CA1293928C - Thermofiltration of plasma - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A method for more selectively removing macromolecules from a plasma solution, whereby plasma containing the macromolecules to be removed is provided and heated to a temperature near or above normal body temperature but below the boiling point of the plasma solution. The heated plasma solution is filtered while at a temperature near or above normal body temperature but below its boiling point with a membrane filter to remove selectively macromolecules from the plasma solution. An apparatus for accomplishing the foregoing is also provided.

Description

~ llL.ll~lION O~ YLAS~IA

DACKGROlJN~ OF TIIE INVbN~IuN

The present invelltion relates to the filtration of macromolecules from fluids, and more particularly to ~he removal of undesirable macronnolecules from plasr~a solutions, by ehe process termed thermoEiltration.
The separation of undesirable solutes from blood plasma through plasma filtration is a known method of treating diseases, wherein such diseases have in common undesirable elevated leYels of plasma solutes, such as toxins, excessive antibodies, and other metabolic factors. Successful treatment o such diseases involves the renloval of the undesirable plasnla solutes from the blood plasma by membrane filtration.
Various methods of plasma filtration, including cascade filtration, double filtration, arld cryofiltration have been developed. However, these methods contain a number of ulldesirable characteristics which lioli~ their usage.
Applicants have Iloted a nuolber of parameters associated with performance, including module design, membrane properties, plaslna composition, and plasma and filtration t~mperature. Characteristics o~ the module which affect flow dynamics and in turn performance include, area, fluid and film dimensions, as well as properties of the separatin~ menlbrane, including polynlcr type and microstructural features such as pore size, pore tortuosity, pore len~th, and pore number.
Variations in the plasma's composition also affects its filtration. Plasllla frool patients with ;9a~

diferent disease stutes or with differin~ macromolecule contents have di~ferent filtrati~n characteristics.
Manipula~ion uf the plasloa to effect changes in pH or elee~rl)lyte composition and the addition of anticoagulants such as hep~rin or other macromolecule-aggre~ating additives such as polyethylene glycol will effec~ filtraLion performance. Cenerally such manipulations are carried out for the purpose of augmenting the separation, by macromolecule a~gregation or precipitation, of one or a ~roup of solutes from the plasma .
Because of the number of paral~eters afecting filtration performance, temperature selection and its control has been demonstrated to be a key parameter in fluid separation. In order to au~ment the selective removal in a particular macromolecular range, it is extrelnely important to operate within the proper temperature range. In this respect, significant differences have been noted for comparable conditions of filtration (similar oper~ting flows, modular types a~d plasma types) between cascade and double filtration, which operate at near ambient temperatures, and cryofiltration, which operates at temperatures below a set physiologic temperature.
Temperature control offers many advantages over the other parameters in that temperature control is the easiest controlled physical parameter, and that temperature control Inay be co~lbined with the use of various complexing a~ents to increase the sensitivity of macromolecule removal. A specific exalnple of this can be shown in the case of cryofiltration, where the addltion o~ heparin aids irl the formation of cryogel by forming complexes wi~h fibronectirl and fibrino~en at temperatures below 25C.

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Filtratior1 at sub physiologic telnperaturt- is effective for the removal of plasma constitue~nts silllilar in size but diÇfering in temperature sensitivity. A
number of autoimmune diseases can be treated in this ~ashion, as has been described in the literatureO The effectiYeness of the treatmen~ is attributed to the formation and removal of cryogel, which is composed of high concentrations of the macromolecules associated with autoimmune disease sta~es. Thus, the separation in cryofiltraLion is not based on molecular size a~
physiologic temperature but on molecular size at reduced temperatures.
Ilowever, opera~ion at a reduced tempf~ra~ure can, in fact, reduce the selectivity of molecule separation when size differences are great since aggregation or complex formation of small molecules may also occur at reduced temperatures. Therefore, for separation based on size diferences at physiologic temperatures, i~ may be more advantageo-ls to avoid cryogel formation.
Accor~ingly, it is arl object of the present invention to provide an improved means of removing undesirable macromolecules from fluids in an effective and efficient manner.

