutting the excess material extending out of the tool with a razor
blade to give specified column lengths and diameters.
[0166] Referring to FIG. 7, the first cut 92 was made to the tip 92
of a pipette tube 90 to form a 1.25 mm inside diameter hole 94 on
the lower column body, and a second cut 96 was made to form a lower
column body segment 98 having a length of 15.0 mm.
[0167] Referring to FIG. 8, a cut 102 was made to the separate
pipette tip 100 to form the upper column body 104. To make a 10
.mu.L bed volume column, the cut 102 was made to provide a tip 106
outside diameter of 2.09 mm so that when the upper body was
inserted into the lower body, the column height of the solid phase
media bed 114 (FIG. 10) was 4.5 mm. To make a 20 .mu.L bed volume
column, the cut 102 was made to provide a tip outside diameter of
2.55 mm cut so that when the upper body was inserted into the lower
body, the column height of the solid phase media bed 114 (FIG. 10)
was 7.0 mm.
[0168] Referring to FIG. 9, a 43 .mu.m pore size Spectra/Mesh.RTM.
polyester mesh material (Spectrum Labs, Ranch Dominguez, Calif., PN
145837) was cut into discs by a circular cutting tool (Pace
Punches, Inc., Irvine, Calif.) and attached to the ends 106 and 108
upper column and lower column bodies to form the membrane screens
110 and 112. The membrane screens were attached using PLASTIX.RTM.
cyanoacrylate glue (Loctite, Inc., Avon, Ohio). The glue was
applied to the polypropylene body and then pressed onto the
membrane screen material. Using a razor blade, excess mesh material
was removed around the outside perimeter of each column body
end.
[0169] Referring to FIG. 10, the upper column body 104 is inserted
into the top of the lower column body segment 98 and pressed
downward to compact the solid phase media bed 114 to eliminate
excess dead volume above the top of the bed.
Example 2
Preparation of SEPHAROSE.TM. Protein G and MEP HYPERCEL.TM.
Extraction Columns
[0170] Referring to FIG. 9, a suspension of Protein G SEPHAROSE.TM.
4 Fast Flow, 45-165 .mu.m particle size, (Amersham Biosciences,
Piscataway, N.J., PN 17-0618-01) in water/ethanol was prepared, and
an appropriate amount of material 114 was pipetted into the lower
column body 98.
[0171] Referring to FIG. 10, the upper column body 104 was pushed
into the lower column body 98 so that no dead space was left at the
top of the bed 114, that is, at the top of the column bed. Care was
taken so that a seal was formed between the upper and lower column
bodies 104 and 98 while retaining the integrity of the membrane
screen bonding to the column bodies.
[0172] Several tips of 10 .mu.L and 20 .mu.L bed volumes were
prepared. Several MEP (Mercapto-Ethyl-Pyridine) HYPERCEL.TM.
(Ciphergen, Fremont, Calif., PN 12035-010) extraction columns were
prepared using the same procedure. MEP HyperCel.TM. resin is a
sorbent, 80-100 .mu.m particle size, designed for the capture and
purification of monoclonal and polyclonal antibodies. The
extraction columns were stored with an aqueous solution of 0.01%
sodium azide in a refrigerator before use.
Example 3
Purification of Anti-Leptin Monoclonal Antibody IgG with 10 .mu.L
and 20 .mu.L Bed Volume Protein G SEPHAROSE.TM. Extraction
Columns
[0173] A Protein G SEPHAROSE.TM. 4 Fast Flow (Amersham Biosciences,
Piscataway, N.J., PN 17-0618-01) extraction column was prepared as
described in Example 2.
[0174] Five hundred .mu.L serum-free media (HTS Biosystems,
Hopkinton, Mass.) containing IgG (HTS Biosystems, Hopkinton, Mass.)
of interest was combined with 500 .mu.L standard PBS buffer. The
resulting 1 mL sample was pulled into the pipette tip, through the
Protein G packed bed at a flow rate of approximately 1 mL/min) or
roughly 15 cm/min). The sample was then pushed out to waste at the
same approximate flow rate. Extraneous buffer was removed from the
bed by pulling 1 mL of deionized water into the pipette column at
about 1 mL/min and pushing it out at about 1 mL/min. The water was
pushed out as much as possible to achieve as dry of a column bed as
is possible. The IgG was eluted from the column bed by drawing up
an appropriate eluent volume of 100 mM glycine-HCl, pH 2.5 (20
.mu.L eluent in the case of a 20 .mu.L bed volume, 15 .mu.L eluent
in the case of a 10 .mu.L bed volume). When the eluent was fully
drawn into the bed, it was "pumped" back and forth through the bed
five or six times, and the IgG-containing eluent was then fully
expelled from the bed. The eluted material was then neutralized
with 100 mM NaH.sub.2PO.sub.4/100 mM Na.sub.2HPO.sub.4 (5 .mu.L
neutralization buffer in the case of a 20 .mu.L bed volume, 4 .mu.L
neutralization buffer in the case of a 10 .mu.L bed volume). The
purified and enriched antibodies were then ready for arraying.
