Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process...
Reexamination Certificate
1998-10-13
2003-05-27
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
C435S029000, C435S267000
Reexamination Certificate
active
06569640
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods for extracting fractions from material of biological origin. The fractions potentially contain one or more compounds which exhibit biological activity. The methods feature supercritical and near critical fluids.
BACKGROUND OF THE INVENTION
At the present time, a number of major pharmaceutical companies are actively carrying out programs for the discovery of drugs from natural products. Typically, such programs involve screening of a large number of natural materials for therapeutic or other biological activity. These biomass materials may be obtained or derived from plant, animal, or microbial sources. Screening is carried out by assaying samples for indications of, for example, cytotoxicity, antibacterial activity, or antiviral activity.
In preparation for screening, the biomass is typically exposed to an extraction step. In many cases, however, the bioactive materials of interest may be sequestered within the substrate and not accessible to extraction. Thus, depending upon the particular biomass, the extraction may be facilitated by a preliminary size reduction (comminution) or disruption step. Comminution/disruption methods include for example grinding, sonication, and homogenization. Conventional comminution/disruption methods may have the disadvantages of incomplete disruption and/or product deterioration. They may also be time-consuming and expensive.
For the extraction, the biomass is contacted with a solvent such as butanol or ethyl acetate, so that compounds of potential interest may migrate from the biomass substrate to the solvent phase. Sometimes multiple extraction steps may be carried out on a single batch of biomass. A “fraction” refers to the material recovered from the biomass in a single one of these extraction steps. Fractions from the extraction steps are further processed so that they may be assayed for the activity of interest. Because different extraction methods will produce different extract profiles, there is a continuing interest in the development of new extraction techniques.
Conventional solvents are not always ideal for biomass extractions. These solvents can be difficult to remove from the compounds potentially exhibiting bioactivity, and also may extract a mixture of compounds which can mask bioactivity in an assay. The solvents may not penetrate membranes or other cellular structures surviving disruption. The solvation properties of conventional solvents cannot be readily modified by changing temperature, pressure, or the concentration of modifying cosolvents, and thus may be cumbersome to use when it is desired to carry out certain types of fractionations such as the selective extraction of compounds of varying polarity.
It would be highly desirable to have a method for fractional extraction of biomass constituents, including those which are sequestered within cells. It would be highly desirable to have a solvent system which can be readily modified by physical parameters and the addition of modifying cosolvents to selectively extract compounds of varying polarity, volatility, or hydrophilicity. It would be highly desirable to have a physical-chemical disruption process that provides for a high level of disruption without loss of active material. It would be desirable to disrupt biomass and form one or more fractions using a single apparatus.
SUMMARY OF THE INVENTION
Aspects of the present invention employ materials known as supercritical fluids. A material becomes a supercritical fluid at conditions which equal or exceed both its critical temperature and critical pressure. These parameters are intrinsic thermodynamic properties of all sufficiently stable pure compounds and mixtures. Carbon dioxide, for example, becomes a supercritical fluid at conditions which equal or exceed its critical temperature of 31.1° C. and its critical pressure of 72.8 atm (1,070 psig). In the supercritical fluid region, normally gaseous substances such as carbon dioxide become dense phase fluids which have been observed to exhibit greatly enhanced solvating power. At a pressure of 3,000 psig (204 atm) and a temperature of 40° C., carbon dioxide has a density of approximately 0.8 g/cc and behaves much like a nonpolar organic solvent, having a dipole moment of zero debyes. A supercritical fluid uniquely displays a wide spectrum of solvation power as its density is strongly dependent upon temperature and pressure. Temperature changes of tens of degrees or pressure changes by tens of atmospheres can change a compound's solubility in a supercritical fluid by an order of magnitude or more. This unique feature allows for the fine-tuning of solvation power and the fractionation of mixed solutes. The selectivity of nonpolar supercritical fluid solvents can also be enhanced by addition of compounds known as modifiers (also referred to as entrainers or cosolvents). These modifiers are typically somewhat polar organic solvents such as acetone, ethanol, methanol, methylene chloride or ethyl acetate. Varying the proportion of modifier allows a wide latitude in the variation of solvent power.
In addition to their unique solubilization characteristics, supercritical fluids possess other physicochemical properties which add to their attractiveness as solvents. They can exhibit liquid-like density yet still retain gas-like properties of high diffusivity and low viscosity. The latter increases mass transfer rates, significantly reducing processing times. Additionally, the ultra-low surface tension of supercritical fluids allows facile penetration into microporous materials, increasing extraction efficiency and overall yields.
While similar in many ways to conventional nonpolar solvents such as hexane, it is well-known that critical fluid solvents can extract a different spectrum of materials than conventional techniques. Product volatilization and oxidation as well as processing time and organic solvent usage can be significantly reduced with the use of supercritical fluid solvents.
A material at conditions that border its supercritical state will have properties that are similar to those of the substance in the supercritical state. These so-called “near critical” fluids are also useful for the practice of this invention. For the purposes of this invention, a near critical fluid is defined as a fluid which is (a) at a temperature between its critical temperature (T
c
) and 75% of its critical temperature and at a pressure at least 75% of its critical pressure, or (b) at a pressure between its critical pressure (P
c
) and 75% of its critical pressure and at a temperature at least 75% of its critical temperature. In this definition, pressure and temperature are defined on absolute scales, e.g., Kelvins and psia. Table 1 shows how these requirements relate to some of the fluids relevant to this invention. To simplify the terminology, materials which are utilized under conditions which are supercritical, near critical, or exactly at their critical point will jointly be referred to as “critical” fluids.
TABLE 1
Physical properties of critical fluid solvents.
P
vap
,
BP,
psia @
T
c
,
P
c
,
0.75T
c
,
0.75P
c
,
Fluid
Formula
° C.
25° C.
° C.
psia
° C.
psia
Carbon
CO
2
−78.5
860
31.1
1070
−45.0
803
dioxide
Nitrous
N
2
O
−88.5
700
36.5
1051
−41.0
788
oxide
Propane
C
3
H
8
−42.1
130
96.7
616
4.2
462
Ethane
C
2
H
6
−88.7
570
32.3
709
−44.1
531
Ethylene
C
2
H
4
−103.8
NA
9.3
731
−61.4
548
Freon 11
CCl
3
F
23.8
15
198.1
639
80.3
480
Freon 21
CHCl
2
F
8.9
24
178.5
750
65.6
562
Freon 22
CHClF
2
−40.8
140
96.1
722
3.8
541
Freon 23
CHF
3
−82.2
630
26.1
700
−48.7
525
Table 1 Notes:
BP = Normal boiling point; P
vap
= Vapor pressure
The present invention utilizes critical fluids to fractionate biomass materials in two steps. In the first step, the biomass is disrupted by exposure to the critical fluid. It is hypothesized that this disruption involves at least two mechanisms, the first being liberation of structural constituents to cause permeabil
Castor Trevor Percival
Hong Glenn Thomas
Aphios Corporation
Gaudet Stephen J.
Naff David M.
Perkins Smith & Cohen LLP
Ware Deborah K.
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