SU~1hlARY OP THE INVENTION
~ . ._ In one aspect, the present invention relates to a method of selectively removing macronlolecules from a plasma solution including the steps of providing a plasma solution containing the macromolecules to be removed, heating and/or maintaining the plasma at a temperature near or above normal body temperature but below the boilin~ point oL the plasrllu solution, and filtering the warmed plasma solution while at a temperature near or above normal body tenlperature but below the boiling point with a membrane filter to remove more selectively rnacromoleeules from the plasma solution. In addition, by heating the plasma solution, various macromolecules present within the plasma solution may ~ecome inactivate~ or denatured, aiding in their selective removal throu~h plasma filtration.
In another aspect, the present invention concerns a method of selectively removing macromolecules from a plasrna solution including the steps o~ securing a blood flow from a specimen, separating the blood flow into a concentrated cellular element strealn and a plasma stream containing the macromolecules to be removed, heating or maintaining the plasma stream containing macromolecules to be rernoved to a temperature near or above normal body temperature but below its boiling point, filtering the warmed plasma stream while at a temperature near or above normal body temperature but below its boiling point with a Inembrane filter to remove more selectively macromolecules from the plasma solution to form a filtered plasma stream, combining the filtered plasma stream and said cellular element stream to form a processed plasma stream, and cooling and/or returning the processed stream plasma to the specimen.
In still another aspect, the pIesent invention concerns a l~ethod of controlling conditions o~
lipoproteirl abnormalities in a living organism by selectively removing nlacrolrlolecules from a plasma solution including the step of securing a blood flow from a living organism, separating the blood flow into a concentrated sellular element stream and a plasma stream 1;~93g~
- s-containing ~he macrolllolLcules to be relnoved, heating or maintaining the plasma strealn containing macromolecules to be removed to a ~emperature near or above normal body temperature but below its boiling point, filtering the heated plasma streanl while at a temperature near or above normal body temperature but below its boiling point with a membrane filter to remove more selectively macromolecules from the plasma solution to form a filtered plasma stream, sombinin~ the filtered plasma stream and said cellular element strea~ to for~ a processed plasma stream, and cooling and/or returning the processed plas~a stream to the living organism.
In still another aspect, the present invention concerns an apparatus for relnoving nlore selectively macromolecules from a plasma solution co~prising a means of receiving and dividing a plasma containing solution containing macromolecules procured from a speciQen into a concentrated cellular element stream and a plasma stream, a means of receiving, heatin~ and/or maintaining the plasma strea~ to a temperature near or above normal body temperature but below its boiling point, a means of receiving and filtering the heated plasma stream to remove selective macromolecules, a means for receiving the filtered plasma strea~ from the filter ~eans and for receiving the concentrated cellular element stream and combining the streams to form a processed strea~
substantially free of the macromolecules intended to be remove and a means of receiving and/or cooling the combination stream to normal body temperature for returning said fluid to the specimen~

3~2~3 BRIF~ DESCRIPTION OF THE DRAWlNGS

The following is a brief description of the drawings which are pr~sented for the purpose of illustrating the invention and not for the purpose of limiting same.
Figure 1 shows the circuit used in in vitro filtration;
Figure 2 shows the extracorporeal circuit used in ex vivo or clinical filtration;
Figure 3 shows the amount of cholesterol removed at varying temperatures for in vitro filtration;
Figure 4 shows the mean sieving coefficients with various kinds of plasma. ~Kuraray Eval 4A module;
In vitro at 37C);
Figure 5 shows the particle size distribution of the lipoprotein for the FHC plasma and the dog plasma with various cholesterol levels; and Figure 6 shows the pos~ treatment recovery in a dog of LDL-VLDL and HDL cholesterol.