Example 4
Purification of Anti-Leptin Monoclonal Antibody IgG with 10 .mu.L
and 20 .mu.L Bed Volume Protein G SEPHAROSE.TM. Extraction
Columns
[0175] A Protein G SEPHAROSE.TM. 4 Fast Flow (Amersham Biosciences,
Piscataway, N.J., PN 17-0618-01) extraction column was prepared as
described in Example 2.
[0176] Five hundred .mu.L serum-free media (HTS Biosystems,
Hopkinton, Mass.) containing IgG (HTS Biosystems, Hopkinton, Mass.)
of interest was combined with 500 .mu.L standard PBS buffer. The
resulting 1 mL sample was pulled into the pipette tip, through the
Protein G packed bed at a flow rate of approximately 1 mL/min (or
roughly 150 cm/min linear velocity). The sample was then pushed out
to waste at the same approximate flow rate. Extraneous buffer was
removed form the bed by pulling 1 mL of deionized water into the
pipette column at about 1 mL/min and pushing it out at about 1
mL/min. The water was pushed out as much as possible to achieve as
dry of a column bed as is possible. The IgG was eluted from the
column bed by drawing up an appropriate eluent volume of 10 mM
phosphoric acid (H.sub.3PO.sub.4), pH 2.5 (20 .mu.L eluent in the
case of a 20 .mu.L bed volume, 15 .mu.L eluent in the case of a 10
.mu.L bed volume). When the eluent was fully drawn into the bed, it
was "pumped" back and forth through the bed five or six times, and
the IgG-containing eluent is then fully expelled from the bed. The
eluted material was then neutralized with a specially designed
phosphate neutralizing buffer of 100 mM H.sub.2NaPO.sub.4/100 mM
HNa.sub.2PO.sub.4, pH 7.5 (5 .mu.L neutralization buffer in the
case of a 20 .mu.L bed volume, 4 .mu.L neutralization buffer in the
case of a 10 .mu.L bed volume). The purified and enriched
antibodies were then ready for arraying.
Example 5
Analysis of Purified IgG with Grating-Coupled Surface Plasmon
Resonance (GC-SPR)
[0177] The anti-leptin monoclonal antibody IgG purified sample from
Example 4 was analyzed with GC-SPR. The system used for analysis
was a FLEX CHIP.TM. Kinetic Analysis System (HTS Biosystems,
Hopkinton, Mass.), which consists of a plastic optical grating
coated with a thin layer of gold on to which an array of
biomolecules is immobilized. To immobilize the purified IgG, the
gold-coated grating was cleaned thoroughly with EtOH (10-20 seconds
under a stream of EtOH). The gold-coated grating was then immersed
in a 1 mM solution of 11-mercaptoundecanoic acid (MUA) in EtOH for
1 hour to allow for the formation of a self-assembled monolayer.
The surface was rinsed thoroughly with EtOH and ultra-pure water,
and dried under a stream of nitrogen. A fresh solution of 75 mM EDC
(1-Ethyl-3-(3-Dimethylaminopropyl) carbodiimide hydrochloride) and
15 mM Sulfo-NHS(N-Hydroxysulfo-succinimide) was prepared in water.
An aliquot of the EDC/NHS solution was delivered to the surface and
allowed to react for 20-30 minutes, and the surface was then rinsed
thoroughly with ultra-pure water. An aliquot of 1 mg/mL Protein A/G
in PBS, pH 7.4 was delivered to the surface. The surface was placed
in a humid environment and allowed to react for 1-2 hours. The
surface was allowed to air dry, was rinsed with ultra-pure water
and then dried under a stream of nitrogen. Immediately prior to
arraying of the IgGs, the surface was rehydrated by placing in a
humidified chamber, such as available with commercial arraying
systems (e.g. Cartesian MicroSys synQUAD System). The purified
anti-leptin IgG was arrayed onto the surface as described
previously (J. Brockman, et al, "Grating-Coupled SPR: A Platform
for Rapid, Label-free, Array-Based Affinity Screening of Fabs and
Mabs", 12.sup.th Annual Antibody Engineering Conference, Dec. 2-6,
2001, San Diego, Calif.) and the surface was introduced to the HTS
Biosystems FLEX CHIP System. 150 nM leptin in PBS, pH 7.4 was
introduced to the surface through the FLEX CHIP System, and
real-time binding signals were collected as described previously
(ibid.). These real-time binding signals were mathematically
processed in a manner described previously (D. Myszka, "Kinetic
analysis of macromolecular interactions using surface plasmon
resonance biosensors", Current Opinion in Biotechnology, 1997, Vol
8, pp. 50-57) for extraction of the association rate (k.sub.a),
dissociation rate (k.sub.d), and the dissociation affinity constant
(K.sub.d=k.sub.d/k.sub.a). The kinetic data obtained is shown in
Table II below.
