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United States Patent [191

[11] Patent Number:

van Reis

[45]

[54]

Date of Patent:

TANGENTIAL FLOW FILTRATION

4,971,696 11/1990 Abe et a1- -

PROCESS AND APPARATUS

5,256,294

[75] Inventor: Robert D. van Reis, Redwood City,

[73] Assignee: Geneutech, Inc., South San Francisco,

10/1993 van Reis ............................... .. 210/637

0069523

1/1983

European Pat. 01f. .

0112510

7/1984

European Pat. Off. .

0220749

5/1937 EulfopeanPat- Off- -

2065129

6/1981

Ul'llt?d Kingdom .

8704169

7/1987

WIPO .

The portion of the term of this patent

OTHER PUBLICATIONS

subsequent to Oct. 26, 2010, has been

disclaimed

* Feb. 13, 1996

FOREIGN PATENT DOCUMENTS

Calif.

[*1 Notice:

5,490,937

Baeyer et al., J. Membr. Sc1., 22: 297315 (1985), Cascade

Plasmapherasis with Online Membrane Regeneration: Labo~

ratory and Clinical Studies.

[211 APPL NO" 271,223

Castino et al., Arti?cial Kidney, Arti?cial Liver, and Arti?

[22] Filed:

cial Cells, Chang, ed., Pleneum Publishing Corp., N.Y., pp.

Jul. 6, 1994
Related U_S_ Application Data

259-266, (1978), The Filtration from Whole Blood: A Novel

Approch to Clinical Detoxi?cation.

Chakrovorty et al., Desalination, 78(2):279286 (1990),

[63] Continuation of seam), 91,945,Ju1_ 15, 1993, abandoned,
which is a continuation of Ser. No. 583,886, Sep. 17, 1990,
Pat. No. 5,256,294.

[511

Int. Cl.6 ................................................... .. B01D 61/22

[58]

US. Cl. ......................... .. 210/637; 210/641; 210/651

Field of Search ................................... .. 210/637, 641,

210/137, 321.65, 651, 109, 321.84

References Cited

[56]

U.S. PATENT DOCUMENTS

3,744,642
4,105,547
4,191,182
4,276,172
4,350,156
4,420,398
4,435,289
4,654,265
4,689,267
4,741,829
4,746,436
4,789,489
4,802,942
4,828,705
4,874,516
4,879,040
4,935,139

7/1973
8/1978
3/1980
6/1981
9/1982
12/1983
3/1984
3/1987
8/1987
5/1988
5/1988
12/1988
2/1989
5/1989
10/1989
11/1989
6/1990

Concentration and Puri?cation of Gelatin Liquor by Ultra

?ltration.

Cheryan, Ultra?ltration Handbook, (Technomic Publ. Co.,

Inc., Pennsylvania, 1986) pp. 21 8221 , p. 311, Fractionation
of Macromolecules.
P1achel et al., Advances in Biochemical Engineering/Bio

technology, 26z73-142, (Downstream Processing, 1983),

Ultra?ltration for the Separation of Biocatalysts."Gabler,
ASM News, 50(7):299304 (1984), Tnagential Flow Filtra
tion for Processing Cells, Proteins, and Other Biological

Components.
(List continued on next page.)

Scala et a1. .
Sandblom .

Popvich et al. .

Primary ExaminerFrank Spear

Henne et a1. .

Attorney, Agent, or FirmJanet E. Hasak

Makchesky et a1. .

[57]

Castino .

Breslau et a1. .

ABSTRACT

Processes and apparati are provided for separating species of

Breslau .

Kopp et al. .
Di Leo et al. .

interest from a mixture containing them which comprises

subjecting the mixture to tangential-?ow ?ltration, wherein
the ?ltration membrane preferably has a pore size that
retains species with a size up to about 10 microns, and the

Takemura et a1. .

?ux is maintained at a level ranging from about 5 % up to

Thakore et a1. ...................... .. 210/636

100 % of transition point ?ux.

Takarnizawa et a1. .
Takemura et a1. .

Kondo .

Prince et a1. .

12 Claims, 11 Drawing Sheets

Davidson et a1. .

/
J; (flux) _ _ _ _ _ i _ _ _-

50% 0i transition

_____ __

point ?ux
540? transition [
TMP

J max

5,490,937
Page 2
OTHER PUBLICATIONS

Michaels, Chem. Eng. Prog., 64(l2):3144 (1968), New

Separation Technique for CPI.
Michaels, Chem. Tech., pp. 3643 (Jan. 1981), Ultra?ltra
tion: An Adolescent Technology.
Michaels, Polymer Science and Technology, vol. 13, Ultra
?ltration Membraines and Applications, pp. 119, (Plenum
Press, N.Y., 1979), Fifteen Years of Ultra?ltration: Prob

Nelson, Proceeding of the International Workshop on Tech

nology for Protein Separation and Improvement of Blood

Plasma Fractionation, Reston, Va., Sep. 7-9, 1977, pp.
133-137, Ultra?ltration in Plasma Fractionation.
Porter, ed., Handbook of Industrial Membrane Technol

ogy, (Noyes Publ., Park Ridge, N.J., 1990) pp. 164-173.

Porter & Michaels, Chem. Tech, pp. 56-63 (Jan. 1971),
Membrane Ultra?ltration.

Michaels et al., Desalination, 53:231-258 (1985), Mem

branes in Biotechnology: State of the Art.
Millipore 1990 Catalog, p. 213, Militan High Resolution

Rautenbach et al., Chem.-Ing., Tech. 54(3), 229240,

(1982) Stoif.
van Reis et al., J. Interferon Res., 2(4):533541 (1982),
Production and Recovery of Human Leukocyte-Derived

Tangential Flow (HRTF) System.

Alpha Interferon Using a Cascade Filtration System.

lems and Future Promises of an Adolescent Technology.

US. Patent

Feb. 13, 1996

Sheet 2 0f 11

5,490,937

US. Patent

Feb. 13, 1996

85

Sheet 3 of 11

FIG. 3

5,490,937

US. Patent

Feb. 13, 1996

Sheet 4 0f 11

5,490,937

US. Patent

Feb. 13, 1996

Sheet 5 of 11

axon53mI

5,490,937

13./ wN.o+m_N
.532
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0E1 - Q2
FIVUJ

TEES20:E.S50Q.:M

US. Patent

Feb. 13, 1996

Sheet 6 of 11

5,490,937

FIG.6

+
52u652I30a 3

AC:5:5EO S

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M.
%
Ma
MW
B
Mu
R
M
7.
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0682

543204Q/|4365.8720l .