DETAILED DESCRIPTION OF THE INVENTION

Applicant has discovered that thermofiltration offers many advantages over the conventionally known methods of plasma filtration. Thermofiltration is the removal of macromolecules from plasma by warming the plasma to a selective temperature near or above the normal pllysiologic temperature but not above its boiling point and filtering the warmed plasma with a membrane filter having a porosity that will remove the ~~3~

desired macromolecules. The critical a~lvantages demonstrate~ by thermoEiltration include the abili~y to filter a mucll greater volume of plasma at higher tempera~ures because plasma exposed to the higher temperature has less of a tendency to form deposits o undesirable solutes on the membrane media and the more selective removal of macromolecules based upon differences in sieving coefficients at higher temperatures.
Evaluations of multiple membrane filters or plasma filtration based on filter material, pore size, ~` and structure indica~e that the Kuraray Eva ~4A module (ethylene and vinyl alcohol copolymer, Kuraray Co., Japan) and other modules of similar properties are particularly well suited for plasma solute fractionation by thermofiltration. Other suitable filters include those which utilize filter media consisting of polysulfone, polypropylene, nylon, polyester, cellulose acetate~ collagen and the like~
Applicants have demonstrated that sieving coefficients of some macromolecules are significantly higher at 37C and 42C than at 25C Eor the Kuraray Eval 4A module. (Table I below). Particularly noteworthy are the higher sieving of HDL cholesterol, IgG, ~ibringen, total protein, and albumin at 37 to 42C. In addition, as a result of the reduction of cryogel formation at these higher temperatures, a much greater volume of plasma can be filtered. This is because at near or above normal physiologic temperatures solute aggregatiorl is kept to a minimulll, and separation is due to the molecular si~e differences of the solutes and not the aggregate compositions.

rra~e mar~

Table I: Sieving coefficents for various macramolecules.

_ . , . . _ .
Volume TP Alb Glb ~ib I~G IgA IgM PIocessed (ml) 25C 0.61 0.71 0,48 <0.06 0.36 0.21 0.15 1,000 37C 0.74 ~.82 0.59 0.13 0.55 0.51 0.17 1,000~3,000 42C 0.~ 0.86 0.59 0.26 0.57 0.46 0.18 1,000~3,000 Volume T Chol LDL HDL TG Processed (ml) 25C 0.07 0.03 0.71 0.13 <11000 37UC 0.06 0.02 0.84 ~.11 1,000~3,000 42C 0.04 0.03 0.97 0.13 1,000~3,000 All macromolecules are from the same plasma source. ~eparin dosage: 1,000 U/L.
TP: total protein; Alb: albumin; Glb: globulin; Fib:
fibrinogen; T Chol: total cholesteroli LDL: low density lipoprotein; HDL: high density lipoprotein; TG: triglycerides.

Moreover, thermofiltration is an effective method of removing pyroglobulins from plasma solutions.
Pyroglobulins are serum globulins that precipitate or gel upon heating. Normally, pyroglobulins are not found in serum of norlnal individuals. Rather they are readily associated with macroglobulinemia and other lymphoproliferative or multiple myeloma disorders.

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g HeatinB a serum col~taining pyroglobulins to 55-56C
results in gel forma~ioll, wllicll can be cf~ectively removed froln the serum through plasma ~iltration.
Similarly, proteins and other immunogloblJlins which ef~ectively denature or coagula~e upon heating may also be selectively removed throu~h ther~ofiltration.
As a consequence of the above advan~ages of thermofiltration over conventionally known methods of plasma separation, ~hermofiltration can be used to selectively remove pathologic macromolecules from blood in on-line and off-line plasma treatments, while at the same time allowing for the passage or return of beneficial plasma pro~eins. The advantage of this type of treatment can be clearly shown in the therapeutic control of cholesterol.
Cholesterol has been determined to be an impor~ant component of arterial plague formation in artherosclerosis as well as in hypercholosterole~ia.
Cholesterol circulates in the blood liJIked to large protein molecules. One form of cholesterol carrying protein, called low-density lipoprotein (LDL~ 9 is known to promote atherosclerosis. About two-thirds or more of total blood cholesterol is transported in LDL. Another orm, called high-density lipoproteiil (HDL), is known to be protective against the disease process. Therefore, the selective removal of LDI. and maintenance of HDL is important in the treatment of atherosclerosis and the therapeutic control of hypercholesterolemia.
Recently, plasma exchange has been utillzed for the removal of plasma and its replacement with electrolyte solutions and/or plasma products in familial hypercholesterolemia patients. However, these methods ~LZ93~

