Note: Descriptions are shown in the official language in which they were submitted.
~ WO92/1~36 PCT/US92/0~7
~ 2102~3~
METHOD AND APPARATUS FOR IMMOBILIZED ENZYME REACTIONS
Field of the Invention
This invention relates generally to methods
and apparatus for conducting enzymatic reac_ions. In
particular, the invention relates to methods ~nd
apparatus for conducting highly productive enzymatic
reactions utilizing enzymes immobilized on a perfusive
matrix.
Backqround of the Invention
The art has placed increasing emphasis on the
use of immobilized enzymes and on developments in
immobilized enzyme technology. Enzyme immobilization
provides a reaction system with high specificity and
sensitivity, and generally increases enzyme stability,
thereby converting enzymes into versatile tools useful
in a wide variety of applications. Immobilized enzyme
reactors ("IMER"s) are used in many industrial
processes, including synthesis and analysis in the
chemical, pharmaceutical, food, and bioremediation
industries, as well as for clinical analysis and
therapeutic purposes in medicine.
Currently there are a number of limitations to the
enzyme reactors in the art. Chief among these are the
current methods of enzyme immobilization, which affect
IMER capacity, cost and speed.
WO92/1~ ~ - 2 PCT/US92/02~87
21~2237
Of the various ways known in the art for
immobilizing enzymes, one of the most common and
potentially versatile methodologies useful in IMER
t~chnology is the use of matrix-bound enzymes. The
nature of the support matrix for an immobilized enzyme
system is important to its function. In continuous
flow reactors, ~or example, materials with poor
dimensional stability should be avoided. In addition,
a large surface area-to-volu~e ratio generally is
desired. The ideal support matrix is one which
promotes substrate binding, decreases product
inhibition, shifts the apparent pH optimum to the
desired value, increases enzyme stability, and
discourages microbial growth. Cost of production and
ease of preparation also should be considered.
Typically, matrix supports useful in liquid
chromatography are used. Particularly useful matrices
comprise porous particles which provide the desired
high surface area-to-volume ratio. Traditional
materials for liquid chromatography also are
characterized by operational constraints based on their
geometric, chemical, and mechanical properties. Soft,
porous particles, for example, cannot be subjected to
pressure drops exceeding about 50 psi because they are
easily crushed. This implies that there are similar
constraints in effecting enzymatic reactions by passing
substrate solutions through enzymes immobilized on such
matrix materials.
In high performance liquid chromatography (HPLC),
instead of employing as matrices soft, particulate,
gel-like materials having mean diameters on the order
of 100 ~m, one employs smaller, rigid, porous and
substantially homogeneous beads of about 10 to 20 ~m in
~ W092/1~36 _ 3 _ 2 ~ Q ~ 2 3 7 PCT/USg2/02~7
.
diameter and made of an inorganic material such as
silica, or a rigid polymer such as a styrene
di~inylbenzene. R~c~use the dense ~ACki ng of these
smaller beads creates a high resistance to liquid flow,
the equipment is designed to operate at hiqh pressures,
which allows rapid fluid transfer.
Unfortunately, flowthrough speeds of solutes
other than small molec~ r weight solutes become
limiting in conv~ntion~ HPLC matrices, prim~rily
because mass transfer within the pores of the ~PLC
particles is diffusive, as compared to the mass
transfer between particles, which is convective.
Accordingly, the time it takes for an enzyme's
substrate to diffuse to an enzyme locus within a pore
from a region where convective transport dominates, and
for the product to diffuse back, becomes a reaction
rate-limiting factor. Beyond a certain opt~mal flow
velocity, further increases serve only to increase
breakthrough of unreacted substrate. In addition, these
diffusion limitations within pores can result in
significant substrate and end product inhi~ition. To
overcome this inhibition, enzyme reactors of the art
often employ a "stop-flow" protocol and/or multiple
sequential subtrate injections, to limit the substrate
concentration provided to the enzyme sites at any given
time. While these protocols can limit the effects of
inhibition, they are time-consuming and can introduce
unwanted errors into the analysis. The limiting
flowthrough speeds also substantially limit the speed
with which enzymes can be loaded onto the particle
surfaces. Currently, this step alone can takes hours
or days to complete.