Average TMP (psi)

US. Patent

Feb. 13, 1996

Sheet 7 of 11

F I G. 7
0 High Periormance Yield
Conventional Yield

[I High Periormance Purification

I Conventional Purification

2 AU 0

80
60
w0

130

:51

12E232Q;
.m
w
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Diavolumes

5,490,937

US. Patent

Feb. 13, 1996

Sheet 8 0f 11

F IG. 8
0 High Performance Yield
0 Conventional Yield

[1 High Penormanco Purification

I Conventional Purification
250*

200'

w O0

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Diavotumes

5,490,937

U.S. Patent

Feb. 13, 1996

Sheet 9 of 11

5,490,937

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0.0K

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US. Patent

Feb. 13, 1996

Sheet 10 of 11

5,490,937

F 16.10
I Cytochrome C

AB

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5 :32

O0 5.6

0 4.

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d

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TMP [psig]

40

US. Patent

Feb. 13,1996

Sheet 11 0f 11

5,490,937

F | G. I I
o HP-TFF. TMP=17psi
o C-TFF, TMP=35psi
1.0 -

o
o

rt-PA

0.9
rhGH
o

.8 -

cytochrome-C

0.7 -

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5,490,937
1

TANGENTIAL FLOW FILTRATION

PROCESS AND APPARATUS

solute discrimination is poor. See, e.g., Porter, ed., Hand

book Of Industrial Membrane Technology (Noyes Publica

tions, Park Ridge, N.J., 1990), pp. 164173.

This is a continuation of serial No. 08/091,945 ?led Jul.

15, 1993, now abandoned which is a continuation of Ser. No. 5
07/583,886 ?led on 17 Sep. 1990, now U.S. Pat. No.

5,256,294.

example, under highly polarized conditions, ?ltration rates

may increase only slightly with increasing pressure, in

1. Field of the Invention

This invention relates to the puri?cation and separation of

contrast to unpolarized conditions, where ?ltration rates are

usually linear with pressure. Use of a more open, higher-?ux
membrane may not increase the ?ltration rate, because the

moieties, particularly those of biological interest, from mix

tures containing them utilizing improved tangential-?ow
15

A result of concentration polarization and fouling pro

cesses is the inability to make eifective use of the macro
20

selective binding of the desired molecules to an a?inity

matrix or gel. Af?nity gels typically consist of a ligand

binding moiety immobilized on a gel support. For example,

GB 2,178,742 utilizes affinity chromatography to purify

hemoglobin and its chemically modi?ed derivatives based

polarized layer is providing the limiting resistance to ?ltra

tion. The situation is further complicated by interactions
between retained and eluted solutes.

Several methods are currently available to separate mol

ecules of biological interest, such as proteins, from mixtures

thereof. One important such technique is af?nity chroma
tography, which separates on the basis of speci?c and

resistance to the ?ltration of solvent. The degree of polar

ization increases with increasing concentration of retained
solute in the feed, and can lead to a number of seemingly
anomalous or unpredictable eifects in real systems. For

BACKGROUND OF THE INVENTION

?ltration processes and apparati.

2. Description of Related Disclosures

A polarized layer of solutes acts as an additional ?lter in

series with the original ultra?lter, and provides signi?cant

molecular fractionation capabilities of ultra?ltration mem

branes for the large-scale resolution of macromolecular
mixtures such as blood plasma proteins See Michaels,
Fifteen Years of Ultra?ltration: Problems and Future Prom

25

on the fact that native (oxy)hemoglobin binds speci?cally to

polyanionic moieties of certain a?inity gels. In this process,

unmodi?ed hemoglobin is retained by the a?inity gel, while

ises of an Adolescent Technology in Anthony R. Cooper,

ed., Ultra?ltration Membranes and Applications, Polymer

Science and Technology, 13(Plenum Press, N.Y., 1979), pp.
1-19, at p. 9. Consequently, the potentially exciting utiliza
tion of membrane ultra?ltration for large-scale complex

modi?ed hemoglobin, which cannot bind to the gel because

macromolecular mixture-separations currently performed

its polyanion binding site is covalently occupied by the

modifying agent, is eluted. Af?nity chromatography col
umns are highly speci?c and thus yield very pure products;
however, a?inity chromatography is a relatively expensive

by such techniques as gel permeation, adsorption, or ion

process.

?ltration schemes have been examined on paper, with a

exchange chromatography, selective precipitation, or elec

trophoresis is considered elusive.
The merits of various multi-stage and cascaded cross-?ow

Another known separation method is membrane ?ltration,

which separates dissolved and suspended solutes on the
basis of their size. In the simplest form of this process, a
solution is forced under pressure through a ?lter membrane
with pores of a de?ned size. Solutes larger than the pore size
of the membrane ?lter are retained, while smaller solutes are

carried convectively through the membrane with the solvent.

Such membrane ?ltration processes generally fall within
the categories of reverse osmosis, ultra?ltration, and micro
?ltration, depending on the pore size of the membrane.
Conventionally, ultra?ltration employs membranes rated for
retaining solutes between approximately 1 and 1000 kDa in
molecular weight, reverse osmosis employs membranes
capable of retaining salts and other low molecular weight
solutes, and micro?ltration, or microporous ?ltration,
employs membranes in the 0.1 to 10 micrometer (micron)
pore size range, typically used to retain colloids and micro

slightly improved e?ect. See Michaels and Matson, Desali

nation, 53: 231-258 (1985), p. 235 in particular. See also the
experimental ?ow circuit in FIG. 1 on p. 535 of van Reis et

al., J. Interf Res., 2: 533541(1982), and M. Cheryan,

40

Ultra?ltration Handbook, (Technomic Publishing Co., Inc.:

Penn, 1986), p. 311, where a cascade membrane recycle

bioreactor system for producing protein hydrolyzate frac

tions of different molecular sizes is described.
To circumvent the effects of concentration polarization,
45

several processes have been developed that modify the feed

plasma source to improve selectivity and ?ux (de?ned as the

?ltration rate divided by the membrane area). For example,

UK Appln. No. 2,065,129 describes a separation technique
wherein serum is diluted to reduce total protein and salt

operation capable of processing thousands of liters per hour.