TABLE-US-00002 TABLE II Serum-free medium PBS No processing Mean
K.sub.d 18 nM 3.2 nM (Adequate [IgG]) Starting 500 .mu.g/mL 500
.mu.g/mL [IgG] With processing Mean K.sub.d 6.6 nM 5.9 nM*
(Insufficient [IgG] Starting 20 .mu.g/mL 500 .mu.g/mL* [IgG] *500
.mu.g/mL IgG in PBS was not processed, but was included in the SPR
analysis for the purpose of comparing dissociation affinity
constants calculated for each
[0178] The first set of data for "No processing" indicates that
when sufficient IgG is present for detection (500 .mu.g/mL) that
the constituents from the serum-free medium can contribute to
inaccuracies. These data indicate for equal concentrations of IgG
spotted within an experiment, the calculated dissociation affinity
constant can be nearly six-fold different from one another (18 nM
vs. 3.2 nM). This can only be a result of components within the
serum-free medium being co-arrayed with the IgG, since the
concentration and composition of IgG is identical for each sample.
Therefore, there is a demonstrated need for removal of any
extraneous components prior to arraying, which is independent of
IgG concentration.
[0179] The second set of data for "With processing" indicates that
when insufficient IgG quantities are present for detection (20
.mu.g/mL) that sample processing not only allows for generation of
sufficient processable signals, but also eliminates the
inaccuracies generated from extraneous components. These data
indicate that the dissociation affinity constants are virtually
identical for 500 .mu.g/mL purified IgG in PBS (unprocessed) as
those calculated from 20 .mu.g/mL IgG in serum-free medium once
processed with the current invention (5.9 nM vs. 6.6 nM).
Example 6
Purification of Nucleic Acids with an Extraction Column
[0180] Columns from Example 1 are bonded with a 21 .mu.m pore size
SPECTRA/MESH.RTM. polyester mesh material (Spectrum Labs, Ranch
Dominguez, Calif., PN 148244) by the same procedure as described in
Example 2. A 10 .mu.L bed volume column is filled with PELLICULAR
C18 (Alltech, Deerfield, Ill., PN 28551), particle size 30-50
.mu.m. One end of the extraction column is connected to a pipettor
pump (Gilson, Middleton, Wis., P-1000 PipetteMan) and the other end
is movable and is connected to an apparatus where the materials may
be taken up or deposited at different locations.
[0181] The extraction column consists of a 1 mL syringe (VWR,
Brisbane, Calif., PN 53548-000), with one end connected to a
pipettor pump (Gilson, Middleton, Wis., P-1000 PipetteMan) and the
other end is movable and is connected to an apparatus where the
materials may be taken up or deposited at different locations.
[0182] A 100 .mu.L sample containing 0.01 .mu.g of DNA is prepared
using PCR amplification of a 110 bp sequence spanning the allelic
MstII site in the human hemoglobin gene according to the procedure
described in U.S. Pat. No. 4,683,195. A 10 .mu.L concentrate of
triethylammonium acetate (TEAA) is added so that the final volume
of the solution is 110 .mu.L and the concentration of the TEAA in
the sample is 100 mM. The sample is introduced into the column and
the DNA/TEAA ion pair complex is adsorbed.
[0183] The sample is blown out of the column and 10 .mu.L of 50%
(v/v) acetonitrile/water is passed through the column, desorbing
the DNA, and the sample is deposited into a vial for analysis.
Example 7
Desalting Proteins with an Extraction Column
[0184] Columns from Example 1 are bonded with a 21 .mu.m pore size
SPECTRA/MESH.RTM. polyester mesh material (Spectrum Labs, Ranch
Dominguez, Calif., PN 148244) by the same procedure as described in
Example 2. A 10 .mu.L bed volume column is filled with PELLICULAR
C18 (Alltech, Deerfield, Ill., PN 28551), particle size 30-50
.mu.m. One end of the extraction column is connected to a pipettor
pump (Gilson, Middleton, Wis., P-1000 PipetteMan) and the other end
is movable and is connected to an apparatus where the materials may
be taken up or deposited at different locations.