W092/1~36 PCT/US92/0~7
- 4 -
- 21û22~
Efforts to reduce these diffusional limitation
effects have led to the development of methods for
loading the enzyme only onto the outer edge of a porous
particle. While this can reduce the enzyme loading
timeframe and limit diffusional pa~hlengths~ it can
limit the capacity of the system substantially.
Alternatively, the particle size may be enlarged to
increase capacity, but this generally only results in
longer diffusional path lengths within the particle.
Reducing or eliminating the ~er~nde~ce of Lmmobilized
enzyme reactors on the diffusive flow rate of solutes
would enh~nce their productivity.
It is therefore an object of this invention to
provide an enzyme reactor that can be rapidly loA~d,
and which effectively is not diffusion limited. It is
another object of this invention to provide a method
and apparatus for immoblized enzyme reactions wherein
substrate and product inhibition is limited. Still
another object of the invention is to provide a method
and apparatus for immobilized enzyme reactions that is
adaptable and cost-efficient to perform.
~ WO92/18636 PCT/US92/0~7
~ 21~%37
Summary of the Invention
This invention pertains to perfusive matrices
capable of immobilizing enzymes and to the use of such
matrices to perform enzymatic reactions. Perfusive
matrices comprise rigid, porous, high surface area
materials such as particles which may be of the same
mean diameter as are employed in conventional
chromatography matrices. The geometry of perfusive
matrices are configured to allow convective fluid
transfer both within and between the par~ s.
Typically, 10-20 ~m diameter particles of per~usive
matrices have throughpores of relatively large mean
diameter (e.g., 6,000 to 8,000 A) and a high surface
area network of internal, blind subpores of smaller
mean diameter (500 to 1500 A) within the throughpores.
The enzymes can be immobilized on all avA;~hle surface
areas, including within the throughpores and the
subpores.
Perfusive matrices are characterized by a
relatively small ratio of the mean diameters of the
interparticle flow paths to the intraparticle
throughpores, thereby permitting intraparticle
convective flow at accessible fluid flow velocities.
The resulting network limits the diffusional path
lengths within the particles so that mass transfer
within the particle pores is governed by convection
rather than diffusion over a large range of high flow
rates. Where the perfusion matrix comprises packed
particles, the diameter of the particles determines the
mean diameter of the interparticle spaces in a packed
bed. In preferred embodiments, the ratio of the mean
particle diameter to the mean diameter of the
intraparticle throughpores is less than 70, most
~ 21 ~2~37
W0 92/18636 PCT/US92/02887
-- 6
preferably less than 50. Preferred subpore diameters are
within the range of about 300-700 A . In addition, the
low ratio (and correspondingly larger intraparticle pore
size) substantially reduces particle pore effects.
Preferred ratios of convective flow velocities through
the interparticle and intraparticle pores are between
about 10:1 to 100:1.
In a perfusive matrix containing immobilized
enzymes, the velocity of a mobile phase can be increased,
for example, to greater than 10 to 100 times that of
conventional HPLC systems without substantial loss of
enzyme binding capacity or substrate conversion
efficiency. Typically, mobile phase velocities of
greater than 1000 cm/hr can be achieved. In addition,
the significantly reduced diffusional pathlengths that
characterize perfusive matrices substantially eliminate
substrate or product inhibition even when the system is
operated in a non-perfusive mode. A description of
perfusive matrix material in chromatogra~hic contexts is
provided in co-pending Canadian patent application serial
number 2,018,393 filed June 16, 1990 and in Afeyan et
al., ~1990) Bio Technoloqy 8:203-206. Perfusive matrix
materials are available commercially from PerSeptive
Biosystems, Inc. of Cambridge, Massachussetts, U.S.A.
In one aspect, this invention is a method for
conducting an enzymatic reaction using a perfusive
matrix formed by packing a multiplicity of particles
defining therewithin through pores and substrate
interactive surface regions within the throughpores
comprising immobilized enzymes. A solution of enzyme
substrate is passed through the matrix at a velocity
WO92/1~36 PCT/US92/02~7
-- 7
~ 7
sufficient to cause convective fluid flow in the
throughpores at a rate greater than the rate of
substrate diffusion through the throughpores.