Ultra?ltration is widely used for protein concentration and

concentration while the pH is adjusted to between 3.8 and

4.7 prior to ultra?ltration. Baeyer et al., J. Membrane Sci.,
22:297315(1985) describes a process wherein the plasma is
?rst diluted by a factor of 12 prior to ultra?ltration. U.S. Pat.
No. 4,350,156 discloses a process for removing macromol
ecules from plasma by cooling the plasma to about 10 C.
and then ?ltering the macromolecules from the cooled
plasma to form a ?ltered low molecular weight plasma

removal of salts and alcohols, as well as in depyrogenation

of process and rinse water, saline solutions, and low molecu

stream. This process does not employ an ultra?ltration

membrane.

lar weight additives. The advantages of ultra?ltration

include low energy cost, low capital equipment outlay, and
e?icient and controllable operation with very low denatur
ation of product.
However, limitations exist on the degree of protein puri

One major approach to address concentration polarization

50

organisms.
Over the past 25 years, ultra?ltration has progressed from
a small-scale laboratory tool to a fully established unit 55

in ultra?ltration systems has been to control the ?uid ?ow

pattern so as to enhance transport of the retained solute away
from the membrane surface and back into the bulk of the
feed. In a process known as tangential-?ow ultra?ltration
?cation achievable in ultra?ltration. These limits are due 65 (TFF), the feed stream is recirculated at high velocities
tangential to the plane of the membrane to increase the
mainly to the phenomena of concentration polarization,
mass-transfer coe?icient for back diffusion. Gabler, ASM
fouling, and wide membrane pore size distribution. Hence,

5,490,937
3

News, 50:299(1984). The ?uid ?owing in a direction parallel

over the ?lter membrane, but on the opposite side of the

membrane from the ?ow of whole blood, to obtain a
substantially uniform TMP across the entire length of the
membrane. The purpose of this embodiment is to increase
?ltration rates to twice that when plasma ?ltrate is removed

to the ?lter membrane acts to clean the ?lter surface con

tinuously and prevents clogging by non-?lterable solutes.

Another ?ltration device that achieves the same e?ects as
TFF is a rotary ?ltration device containing an outer and inner
cylinder, where the inner cylinder is rotated to create a

vortex to obtain high velocity without a change in pressure.

In TFF, a pressure di?erential gradient, called transmem

brane pressure (TMP), is applied along the length of the

membrane to cause ?uid and ?lterable solutes to ?ow 10

through the ?lter. Flux is independent of TMP above a

certain minimum value that can be determined empirically.
To achieve maximum ?ux, ultra?ltration systems are typi
cally run with an outlet pressure equal to or greater than this
minimum value. Hence, ?ux is constant along the length of
the membrane, while the TMP varies. Both laminar and
turbulent ?ow approaches have been used with some suc
cess; however, conventional TFF still aifords only poor
molecular size resolution.

from the ?ltrate chamber without such recycling. Also, it

allows for the use of long ?ltering chamber ?ow paths and
for the design of coil-type ?ltration cells.
More recently, U.S. Pat. No. 4,789,482 issued Dec. 6,
1988 describes a process for separating blood plasma into
high and low molecular weight streams. The blood plasma
is introduced at a certain shear rate into an inlet portion of

a separation unit containing several thin channels or hollow

?bers with walls through which ultra?ltration is carried out.
The high and low molecular weight streams are separated

from the unit, the high molecular weight stream is recircu

Attempts have been made to combine a?inity separation

lated to an inlet portion of the separation unit to form a

recirculation stream, and the ratio of the recirculation stream
?ow rate to the permeate stream ?ow rate is controlled
between 5 and 100, with the ratio of TMP at outlet to inlet
of the separation unit between about 0 and 0.85. This method

with TFF to increase the ability of TFF to separate selec

tively based on biological differences. See WO 87/04169
published Jul. 16, 1987. In TFF ultra?ltration, a soluble

that the outlet and inlet TMPs must be ditferent; thus, it does
not overcome the concentration polarization problem inher

uses the conventional thinking regarding ultra?ltration, i.e.,

ent in ultra?ltration.

a?inity polymer is placed in the mixture containing the

fractions to be separated, on the upstream side of the ?lter 25

membrane. Since the polymer is much larger than the solute

particles, a ?lter can be selected that will allow unbound

solutes to pass through the ?lter but prevent the passage of

polymer and any substance bound to the polymer. Due to the

Despite all of these attempts at improvement, it is still the

case that although concentration polarization can be modi
?ed, and quite high ?ltration rates can be achieved even
from very concentrated solutions if appropriate ?ow condi
tions are supplied, the polarized layer can never be com
pletely eliminated. This is observed from a number of

problems posed by conventional TFF, however, even this

protein mixtures, and a rule of thumb has evolved that

approach has met with limited success.

fractionation of protein mixtures by simple ultra?ltration

U.S. Pat. No. 4,105,547 issued Aug. 8, 1978 discloses a

will probably be highly ine?icient unless the species are at

?ltering process, designed especially for ultra?ltration, in

least a factor of ten dilferent in molecular weight. Although

which a ?lterable ?uid is caused to ?ow under pressure 35

some closer separations have been occasionally reported,

through a ?ltering passage extending along one side of a

they have always been under extremely and impractically

?lter, in such a way that a considerable pressure drop arises

along the ?lter area in the ?ow direction. In the apparatus

dilute conditions. Nelsen, Ultra?ltration in Plasma Frac

tionation, in Proceedings of the Internat. Workshop on

employed, the pressure di?erence between both sides of the
?lter is maintained substantially constant throughout the 40 Technology for Protein Separation and Improvement of
Blood Plasma Fractionation, Reston Va., Sep. 79, 1977,
entire ?lter area. The patentees teach that clogging of the
Sandberg, ed., NIH, DHEW Pub. No. NIH 78-1422, p. 137;
?lter tending to reduce the ?ow can be compensated for by
Flaschel et al, Ultra?ltration for the Separation of Biocata
successively raising the driving pressure to a level that is
lysts, in Fiechter, ed., Advances in Biochemical Engineer
below the pressure-independent region of the ?ux v. TMP
curve. This raising of the driving pressure results in constant 45 ing/Biotechnology, Vol. 26(Downstream Processing), pp.
73-142 (1983), particularly, p. 124; Cheryan, supra, p.
?ltration rate, but not higher selectivity. In addition, the
2182l9.
patentees disclose that the transmembrane pressure should
It is an object of the present invention to provide tangen
be increased as the ?ltration is being carried out. Other
tial-?ow ?ltration processes for separating species such as
features taught by the patentees include better membrane
particles and molecules by size, which processes are selec
cleaning.
50

U.S. Pat. No. 4,191,182 issued Mar. 4, 1980 discloses in

one embodiment a process and apparatus for continuously

separating blood into plasma and cellular component frac

tions. The process involves withdrawing whole blood and
pumping it into a ?ltering chamber of a ?ltration cell, and

55

continuously ?ltering the whole blood by passing it in a ?ow

over and parallel to a membrane of a certain pore size range
and at a ?ow rate sufficient to provide a speci?ed shear stress

range at the membrane interface within particular TMP

con?nes. Then, the cellular component fraction is continu
ously mixed with an amount of replacement ?uid substan

tially equal to the separated plasma fraction, and the cellular

tive for the species of interest, resulting in higher-fold

puri?cation thereof.