[0185] The sample is a 100 .mu.L solution containing 0.1 .mu.g of
Protein kinase A in a phosphate buffer saline (0.9% w/v NaCl, 10 mM
sodium phosphate, pH 7.2) (PBS) buffer. Ten .mu.L of 10% aqueous
solution of trifluoroacetic acid (TFA) is added so that the final
volume of the solution is 110 .mu.L and the concentration of the
TFA in the sample is 0.1%. The sample is introduced into the column
and the protein/TFA complex is adsorbed to the reverse phase of the
bed.
[0186] The sample is blown out of the column and 10 .mu.L of 50%
(v/v) acetonitrile/water is passed through the column, desorbing
the protein from the bed of extraction media, and the sample is
deposited into a vial for analysis.
[0187] Alternatively, the bed may be washed with 10 .mu.L of
aqueous 0.1% TFA. This solution is ejected from the column and the
protein is desorbed and deposited into the vile.
[0188] If necessary, alternatively 1% heptafluorobutyric acid
(HFBA) is used instead of TFA to reduce ion suppression effect when
the sample is analyzed by electrospray ion trap mass
spectrometry.
Example 8
Straight Connection Configuration
[0189] This example describes an embodiment wherein the column body
is constructed by engaging upper tubular members and membrane
screens in a straight configuration.
[0190] Referring to FIG. 11, the column consists of an upper
tubular member 120, a lower tubular member 122, a top membrane
screen 124, a bottom membrane screen 126, and a lower tubular
circle 134 to hold the bottom membrane screen in place. The top
membrane screen is held in place by the upper and lower tubular
members. The top membrane screen, bottom membrane screen and the
channel surface 130 of the lower tubular member define an
extraction media chamber 128, which contains a bed of extraction
media 132 (i.e., packing material). The tubular members as depicted
in FIG. 11 are frustoconical in shape, but in related embodiments
could take other shapes, e.g, cyclindrical.
[0191] To construct a column, various components are made by
forming injected molded members from polypropylene or machined
members from PEEK polymer to give specified column lengths and
diameters and ends that can fit together, i.e., engage with one
another. The configuration of the male and female portions of the
column body is shaped differently depending on the method used to
assemble the parts and the method used to keep the parts
together.
[0192] The components are glued or welded. Alternatively, they are
snapped together. In the case of snapping the pieces together, the
female portion contains a lip and the male portion contains a ridge
that will hold and seal the pieces once they are assembled. The
membrane screen is either cut automatically during the assembly
process or is trimmed after assembly.
Example 9
End Cap and Retainer Ring Configuration
[0193] This example describes an embodiment where an end cap and
retainer ring configuration is used to retain the membrane screens
containing a 20 .mu.l bed of column packing material. The
embodiment is depicted in FIG. 12.
[0194] Referring to the figure, pipette tip 140 (VWR, Brisbane,
Calif., PN 53508-987) was cut with a razor blade to have a flat and
straight bottom end 142 with the smooth sides such that a press fit
can be performed later. An end cap 144 was machined from PEEK
polymer tubing to contain the bottom membrane screen 146.
[0195] Two different diameter screens were cut from polyester mesh
(Spectrum Labs, Ranch Dominguez, Calif., PN 145836) by a circular
cutting tool (Pace Punches, Inc., Irving, Calif.), one for the top
membrane screen 148 and the other for the bottom membrane screen
146. The bottom membrane screen was placed into the end cap and
pressed onto the end of the cut pipette tip.
[0196] A 20 .mu.L volume bed of beads 150 was formed by pipetting a
40 .mu.L of 50% slurry of protein G agarose resin into the column
body.
[0197] Two retainer rings were used to hold the membrane screen in
place on top of the bed of beads. The retainer rings were prepared
by taking 1/8 inch diameter polypropylene tubing and cutting thin
circles from the tubing with a razor blade. A first retainer ring
152 was placed into the column and pushed down to the top of the
bed with a metal rod of similar diameter. The membrane screen 148
was placed on top of the first retainer ring and then a second
retainer ring 154 was pushed down to "sandwich" the membrane screen
while at the same time pushing the whole screen configuration to
the top of the bed and ensuring that all dead volume was removed.
The membrane is flexible and naturally forms itself to the top of
the bed.
[0198] The column was connected to a 1000 .mu.L pipettor (Gilson,
Middleton, Wis., P-1000 PipetteMan) and water was pumped through
the bed and dispensed from the bed. The column had low resistance
to flow for water solvent.
[0199] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover and variations, uses, or adaptations of the invention that
follow, in general, the principles of the invention, including such
departures from the present disclosure as come within known or
customary practice within the art to which the invention pertains
and as may be applied to the essential features hereinbefore set
forth. Moreover, the fact that certain aspects of the invention are
pointed out as preferred embodiments is not intended to in any way
limit the invention to such preferred embodiments.
* * * * *