In another embodiment, a matrix is provided
defining inter-~o~n~cted first and second throughpore
sets wherein the members of the first throughpore set
have a greater mean diameter than the members of the
second throughpore set. Enzymes are immobilized, using
chemistries known per se, or novel methods, on all
surfaces ~f the matrix, including on surface regions in
fl~id communication with the members of the second
throughpore set. A solution of enzyme substrate is
passed through the matrix at a rate sufficient to
induce convective flow through both throughpore sets,
and to induce a rate of convective flow through the
second throughpore set that is greater than the rate of
diffusion of the substrate within that set.
In another aspect, the invention provides a
method for conducting an enzymatic reaction utilizing a
matrix defining interconnected first and second
throughpore sets dimensioned to allow convective fluid
flow through both throughpore sets. Both throughpore
sets comprise a multiplicity of throughpores for
channeling through the matrix an enzyme solution and a
substrate solution reactive with that enzyme. The
matrix also includes interactive surface regions
capable of immobilizing enzymes and which are in fluid
communication with the members of the second pore set.
Enzymes preferably are immobilized on the
surfaces of the matrix by passing a solution containing
the enzyme through the matrix. In preferred
embodiments of this invention, the fluid mixture is
WO92/1~36 PCT/US92/02~7
2 10~;~3-7 8 -
passed through the matrix at a rate sufficient to
produce convective fluid flow through both pore sets,
the velocity through the first set being greater than
the velocity through the second set, and the convective
fluid flow velocity through the second pore set being
greater than the diffusive flow rate of the enzyme
within the second pore set. The dimensions of the
members of the second pore set and the interactive
surface regions permit flow through the members of the
second pore set at a rate such that the time for a
solu~e to diffuse to and from the interactive surface
regions is comparable to or shorter than the time for
the solute to flow convectively past the region.
To bring about reaction between ~he enzyme
substrate and the immobilized enzyme, the substrste
solution then is passed through the matrix at a fluid
flow rate sufficient to allow catalysis of the
substrate by the immoblized enzyme. In preferred
embodiments, this fluid flow r~te is sufficient to
produce convective fluid flow through both pore sets.
This allows the enzymatic reaction to take place under
kinetically very favorable conditions. Specific
dimensions and flow velocities are described in greater
detail below.
In yet another aspect, the invention provides
a method for performing a series of enzyme reactions in
successive zones within the matrix. In this method
each enzyme is loaded at a perfusive fluid flow
velocity and at a concentration insufficient to
saturate all available binding sites on the matrix. In
addition, the enzymes are loaded in the order in which
they are to be used. Because the perfusive flow rate
allows the enzyme solution to encounter all available
WO92/1~36 PCT/US92/02~7
21~237
binding sites it flows past, the first enzyme added
will saturate the avaliable binding sites it first
encounters, forming a discrete zone of immobilized
enzyme. The second enzyme, added subsequently, then
will flow past this zone of saturated binding sites
cont~i n i ng the immobilized first enzyme and will begin
binding at the first available binding sites
encountered below, forming a second discrete zone
composed of immobilized second enzyme. Similarly, the
next enzyme added will flow past both these first ~nd
second zones and wili occupy available bin~ing sites
further down the column. In this way multiple,
different enzymes may be bound in successive, discrete
zones. The method is particularly useful where
multiple, different enzymatic reactions with ~ given
substrate are desired. For example, the IMER may be
constructed to mimic the actions of an enzyme complex
found in nature. Alternatively, multiple different
reactions may be required to obtain a product of
interest. In these cases the product formed by
reaction of a substrate with the first enzyme becomes
the substrate for the reaction with the immobilized
enzyme in the zone below. In preferred embodiments,
optimal kinetics for all reactions in the system can be
achieved with one given flow rate.
Alternatively, the multiple enzyme system may
be used to perform catalytic reactions on different
substrates. A particularly useful application of this
method is in clinical analyses, to assay the presence
and/or concentration of different solutes in a body
fluid sample. In this case the different enzymes need
not be se~regated into discrete zones.