It is another object to provide improved ?ltration pro

cesses, including ultra?ltration processes, for separating
biological macromolecules such as proteins which processes
minimize concentration polarization and do not increase
?ux.
It is another object to provide a ?ltration process that can

60

separate by size species that are less than ten-fold di?erent

in size and does not require dilution of the mixture prior to
?ltration.
These and other objects will become apparent to those

component fraction and replacement ?uid mixture are con

skilled in the art.
tinuously returned to a blood vessel of the donor.
SUMMARY OF THE INVENTION
In one embodiment a portion of the plasma fraction 65
These objects are achieved in a process for separating
separated from the whole blood is recycled in a ?ow parallel
to and in the same direction as the ?ow of the whole blood
species of interest from a mixture, which process comprises

5,490,937
5

?ltering the mixture by tangential-?ow ?ltration through a

pore size that retains species of sizes in descending order

from the ?rst to the last of the middle layered membrane(s).

membrane having a pore size that separates the species of

interest from the mixture, while maintaining ?ux at a level
ranging from about 5 to 100% of transition point ?ux.
Preferably, transmembrane pressure for the ?ltration is
maintained substantially constant along the membrane at a

In a still further aspect, the invention provides a tangen

tial-?ow ?ltration apparatus comprising:

5

?ltration unit having a ?rst ?ltration membrane that

separates said unit into a ?rst ?ltering and ?ltrate
chamber, said ?ltering and ?ltrate chambers each hav

level no greater than the transmembrane pressure at the

transition point of the ?ltration.

In a more speci?c aspect, the species of interest has a size
of up to about 10 microns. In an even more speci?c aspect,

ing an inlet and an outlet,

(b) means connecting an inlet of the ?rst ?ltering chamber

to the ?rst vessel, which contains means for pumping
?uid from the ?rst vessel to the inlet of the ?rst ?ltering

this invention provides a process for separating species of

interest having a molecular weight of about 1 to 1000 kDa
from a mixture which process comprises ?ltering the mix
ture by tangential-?ow ?ltration through a ?ltration mem
brane having a pore size that separates said species of
interest from the mixture, while maintaining ?ux at a level
ranging from about 5 to 100% of transition point ?ux,

chamber,
(c) means for generating a pressure gradient within the
?rst ?ltrate chamber,
(d) a second vessel in ?uid communication with a second
?ltration unit having a second ?ltration membrane that
separates said unit into a second ?ltering and ?ltrate

whereby the species of interest are selectively separated

from the mixture.

chamber, said ?ltering and ?ltrate chambers each hav

In yet another aspect, this invention provides a process for

separating species of interest having a size of about 0.1 to 10
microns from a mixture which process comprises ?ltering
the mixture by tangential-?ow ?ltration through a ?ltration
membrane having a pore size that separates said species of
interest from the mixture, while maintaining ?ux at a level

ing an inlet and an outlet,

(e) means for circulating the ?ltrate from the outlet of the
?rst ?ltrate chamber to the second vessel,
(f) means connecting an inlet of the second ?ltering
25

ranging from about 5 to 100% of transition point ?ux,

the second ?ltering chamber,

from the mixture.

In a still further aspect, this invention supplies a tangen

(g) means for generating a pressure gradient within the

second ?ltrate chamber,
30

(a) a vessel in ?uid communication with a ?ltration unit

comprising a plurality of adjacent, parallel ?ltration

ing an inlet and an outlet,

(i) means for circulating the ?ltrate from the outlet of the
second ?ltrate chamber to the third vessel,
(j) means connecting an inlet of the third ?ltering chamber
to the third vessel, which contains means for pumping

an outlet,

(b) means connecting an inlet of the ?ltering chamber to

the vessel, which contains means for pumping ?uid
from the vessel to the inlet of said ?ltering chamber,
(0) means for generating a pressure gradient within the

?uid from the third vessel to the inlet of the third

?ltering chamber, and

?ltrate chamber, and

45

and more preferably a pump.

In yet another aspect, this invention provides a tangential

?ow ?ltration apparatus comprising a ?ltration unit having
a plurality of layered ?ltering and ?ltrate chambers, all but
the last ?ltrate chamber in the layering order having inlet and

(h) a third vessel in ?uid communication with a third

?ltration unit having a third ?ltration membrane that

separates said unit into a third ?ltering and ?ltrate

chamber, said ?ltering and ?ltrate chambers each hav

membranes having the same pore size, which separate

said unit into a ?ltering and ?ltrate chamber, said
?ltering and ?ltrate chambers each having an inlet and

((1) means for collecting ?ltrate from the outlet of said

?ltrate chamber.
Preferably, the membrane pores range in size from 1 kDa
to 10 microns. Also preferably, the generating means is a
means for recirculating ?ltrate through the ?ltrate chamber
parallel to the direction of the ?uid in the ?ltering chamber,

chamber to the second vessel, which contains means

for pumping ?uid from the second vessel to the inlet of

whereby the species of interest are selectively separated

tial-?ow ?ltration apparatus comprising:

(a) a ?rst vessel in ?uid communication with a ?rst

50

(k) means for circulating the ?ltrate from the outlet of the
third ?ltrate chamber to the ?rst vessel, wherein the ?rst
?ltration membrane has a pore size that retains species
of the largest size, the second ?ltration membrane
retains species of a size intermediate to the pore size of
the ?rst and third ?ltration membranes, and the third
?ltration membrane has a pore size that retains species
of the smallest size.
Optionally, the latterrnost apparatus also contains means

for generating a pressure gradient within the third ?ltrate

chamber. Preferably, all the generating means are means for

recirculating ?ltrate through the ?ltrate chamber parallel to

outlet means each in ?uid communication with a separate

the direction of the ?uid in the ?ltering chamber.
vessel, and the last ?ltrate chamber having an outlet means 55
Also, preferably all the membranes of this latter apparatus
for circulating ?ltrate to the vessel that is in ?uid commu
are ultra?ltration membranes of decreasing pore size in the
nication with the ?rst ?ltering chamber in the layering order,
cascade.
each chamber having a means for pumping ?uid from the
Much of the literature to date operates under the assump~
vessel to the inlet means of the chamber, and each chamber
tion that size separation must take place in the pressure
being separated from its adjacent chamber by a ?ltration
independent region of the curve of ?ux versus TMP. The size

membrane, the apparatus being such that the ?ow of ?uid

from the inlet to the outlet means of all chambers is parallel
and in the same direction, the ?rst ?ltration membrane in the
layering order has a pore size that retains species of the

separation of this invention is achieved by operation of the

tangential-?ow ?ltration in the pressure-dependent region of

the ?ux versus TMP curve. In general, the ?ux is actually

decreased in the process of this invention relative to the rate
largest size, the last ?ltration membrane in the layering order 65 obtained when ?ltration is carried out in the pressure
has a pore size that retains species of the least size, and the
independent region of the ?ux versus TMP curve. Further,
middle ?ltration membrane(s) in the layering order have a
the ?ltration is carried out such that the TMP is approxi

5,490,937
7

mately constant with time or decreases throughout the

?ltration.
The maintenance of the TMP within this pressure-depen
dent region results in a dramatic decrease in retention of
molecules with molecular weights lower than the membrane

TFF) (open circles for yield and open squares for puri?ca

tion).
FIG. 8 depicts a graph of the calculated percent yield and
fold puri?cation versus diavolumes for C-TFF and HP-TFF
using a higher recirculation rate than was used to generate
FIG. 7.
FIG. 9 is a graph of ?ux versus TMP for a short pathlength

rating. In addition, this feature greatly improves the overall

selectivity of the system for the species desired to be
puri?ed, thereby overcoming the barrier of the concentration
polarization layer. Hence, a greater fold puri?cation of the
species of interest is obtained over conventional tangential
?ow ?ltration, where ?ux D is greater than about 100% of
transition point ?ux. An additional advantage of the process
resides in separating species that are less than ten times

experimental TFF module using a 30 kDa regenerated

cellulose membrane.
10

FIG. 11 is a graph of retention versus log molecular

smaller in molecular weight than the larger species of the

weight for a mixture containing Cytochrome-C, rh-GH,

rt-PA, and arginine using C-TFF (solid circles) and HP-TFF

mixture.
All of these desirable attributes are accomplished without
the need to raise the driving pressure to a level just below the

level above which the ?ltrate ?ow is independent of the

driving pressure. These features are also achieved without
the need to dilute the mixture before ?ltration. Thus, the

FIG. 10 is a graph of retention versus TMP for a mixture

containing Cytochrome-C (squares), rh-GH (open triangles),

rt-PA (solid triangles), and arginine (open circles).

(open circles).
20

process can be carried out for the concentration level of the

DETAILED DESCRIPTION OF THE PREFERRED

EMBODIMENTS

A. De?nitions
The term species as used herein generally means par

protein at the stage of the process where the TFF step is

introduced, avoiding a deliberate dilution of the protein
concentration at that stage.

ticles or molecules that are to be separated from a solution

25

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts a graph of ?ux (If) versus TMP for

tangential-?ow ?ltration using a single membrane. On this
graph the range of 5 to 100% of transition point ?ux is
indicated, as well as the transition point (If...) and the lines

or suspension in a ?uid, e.g., a liquid. The particles or

molecules are separated from the ?uid and, in most
instances, from other particles or molecules in the ?uid. The
size of the species of interest to be separated will determine
the pore size of the membrane to be utilized. Preferably, the

species arebiological entities of natural biological or bio

chemical origin or produced by biological or biochemical
processes. Examples of preferred species include mamma
lian cells and microorganisms such as bacteria, fungi, and
yeast (both cells and microorganisms being amenable to

and curves used to determine transition point, de?ned fur~

ther below.

Figure 1B shows the transition points for single and

double membranes.
35 micro?ltration techniques), as well as species of suitable
size for ultra?ltration, including polypeptides, proteins, cel
FIG. 2A depicts a schematic diagram of one apparatus

lular components, DNA, colloids, mycoplasm, endotoxins,

useful for carrying out the high-performance tangential-?ow

viruses, carbohydrates, and other molecules of biological

?ltration process of this invention wherein two pumps are
interest, whether glycosylated or not.
employed on the ?ltrate chamber of the ?ltration unit and
The species of interest for ultra?ltration preferably are
one pump is used in the retentate compartment of the 40
biological macromolecules having a molecular weight of at
least about 1000 daltons, and most preferably polypeptides
and proteins. Also preferred is that the species of interest be
less than tenfold larger than the species from which it is to
be separated, i.e., contaminant, or be less than ten-fold
smaller than the species from which it is to be separated.
As used herein, the term tangential-?ow ?ltration refers

?ltration unit. FIG. 2B depicts the ?ltration unit of FIG. 2A

wherein two layered, parallel membranes are used in place
of one.

FIG. 3 depicts a schematic diagram of an apparatus that

can be used for a three-stage cascade tangential-?ow ?ltra
tion process.
FIG. 4A depicts a schematic diagram of an alternative
apparatus requiring only three pumps that can be used for a

to a process in which the ?uid mixture containing the

components
to be separated by ?ltration is recirculated at
three-stage cascade tangential-?ow ?ltration process. FIG.
4B depicts the ?ltration unit of FIG. 4A wherein two layered, 50 high velocities tangential to the plane of the membrane to
increase the mass-transfer coe?icient for back diffusion. In
parallel membranes are employed for each membrane shown
such ?ltrations a pressure differential is applied along the
in FIG. 4A.
length of the membrane to cause the ?uid and ?lterable
FIG. 5 depicts an HPLC chromatogram of retentate
solutes to ?ow through the ?lter. This ?ltration is suitably
samples from a run similar to that used for FIG. 6 for the

conducted as a batch process as well as a continuous-?ow

start bulk solution (30 kD bulk), l, 5, and 9 diavolumes (DV)

of ?ltration, and the end bulk solution, with the ?rst peak

process. For example, the solution may be passed repeatedly

over the membrane while that ?uid which passes through the
?lter is continually drawn off into a separate unit or the
solution is passed once over the membrane and the ?uid

indicating t-PA and the second peak indicating Cytochrome

C

FIG. 6 illustrates a graph of ?ux at 30 C. (open circles)

and Cytochrome-C retention (solid circles) versus average
TMP for separating t-PA from Cytochrome-C.
FIG. 7 depicts a graph of the calculated percent yield and
fold puri?cation versus diavolumes for conventional tangen

60

passing through the ?lter is continually processed down

stream.

As used herein, the term ultra?ltration is used for

processes employing membranes rated for retaining solutes

having a molecular weight between about 1 kDa and 1000

(solid circles for yield and solid squares for puri?cation) and

kDa.
As used herein, the term reverse osmosis refers to

for high-performance tangential-?ow ultra?ltration (HP

processes employing membranes capable of retaining sol

tial-?ow ultra?ltration (C-TFF) without a constant TMP

5,490,937
10

9
utes of a molecular weight less than 1 kDa such as salts and

B. Modes for Carrying Out the Invention

other low molecular weight solutes.