WO92/1~ ~ PCT/US92/02887
-- 10 -- ~
3 7
In still another aspect, the invention
provides a novel enzyme reactor including a rigid
matrix defining interconnected first and second pore
sets dimensioned to allow convective fluid flow through
both throughpore sets, each pore set comprising a
multiplicity of pores for chAnneling through the matrix
a solution of enzyme or enzyme substrateO The matrix
further defines surface regions comprising immobilized
enzyme and in fluid communication with the members of
the second throughpore set. The relative dimensions of
the members of the first and second throughpore sets
and the surface regions are fixed so that when fluid is
passed through the matrix at a preselected velocity,
there is convective fluid flow through both pore sets.
As a result, even though diffusion is required, the
system is not "diffusion bound" except at very high
flow rates. At practical flow rates, the rate of
diffusion is not rate limiting in the productivity of
the system.
In one embodiment of the apparatus of the
invention, plural different enzymes may be immobilized
on the matrix surfaces. The different enzymes further
may be immobilized on the matrix in successive zones.
In another embodiment of the apparatus of the
invention, the rigid matrix comprises a multiplicity of
interfacing particles defining an interstitial volume
which constitutes the first throughpore set. Each
individual particle defines a plurality of
throughpores, constituting the second pore set, and a
plurality of blind pores in fluid communication with
the throughpores, within which enzymatically active
surface regions are located. Preferably, throughpores,
subpores, and any interconnecting pores are
~ WO~2/1~ ~ 11 - PCT/US92/0~7
~iO22~7
anisotropic. Currently preferred particles have a mean
diameter greater than about 50 ~m, most preferably
greater than about 100 ~m, and have a ratio of me~n
particle diameter to mean intraparticle throughpore
diameter of less than 70. A preferred geometry of the
particles comprises adhered clusters of smaller similar
clusters made up of small interadhered particles called
porons. The preferred material for manufacture of the
particles is polysLylel,e divinyl benzene copolymer.
Such particles are available commercially from
PerSeptive Biosystems, Inc. of Cambridge, MA.
These and other aspects of the invention will
be understood more fully by reference to the following
detailed description in conjunction with the att~ch~d
drawing in which like-referenced characters refer to
like parts throughout the several views.
-
WO92/18636 - 12 - PCT/US92/0~7
21~22~7 ` ~
Brief Description of the Drawing:
FIGURE 1 is a schematic representation of a
particle suitable for use in forming a perfusive matrix
contAini~g immobilized enzymes in accordance with the
ingS of the present invention;
FIGURE 2 is a schematic representation of an
apparatus embodying the present invention; and
FIGURE 3A-C are graphic representations for
perfusive immobilized enzyme reactiong performed under
various reaction conditions.
WO92/1~36 - 13 - PCT/US92/02~7
21~2~7
Detailed Description
The matrix of the enzyme reactors of this
invention is characterized by a geometry which is bi-
modal or multi-modal with respect to its porosity snd
which has interactive surface regions capable o~
immobilizing enzymes. The matrix defines a set of
pores of larger di~meter, such as are defined by the
interstices among a bed of particles, which determine
pressure gradient and fluid flow velocity through the
bed, and a set of pores of smaller diameter (e.g.,
anisotropic throughpores). The smaller pores penmeate
the individual particles. At fluid flow velocities
above a threshold level, these pores serve to deliver
by perfusion a solution of sn enzyme or enzyme
subs~rate to substantially all surface regions within
the particles.
FIGURE 1 shows schematically a matrix p~rticle
10 suitable for use with the present invention. As
illustrated, in addition to extra-particle pores or
channels 12, which have a relatively large mean
diameter (defined by the interstices amon~ particles),
a matrix utilizing the particle 10 also comprises a
second set of throughpores 14, here embodied as pores
defined by the body of the particle 10. Also de~ined
by the particle 10 is a set of diffusive transport
pores 16. The mesn diameter of the pores 12 is larger
than the throughpores 14. In accordance with the
immobilized enzyme reactor of the invention, the ratio
of the mean diameters of pores 12 and 14 is such that,
when the particles are close-packed in a bed, there
exists a threshold of fluid velocity which can
practically be achieved in the bed which induces a
convective flow within pores 14 that is faster than the
WO92/1~36 4 PCT/US92/0~87
-- 1 -- ~
2~ Q2237
rate of diffusion within pores 14. Above the threshold
bed velocity the bed is said to be operating in the
perfusive domain where contact between enzyme and
substrate is no longer bound by diffusive transport.