In its broadest aspect, the high-performance tangential

?ow ?ltration process contemplated herein involves passing

As used herein, the term micro?ltration refers to pro

cesses employing membranes in the 0.1 to 10 micron pore
size range.

the mixture of the species to be separated through one or

As used herein, the expression transmembrane pressure

designed for a type of tangential-?ow ?ltration under certain

or TMP refers to the pressure differential gradient that is

applied along the length of a ?ltration membrane to cause
?uid and ?lterable solutes to ?ow through the ?lter.

the pressure-dependent region of the ?ux v. TMP curve,

more ?ltration membranes in an apparatus or module

conditions of TMP and ?ux. The TMP is held at a range in

namely, at a range that is no greater than the TMP value at

As used herein, the expression substantially constant as

the transition point. Thus, the ?ltration is operated at a ?ux

ranging from about 5% up to 100% of transition point ?ux.

applied to TMP refers to a TMP that does not increase or

decrease along the length of the membrane generally by

See FIGS. 1A and 1B, wherein the ?ux v. TMP curve is

more than about 10 psi of the average TMP, and preferably

depicted along with the transition point. As a result, the

species of interest are selectively retained by the membrane
as the retentate while the smaller species pass through the

by more than about 5 psi. As to the level of the TMP

throughout the ?ltration, the TMP is held constant or is

lowered during the concentration step to retain selectivity at

higher concentrations. Thus, substantially constant TMP

time.
As used herein, the expression same pore size as

membrane as the ?ltrate, or the species of interest pass

through the membrane as the ?ltrate and the contaminants in
the mixture are retained by the membrane.
It is noted that the TMP does not increase with ?ltration

applied to the ?ltration membrane refers to membranes that

time and is not necessarily held constant throughout the

are rated or sold as having the same pore size, even though
the actual pore sizes of the membranes vary somewhat.

?ltration. The TMP may be held approximately constant

As used herein, the expression adjacent as describing

?ltration membranes means substantially adjacent in that the
membranes may be physically layered on top of one another

retained species are being concentrated, then it is preferred

refers to TMP versus membrane length, not versus ?ltration

with time or may decrease as the ?ltration progresses. If the

to decrease the TMP over the course of the concentration

25

or with a slight space between.

step.
Each membrane preferably has a pore size that retains
species with a size of up to about 10 microns, more

As used herein, the expression means for generating a

pressure gradient within the ?ltrate chamber refers to a
mechanism for creating a gradient in pressure so that the
transmembrane pressure can be operated substantially at a
constant level in the pressure-dependent region of the ?ux

preferably 1 kDa to 10 microns. Examples of species that

can be separated by ultra?ltration include proteins, polypep

versus TMP curve.

that can be separated by rnicro?ltration include mammalian

cells and microorganisms such as bacteria.

tides, colloids, mycoplasm, endotoxins, viruses, amino

acids, DNA, RNA, and carbohydrates. Examples of species

As used herein, the expression means for recirculating

?ltrate through the ?ltrate chamber parallel to the direction

Because membrane ?lters are not perfect and may have

holes that allow some intended retentate molecules to slip
through, a preferred aspect herein is to utilize more than one
membrane having the same pore size, where the membranes
are placed so as to be layered parallel to each other,
preferably one on top of the other. Preferably the number of
membranes for this purpose is two.
While the ?ux at which the pressure is maintained in the
above process suitably ranges from about 5 to 100% , the
lower the ?ux, the larger the surface area of the membrane

of the ?uid in the ?ltering chamber refers to a mechanism

or con?guration that directs a portion of the ?uid from the
?ltrate chambers to ?ow parallel to and in substantially the
same direction (allowing for some eddies in ?ow to occur)

as the ?ow of ?uid passing through the adjacent ?ltering

chamber from the inlet to the outlet of the ?ltering chamber.
Preferably, this means is a pumping means.
As used herein, the expression vessel refers to a con

tainer or tank for storing, dispensing, and/or receiving ?uid.

As used herein, the expression transition point refers to

required. Thus, to minimize membrane cost, it is preferred to

a ?xed point on a curve of ?ux versus TMP that is deter 45 operate at a pressure so that the ?ux is at the higher end of

mined as follows: Experimental data of ?ux (Jf) versus TMP

the spectrum. The preferred range is from about 50 to 100%

are collected in either a short-path-length module where the

inlet and outlet IMP are +10% of each other, or in a
full-path-length module where the same conditions are met

, and the more preferred range is about 75 to 100%, of the

transition point ?ux.

While the TMP need not be maintained substantially
constant along the membrane surface, it is preferred to

by using recirculating ?ltrate. The experimental data are ?t

to the curve de?ned by the equation:

maintain the TMP substantially constant. Such a condition is

generally achieved by creating a pressure gradient on the

?ltrate side of the membrane. Thus, the ?ltrate is recycled
through the ?ltrate compartment of the ?ltration device in

JFImuxTMP/(hTMP), (Equation 1)

where Jmax(an asymptotic value) and k are determined by

linear regression of l/Jf versus l/TMP, which yields an

55

lowing criteria: Determine the intercept of JJ=Jmax and the

tangent through the origin to the curve de?ned by equation
1 (the tangent is J]=JmmXTMP/k). A line is then drawn

the same direction and parallel to the ?ow of the mixture in

the retentate compartment of the device. The inlet and outlet

pressures of the recycled material are regulated such that the
pressure drop across the ?ltrate compartment equals the
pressure drop across the retentate compartment.

intercept of l/Jm and a slope of k/jmax. Referring to FIG.

1A, graphically the transition point is de?ned by the fol
60

Several practical means can be used to achieve this ?ltrate

through that intercept and perpendicular to a tangent on the

pressure gradient. One example is the con?guration shown

above curve. The intercept of this latter line and the curve

in FIG. 2A. Thus, the mixture to be separated enters the

device through inlet conduit 1, which communicates with a

de?nes the transition point ?ux. Mathematically, this tran

sition point (jf*) is de?ned as the real solution in the

experimental data range to the following equation:

(JmW/?E/w-P(IN-21f") . (Equation 2)

fermentor tank (not shown) if the products to be separated

65

are in a fermentation broth. It may also communicate with

a vessel (not shown) that holds a cell lysate or a supernatant

after cell harvest. The ?ow rate in conduit 1 is regulated via

5,490,937
12

11

In the con?guration shown in FIG. 2A, the membranes

retentate pumping means 3. The pump is any suitable pump

known to those skilled in the art, and the ?ow rate can be
adjusted in accordance with the nature of the ?ltration as is
known to those skilled in the art.
A pressure gauge 5 is optionally employed to measure the
inlet pressure of the ?ow from the pumping means 3. The
?uid in inlet conduit 1 enters ?ltration unit 7. This ?ltration
unit 7 contains a ?ltering chamber 70 at the top portion
thereof and a ?ltrate chamber 7b at the bottom portion.
These two compartments are divided by a ?ltration mem

brane 9. The inlet ?uid ?ows in a direction parallel to

will need to be placed with respect to chambers 7a and 7b '

to provide the indicated ?ow rates and pressure differences
across the membrane. The membranes useful in the process
of this invention are generally in the form of ?at sheets,

rolled-up sheets, cylinders, concentric cylinders, ducts of

various cross-section and other con?gurations, assembled

10

?ltration membrane 9 within ?ltering chamber 7a. The

upper, ?ltering chamber 7a receives the mixture containing
the species of interest. Smaller-sized species pass through

singly or in groups, and connected in series or in parallel

within the ?ltration unit. The apparatus generally is con
structed so that the ?ltering and ?ltrate chambers run the

length of the membrane.

Suitable membranes are those that separate the desired

species from undesirable species in the mixture without

substantial clogging problems and at a rate su?icient for
continuous operation of the system. Examples are described

the membrane 9 into the lower chamber 7b. The concen

trated retentate passes from the ?ltration unit 7 via outlet

by Gabler, supra. They are typically synthetic membranes of

conduit 11, where it may be collected and processed further,

if necessary, to obtain the desired species of interest. Alter

either the microporous type or the ultra?ltration type. A

microporous membrane has pore sizes typically from 0.1 to

natively, the retentate stream is circulated back to a tank or

10 micrometers, and can be made so that it retains all

ferrnentor from whence the mixture originated, to be

particles larger than the rated size. Ultra?ltration membranes

recycled through inlet conduit 1 for further puri?cation.

20

The solution containing molecules that pass through the

membrane 9 into the ?ltrate chamber 7b leaves the ?ltration

have smaller pores and are characterized by the size of the

protein that will be retained. They are available in incre
ments from 1000 to 1,000,000 Dalton nominal molecular

weight limits.

unit 7 via outlet conduit 13 at the same end of the ?ltration

unit as the retentate ?uid exits via outlet conduit 11. A

Ultra?ltration membranes are most commonly suitable

for
use in the process of this invention. Ultra?ltration
25
membranes are normally asymmetrical with a thin ?lm or
discarded or sent to a second tank or vessel for further
skin on the upstream surface that is responsible for their
processing in a cascade mode of ?ltration through a mem

portion of this ?ltrate leaves the system via conduit 13 to be

separating power. They are commonly made of regenerated

brane of smaller pore size. Optionally, a ?ltrate pumping
cellulose or polysulfone.
means 15 is disposed within conduit 13 for this purpose.
Membrane ?lters for tangential-?ow ?ltration are avail
Another portion is recycled via conduit 17 to a ?ltrate
pumping means 19, which generates the ?ltrate pressure 30 able as units of different con?gurations depending on the
volumes of liquid to be handled, and in a variety of pore
gradient. This portion of the liquid is recycled via inlet
conduit 21 to the inlet end of the ?ltrate chamber 7b such
sizes. Particularly suitable for use in the present invention,
that a ?ltrate ?ow occurs in ?ltrate chamber 7b parallel to,
on a relatively large scale, are those known, commercially
available tangential-?ow ?ltration units.
and in the same direction as, the mixture being separated in
35
In an alternative and preferred apparatus, and for the
?ltering chamber 7a.
reasons presented above, the ?ltration unit 7 of FIG. 2A
The operating rate of the pumping means 19 is adjusted so
that the pressure at which the ?ltrate is introduced into
comprises multiple, preferably two, ?ltration membranes,
shown in FIG. 2B as membranes 9a and 9b, respectively.
chamber 7b (?ltrate inlet pressure) and the pressure at which
These membranes are layered in a parallel con?guration.
the ?ltrate is removed via outlet conduit 13 (?ltrate outlet
The invention also contemplates a multi-stage cascade
pressure) can be controlled to provide substantially constant 40
process wherein the ?ltrate from the above process is passed
TMP along the length of the membrane. The preferred
through a ?ltration membrane having a smaller pore size
procedure is to adjust the difference in pressure across
than the membrane of the ?rst apparatus in a second tan
chamber 7b to equal the pressure drop across chamber 7a of
gential-?ow ?ltration apparatus, the ?ltrate from this second
unit 7 to make the TMP about equal across the entire
membrane. Pressure sensing means 23a and 23b can be 45 ?ltration is recycled back to the ?rst apparatus, and the
employed at the inlet and outlet, respectively, of the recycle
?ow loop of ?ltrate through ?ltrate chamber 7b to monitor
the pressure drop and adjust it as necessary. Pressure sensing
means 23c is optionally employed in outlet conduit 11 to
monitor the pressure drop for the ?ltering chamber 7a. The
?ltrate chamber 7b may include a restrictive ?ow path to

yield an adequate pressure gradient while minimizing the

process is repeated.
In this cascade process, the second ?ltration typically is a
conventional ?ltration, wherein the ?ux is held at a level
50

greater than about 100% of transition point ?ux; also,

generally the 'IMP is not held substantially constant along
the membrane.
In a more preferred embodiment of the cascade process,

?ltrate circulation rate. The result of this con?guration is a

both stages involve tangential-?ow ultra?ltration, wherein
the pore size is from 1 to 1000 kDa.
substantially constant TMP across the entire surface of the
In a three-stage cascade process the ?ltrate from the
membrane.
55
second ?ltration is passed through a ?ltration membrane
The ?ltration unit useful herein is suitably any unit now
having a pore size that is less than that of the second
known or discovered in the future that serves as an appro
membrane in a third tangential-?ow ?ltration apparatus, the
priate ?ltration module, particularly for rnicro?ltration arid
?ltrate from this third ?ltration is recycled back to the ?rst
ultra?ltration. The preferred ?ltration unit is hollow ?bers or
a ?at sheet device. These sandwiched ?ltration units can be
?ltration apparatus, and the process is repeated. If only two
high-performance ?ltrations are needed in the cascade pro
stacked to form a composite cell. One preferred type of

rectangular ?ltration plate type cell is available from Filtron

cess, then the third stage is conventional, i.e., the ?ux is held
at a level greater than about 100% of transition point ?ux in
trade name Centrasette.
this third stage.
In a more preferred embodiment of this three-stage cas
Another suitable ?ltration unit is the Millipore Pellicon 65
cade process, all three stages involve tangential-?ow ultra
ultra?ltration system available from Millipore, Bedford,
?ltration.
Mass.

Technology Corporation, Northborough, Mass, under the

5,490,937
13

14

One tangential-?ow apparatus suitable for conducting the

second ?ltering/?ltrate chamber 95c. The second ?ltering/

cascade process is shown in FIG. 3. Here, a ?rst vessel 31

is connected via inlet conduit 33 to a ?ltering chamber 35a

membrane 43. The ?rst ?ltrate chamber 35b has an outlet

conduit 47 connected to the inlet of chamber 35b with a

?ltrate chamber 95c is connected via an outlet conduit 121

to the third vessel 115. The second ?ltering/?ltrate chamber
95c is separated from a ?ltrate chamber 95d situated directly
below it within ?ltration unit 97 by a third ?ltration mem
brane 123.
The ?ltrate chamber 95d is connected via outlet conduit
125 to the ?rst vessel 91, to allow the ?ltrate to recirculate
to the original vessel. The ?ow of ?uid from the inlets to the
outlets of all chambers is parallel and in the same direction.
Moreover, the ?rst ?ltration membrane 103 has a pore size

?ltrate pumping means 49 disposed in the conduit 47.