Precisely where this threshold of perfusion occurs
depends on many factors, but primarily it ~ep~n~s upon
the ratio of the mean di~meters of the first and second
pore sets, here, pores 12 and 14 respectively. The
smaller th~t ratio, the lower the threshold velocity.
In preferred embodiments, the ratio of the mean
particle diameter to mean intraparticle throughpore
diameter is less than 70, most preferably less than 50.
In accordance with the invention, the
particle 10 inclu~es a large surface region 18 within
the particles onto which enzymes can be immobilized.
Enzymes may be covalently att~ched to the surfaces 18
at high ron~tration using any one of a nu~ber of
techniques well known to those skilled in the art.
DepenAin~ on the composition of the matrix material and
the enzyme, covalent attachment can be through
functional groups such as carboxyl, amino, hydroxyl,
sulfhydryl, hydroxyphenyl groups and/or other groups.
In addition, the matrix first may be derivatized to
create the desired functional group for a given
attachment protocol. Additionally, a pendant chain can
be attached to the surface having a terminal functional
group distal to the surface to which the enzyme can be
coupled resulting in an immobilized enzyme which is
attached by a "leash~' at a distance away from the
surface. Enzymes also may be covalently bound to the
matrix surface by means of difunctional crosslinkers.
Further details on methods for covalent immobilization
of matrices can be found in a number of references in
the art. Included among these are Falb, R . D .
WO92/1~36 15 PCT/US92/02~7
21022'~7
pp. 67-76, in Enzyme Enqineerinq Vol 2, Pye, E. at al.
eds., (1973) Plenum Press, NY, and White et al., (1980)
Enzyme Microb. Technol. 2:82-90.
Alternatively, enzymes may be noncovalently
attached to the matrix support. Noncovalent attachment
provides a number of advantages over covalent
attachment, chief among them being the cost efficiency
and adaptability this approach provides. A given
enzyme can be bound to the matrix using any of a number
of well-known noncovalent interactions. Thereafter the
enzymatic conversion is run, and the enzyme
subsequently eluted. The matrix then can be reloaded
with the same or a different enzyme, thereby
substantially increasing the life of a given enzyme
and/or reactor system. Alternatively, multiple
substrate conversions may be performed with a given
enzyme bound to the matrix, which then is eluted and
replaced. As removal of the enzyme does not require
breaking strong chemical bonds, harsh extraction or
"stripping" conditions are not needed, allowing the
enzyme to be reused as needed. In addition,
noncovalent attachment is thought to minimize
conformational distortion of the enzyme ne~e~ to
achieve binding. It will be understood by those skilled
in the art that substrate conversions performed using
noncovalently immobilized enzymes will need to be run
under conditions that do not induce elution of the
enzyme. This may be achieved by manipulation of
various reaction parameters, such as buffer pH and/or
salt conditions. Particularly useful matrices for non-
covalent enzyme immobilization include ion exchangers,
and matrices that bind by hydrophobic interactions
(e.g., "reverse phase" matrices.)
.
WOg2/18636 - 16 - PCT/US92/02$87
'~ 102237
In any enzyme immobilization procedure, a pri~ary
design criterion is to maximize enzyme activity of the
resulting enzyme reactor. Even though the reactor may
be highly stable and permit repeated use of the enzyme,
a significant loss of enzymatic activity in the
attachment step often can make the reactor economically
unfeasible. Accordingly, the chemistry for
immobilization should be compatible with the enzyme.
The amount of enzymatic activity per unit weight of
reactor matrix is another important factor and, for
most purposes to avoid excessive h~ i ness, a reactor
should have an enzyme activity of at least 100 U/g of
matrix material, where a unit of enzyme activity (U) is
defined as one micromole of substrate converted per
minute.
An important consideration for enzyme reactors
int~ndeA for continuous operation is the suitability of
the matrix for column flow. A high surface area
usually is required to obtain the desired levels of
enzymatic activity. High surface area can be attained
by employing microporous materials, but this can result
in inefficient substrate transport by diffusion.