Conduit 45, which is connected to outlet conduit 47, is

that retains species of a larger size than that of the second

and third ?ltration membranes 113 and 123, respectively,

disposed within a ?ltration unit 37. A ?rst input pumping

means 39 is disposed between the ?rst vessel 31 and ?ltering
chamber 35a. The ?ltering chamber 35a is connected via an
outlet conduit 41 to the ?rst vessel 31. The ?ltering chamber
35a is separated from a ?rst ?ltrate chamber 35b situated
directly below it within ?ltration unit 37 by a ?rst ?ltration

connected also to second vessel 51.

and the second ?ltration membrane 113 has a pore size that
This vessel 51 is connected via inlet conduit 53 to a 15 retains species of a larger size than the third ?ltration
second ?ltering chamber 55a disposed within a second
membrane 123.

?ltration unit 57. A second input pumping means 59 is

disposed between the second vessel 51 and ?ltering chamber
55a. The ?ltering chamber 55a is separated from the second
?ltrate chamber 55b situated directly below it within ?ltra
tion unit 57 by a second ?ltration membrane 63. The second

The cascade apparatus of FIG. 4A allows for high

perforrnance tangential-?ow multiple ?ltrations having all

the advantages therein and requiring only three pumps and
20

devices that would otherwise be required for the high

performance tangential-?ow ?ltration cascade system

?ltrate chamber 55b has an outlet conduit 67 connected to

the inlet of chamber 55b with a ?ltrate pumping means 69

disposed in the conduit 67. Conduit 65, which is connected

to outlet conduit 67, is connected also to third vessel 71.

one device rather than the ?ve or six pumps and three

shown in FIG. 3.

Optionally, the apparatus of FIG. 4A is con?gured so that

25

each ?ltration membrane constitutes a plurality of mem

This vessel 71 is connected via inlet conduit 73 to a third

branes, preferably two, layered parallel and on top of the

?ltering chamber 75a disposed within a third ?ltration unit

57. A third input pumping means 79 is disposed between the
third vessel 71 and ?ltering chamber 75a. The ?ltering
chamber 75a is separated from the third ?ltrate chamber 75b
situated directly below it within ?ltration unit 77 by a third
?ltration membrane 83. The third ?ltrate chamber 75b has an
outlet conduit 87 connected to conduit 85, which is con

other, within ?ltration unit 97, as shown in FIG. 4B (mem

brane 103b for ?rst membrane 103a, membrane 113b for
second membrane 113a, and membrane 123b for third
membrane 123a ).
The cascade apparati shown in FIGS. 3 and 4 are such that
the ?rst membrane 43 or 103 has a pore size with a cut-o?

greater than that of the second membrane 63 or 113, respec

nected to ?rst vessel 31, to allow the ?ltrate to recirculate to

tively, which in turn has a pore size with a cut-off greater

the original tank.
35 than that of the third membrane 83 or 123, respectively.
Optionally, the outlet conduit 87 of the third ?ltrate
Most preferably, these membranes are all ultra?ltration
chamber 75b is connected to the inlet of chamber 75b with
membranes.
a ?ltrate pumping means 89 disposed in the conduit 87. T h
The apparati of FIGS. 3 and 4A are designed to conduct
e ?ow of ?uid from the inlets to the outlets of all chambers
a three-stage separation employing three ?ltration mem
branes. The invention herein also contemplates devices
is parallel and in the same direction. Moreover, the ?rst
wherein only two ?ltration membranes and two vessels are
?ltration membrane 43 has a pore size that retains species of
a larger size than that of the second and third ?ltration
involved, so that, for example, in FIG. 4A the second
membranes 63 and 83, respectively, and the second ?ltration
?ltrating/?ltrate chamber 95c is a ?ltrate chamber with an
membrane 63 has a pore size that retains species of a larger
outlet conduit, and elements 95d, 121, 123, 115, 117, and
45 119 are not present. In other variations, the apparati of FIGS.
size than the third ?ltration membrane 83.
A second tangential'?ow apparatus suitable for conduct
3 and 4A contain more than three ?ltration membranes and
ing the cascade process is shown in FIG. 4A. Here, a ?rst
more than three vessels, so that, for example, the second
vessel 91 is connected via inlet conduit 93 to a ?ltering
?ltrating/?ltrate chamber 95c of FIG. 4A is connected via a
chamber 95a disposed within a ?ltration unit 97. A ?rst input
fourth ?ltration membrane to a third ?ltrating/?ltrate cham
pumping means 99 is disposed between the ?rst vessel 91
ber equipped with an inlet and outlet conduit in ?uid
and ?ltering chamber 95a. The ?ltering chamber 95a is
communication with a fourth vessel, and so on, with the last
connected via an outlet conduit 101 to the ?rst vessel 91. The
or bottommost chamber being a ?ltrate chamber like that of
?ltering chamber 95a is separated from a ?rst ?ltering!
95d in ?uid communication via conduit 125 with the ?rst
?ltrate chamber 95b situated directly below it within ?ltra
vessel 91, as illustrated in FIG. 4A.
55
tion unit 97 by a ?rst ?ltration membrane 103.
The process of the present invention is well adapted for
A second vessel 105 is connected via inlet conduit 107 to
use on a commercial and semi-commercial scale. It can be

the ?rst ?ltering/?ltrate chamber 95b. A second input pump

ing means 109 is disposed between the second vessel 105
and ?rst ?ltering/?ltrate chamber 95b. The ?rst ?ltering/

run in batch or continuous operations, or in a semi-continu

ous manner, e.g., on a continuous-?ow basis of solution

?ltrate chamber 95b is connected via an outlet conduit 111

to the second vessel 105. The ?rst ?ltering/?ltrate chamber

until an entire large batch has thus been ?ltered, with

95b is separated from a second ?ltering/?ltrate chamber 950

situated directly below it within ?ltration unit 97 by a second
?ltration membrane 113.
A third vessel 115 is connected via inlet conduit 117 to the

fresh batches of solution can be treated. In this way, a

second ?ltering/?ltrate chamber 95c. A third input pumping

means 119 is disposed between the third vessel 115 and the

containing the desired species, past a tangential-?ow ?lter,

washing steps interposed between the ?ltration stages. Then
continuous cycle process can be conducted to give large
yields of desired product, in acceptably pure form, over
65

relatively short periods of time.

The unique feature of tangential-?ow ?ltration as
described herein with its ability to provide continuous