Increases in surface area also can be achieved by
reducing particle size, but the column flow properties
of the reactor are usually unsatisfactory.
To circumvent these problems, highly porous, rigid
materials are used. It becomes apparent that a matrix
comprising a multiplicity of particle 10 is
particularly well suited for enzyme immobilization.
The exceptionally high surface area-to-volume ratio
achievable with perfusive matrix particles (typically
on the order of about 30-50 m2/ml for 10-20 ~m
-
W092/1~36 - 17 - PCT/US92/02~7
210~37
particles) allow immobilized enzyme reactions to be
performed in small volumes without compromising
capacity. In addition, the rapid throughput of
perfusive systems allow enzymes to be loaded rapidly
onto a perfusive reactor system (e.g., in seco~ or
minutes) in a fraction o~ the time currently required
without loss of binding capacity. Moreover, as will be
appreciated by those skilled in the art, the rapid
throughput of the system and its small size allow one
to ~ni~ nte reaction parameters rapidly and easily
when developing a reaction protocol.
Virtually any enzyme can be utilized in the enzyme
reactor of this invention for an equally large variety
of applications. In addition, multiple different
enzymes may be immobilized on the matrix to catalize
reactions with multiple, different subtrates. Useful
enzymes include oxido-reductases, trsnsferases,
hydrolases, lyases and isomerases. Useful applications
include a wide range of industrial applications,
including those in the chemical, food and fragrance
industries; in bioremediation, including the treatment
of waste water by immobilization of pesticide-
detoxificstion enzymes; snd in pharmsceutics,
particulsrly for the sepsration of chiral isomers from
a racemic mixture. Among the many useful applications
in medicine are the use of IMERs as part of
extracorporeal devices, and in a variety of clinical
analyses, including assaying the presence and/or
concentration of one or more solutes of interest in a
body fluid sample. In addition, the IMERs of this
invention are useful for the biochemical analysis of
particular enzymatic and metabolic reactions.
WO92/1~ ~ - 18 - PCT/US92/02~7
2~2237
In addition to isolated and purified enzymes,
it should be noted that relatively impure and/or
heterogeneous enzyme preparations, such as those
derived from cell extracts, cell lysates, partiaily
purified enzyme isolates and whole cells can also be
used, albeit at some reduction in the enzymatic
activity. Accordingly, the tenm "enzyme" as used
herein is meant to broadly include catslytic enzymes in
all of these forms.
It also is possible to immobilize plural
enzymes of different types in successive zones on the
matrix surfaces to affect a series of enzymatic changes
in a single pass. For example, enzymes normally
associated as a complex to perfonm sequential enzymatic
reactions in nature may be immobilized in series in a
perfusive matrix in the following manner. A solution
contAin;ng a r~n~Pntration of the first enzyme in the
series insufficient to saturate all available bindinq
sites on the matrix is provided to the perfusive
matrix, e.g., to packed particles in a column. The
solution further is provided at a fluid flow velocity
sufficient to produce perfusion. At or above this flow
rate the enzyme will encounter virtually all available
binding sites it passes, thereby saturating the first
available sites, forming a discrete zone of immobilized
enzyme. A second solution containing a second enzyme,
also at a concentration insufficient to saturate all
available binding sites, then is added. The enzymes in
the second solution, also provided to the matrix at a
perfusive flow rate, will flow past the zone of
saturated binding sites containing immobilized first
enzyme, and will begin binding the first available
sites encountered further down the column, forming a
second zone of immobilized enzyme. Other, different
WO92/1~36 19 PCT/US92/02~7
~ 23~2'~7
enzymes then may be added in the same manner. The
number of enzymes that may be added will be limited
primarily only by the capacity of the column and the
concentration of enzymes needed to effect efficient
catalysis. It will be appreciated by those skilled in
the art that optimal kinetics for enzyme reactions
performed in series will require use of enzymes with
similar reaction rates, and/or limiting the flow r~te
to a rate which will provide optimal kinetics for the
slowest reaction.
A schematic representation of a system for
practicing the invention is illustrated in FIGURE 2. A
solution of enzyme substrate 20, is pumped by a pump 22
to an enzyme reactor 24. The enzyme reactor 24
includes a matrix comprising a packed bed of particles
10. While a matrix comprising a packed bed of
particles 10 is pre~erred, other matrix forms may be
used. It is necessary only that the matrix be rigid,
so that it can withstand substantial pressure drops,
and that it define throughpore sets capable of
convectively ch~nnel 1 ing a solution of enzyme substrate
throughout the column 24 at reasonable, and preferably
low, pressure drops. Immobilized on the interactive
surface regions 18 of the particle 10 are enzymes 25.
In the practice o the invention, the solution of
enzyme substrate 20 is passed through the reactor 24,
thereby inducing an enzymatic reaction resulting in a
product stream 26 exiting from the enzyme reactor 24.
The apparatus also may be part of an automated
system. In this case, the apparatus preferably further
comprises a multi-port sampling valve 28 which may
provide the enzyme(s) to be loaded, as well as all
necessary solvents or buffers, including washing
WO92/18636 20 PCT/US92/0~7
2~Q2~37
solvents, eluting solvents, and recycling or
"stripping~ solvents. A valve 30 at the exit of the
reactor 24 may direct the product stream 26 to a
detector 32, to waste collectors 34, or to product
collectors 36. In addition, valve positions may be
under computer control
As discussed above, the rate at which a
solution (e.g., 20J is passed through the enzyme
reactor 24 and the dimensions of the throughpores 14
and the enzyme interactive surfaces 18 are important to
the practice of the invention. That is, flow rates
sufficient to induce convective flow through both the
interstitisl pores 12 and the throughpores 14 should be
achievable without reguiring excessive pressure drops.
Moreover, for true perfusive operation, the convective
flow rate within the throughpores 14 should be greater
than the ra~e of diffusion of the solution 20 within
the throughpores 14.
In the currently preferred embodiment of this
invention, perfusive particles and their throughpores
are of a mean diameter sufficient to permit substrate
conversion to be performed in perfusion mode. One
measure of the mass transfer of a solute through a pore
is given by a characteristic Peclet number (Pe), a
~ en~ionless quantity equal to VL/D, where V is the
convective velocity through the pore, L is its length,
and D is the diffusivity of the solute through the
pore. In conventional porous matrix systems, under all
regimes, the Peclet number which describes the ratio of
convective to diffusive transport within the pores of a
material is always much less than one. In perfusive
systems, the Peclet number in the second set of pores
(e.g., the throughpores) is always greater than one.
WO92/1~ ~ - 21 - PCT/US92/02~7
~10~7
As should be evident from the foregoing, the size of
the particle (and the related intraparticle
throughpore) will affect the flow rate and Peclet
number. For example, for 10 ~m particles having 4,000A
intraparticle throughpores, and for a solute having a
pore diffusi~ity of 10-7 cm2/sec~ the threshold flow
rate is about 300 cm/hr. Above this threshold, it will
be found that increased pressure drop and velocity
permit increased throu~hput per unit volume of matrix
above levels heretofore achievable. Extrsordinary
productivities are achieved at flow rates of within the
range of 1000-4000 cm/hr.
Particle sizes commonly used in IMERs are
substantially larger than conven~io~l HPLC matrix
particles, often on the order of 100-lOOO~m in
diameter. Perfusive matrices comprising particles in
this size range can have intraparticle throughpores in
the range of aobut 2-20 ~m. Thus, for 200 ~m particles
and a solute having a pore diffusivity on the order of
lo- 6 cm2/sec, to achieve a Peclet number of grester
than one, the threshold flow rate need only be about
0.3 cm/hr to achieve convection within the pores (or,
sssuming only 1% bed to pore velocity, 30 cm/hr).
Accordingly, perfusion can be achieved at very low
pressure drops and at sufficiently slow flow rates that
enzyme conversion reactions are not compromised, even
for reactions requiring flow rates at fractions of a
ml/min.
For smaller particles (e.g., 10 ~m particles),
optimal catalytic kinetics for immbilized emzyme
reactions may require reductions in flow rates to ~elow
the perfusive threshold. However, it will be
appreciated by those skilled in the art that perfusive
WO92/1~36 - 22 - PCT/US92/02~7
2~237
matrices comprising smaller particles and requiring
concomitantly higher threshold flow rates still will be
useful, even when enzyme reactions are run below the
perfusion mode. There are several reasons for this.
First, the enzymes themselves may be loaded in the
perfusion mode, allowing the column to be loaded in
seconds, a fraction of the time required using
conventional systems. S~co~A, the increased
intraparticle throughpore size substantially reduces
pore effects identified with ~ol~ventio~Pl porous
matrices. Third, the r~ c~ ratio of interparticle
and intraparticle pores, and the substantially reduced
intraparticle diffusional pathlengths that characterize
these matrices ~irtually eliminate substrate or enzyme
inhibition due to solute build up in a st~g~Ant mobile
phase.
The invention may be further understood from the
following, nonlimiting example.
Calf intestinal alkaline phosphatase (CI-AP) is
noncovalently attached to a perfusive reverse phase
column (POROS~M RM, PerSeptive Biosystems, Inc.,
Cambridge, MA, 0.1 x 2 cm, 20 ~m diameter particle,
packed at 40 bar). CI-AP, reconstituted to 1 mg/ml
with 10 mM tris(hydroxymethyl)aminoethane, pH 8.0, is
diluted (1:100) in 10 mM phosphate buffer (PB), pH 7.1,
and ng quantities loaded onto the column at a given
flow rate, generally between 0.1 ml/min to 0.5 ml~min.
Typically, the column is loaded in about 10 seconds.
The enzyme substrate, p-nitrophenylphosphate,
(PNPP, Pierce Co., Rockford, IL) ) is dissolved in 10 mM
diethylanolamine, 0.5 mM MgC12, pH 9.~, and loaded at
either 0.1 or 1.0 mg/ml. The substrate reaction is
~ WO92/1~36 - 23 - PCT/US92/02~7
.
2~d2237
run as a continuous flow system at a given flow rate
snd ~oncentration of enzyme. Product is identified by
absorbance at 405 nm.
The column may be stripped by washing with PB,
followe~ by a stripping solvent comprising 80%
acetonitrile/20% acetic acid, and re-equilibrat with
PB. PNPP can be passed over the column before a second
enzyme is loaded, and the effluent tested for residual
enzyme activity.
Fig. 3A and 38 represent plots of product formation
versus enzyme concentration for 10 different flow rates
and two different substrate concentrations (O.1 mg/ml
and 1.0 mg/ml in 3A and 3B, respectively). A lOofold
increase in substrate concentration appears to have no
effect on the system. As expected, slower flow rates
increase sensitivity of the assay. However, while
preferred substrate reaction flow rates (e.g., less
than about 0.2 ml/min) are below the threshold
perfusion flow rate for the size particle used in this
experiment (20 ~m), the linearity of the curves at both
high and low substrate concentrations (R=l.000-0.990)
provides dramatic evidence that no significant
substrate or product inhibition occurs in this system,
even when high substrate concentrations are provided as
a con~inuous flow. As stated above, in the preferred
embodiment of this invention, perfusive particles for
use in IMERs have a diameter on the order of about 100-
1000 ~m, substantially reducing the threshold flow rate
for intraparticle convective flow.
In Fig. 3C, 1650 column volumes have been passed
through the system (containing 25 ng CI-AP) at a
perfusive flow rate (3600 cm/hr) between a first and
WO92/1~ ~ PCT/US92/0~7
- 24 -
2~ 02237
second substrate conversion series (curves 1 and 2,
respectively, using 1 mg/ml PNPP, and a range of flow
ra~es). As is evident from the data plotted in Fig.
3C, no significant leaching of the noncovalently
im~obilized enzyme has occurred in the interim. In yet
another experiment, a second column capable of
~mmobiliz~ng enzyme is placed $n t~ndem with the first,
and the effluent from the fir~t column passed o~er it
to test for leaching enzyme. No significant substr~te
conversion can be identified in thls econd colu~n when
substrate is subseguently passed through this second
column, indicat~ng that substantially no enzyme has
been leAched from the first column..
The invention may be e~bodied ~n other specifie
forms without departing from the spirit and essential
characteristics thereof. Accordingly, the invention is
to be defined not by the preceding description, which
is intended as illustrative, but by the claims that
follow.