are non-selective and remov~ proportionately low density lipoproteins (I.DI.) with high-density lipoproteins (IIDL) and other plasma proteins which are beneficial to the patient. In addition, several methods have been studied for the selective remo~al oE LDL, including anti-LDL-antibody Sepharose columns, and combinations of heparin precipitation and bicarbonate dialysis, however, meIDbrane filtration offers many advantages over these methods in terms of biocompatibility and treat~ent cost effectiveness.
The selective removal of LDL cholesterol from plasma by thermofiltration can be demonstrated under both in vitro and ex vivo conditions. In vitro pertains to conditions of experim~ntation i~ a laboratory setting, whereas ex vivo pertains to conditions of extracorporeal circulation with living organisms.

I. In Vitro ~:iltration . .
Various types of plasma are used in vitro to evaluate the temperature effect on selective cholesterol removal. The in vitro filtration tes~s are carried out with the various types of plasma at varying temperatures according to the extracorporeal circuit demonstrated in Figure 1.
In Figure 1, oIle unit per ml. heparin (heparin sodium injection, Invenex Lab., OH) is added to plasma pool 10 wherein the plasma is kept at approximately 37C
by h~at controller 12 and uIa~netic stirrer 14. Plasma is drawn from plasma pool 10 into line 16 and fed into plasma pump 18 of a plasma flow rate of l5ml/min. The plasma is pumped from plasma punIp 18 into line 20 and then into water bath 22 which is controlled by thermo-re~ulater 24. Within water bath 22, the plasma TrAde ~a~

~33~

passes throlJgh h~at exc~langer 26 an(l passes by pressure gauge 28 into ~ilter 30, where the l.PI. ctlolesterol is retained and the ~II)L cholesterol and albumin substantially pass through. From filter 30, the filtered plasma minus LDL cholesterol flows through line 32 into filtrate collection pan 34.
The following specific examples further illustra~es the practice of the present invention.

EXAMPLE I
Familial type II hypercholes~erolemic plasma (FHC) was procured by repeated centrifugal plasma exchange. The in vitro filtration tests were carried out with the PHC plasma and the Kuraray EVAL 4A membrane filter ae temperatures of 4, 25~ 37, 42 and 47C, respectively, according to the extracorporeal circuit demonstrated in Figure 1 and as described above. The mean sieving coefficients (sc~, the plasma volume processed, and the ~otal amount of LDL and llDL
cholesterol were determined at the above temperatures by the following calculations.
Concentration in ~iltrate sieving coefficient (sc) - (mg/dl) Concentration in filter inlet ~mg/dl~

wherein a sieving coefficient oE
0.9 to 1 indicates little or no separation or removal of the macromolecule from plasma, and a sieving coefficient of O to 0.1 indicstes substantially total renloval of macromolecules from plasma.

~3~3~3 Removal Alnount (g) ~ Concentration in plasma pool (g/~l) X(l-sc) x Processe~ Volume (~1) RL.SUL'!'S
Table Il outlines the volume processe~ and the mean sieving coefficients for total cholesterol, 5IDL
cholesterol, LDL cholesterol, and albumin at varyin~
temperatures. l`he results ~ndicated that greater than 85~ of the total cholesterol and 90~ of LDL cholesterol were removed while oYer 70~ of albuolin and 6a~ of HDL
cholesterol were passed throu~h ~he filter. The sieving coefficients of tlDL cholesterol and albumin increased with increasing temperature while LDL choles~erol was independent of temperature.
ABL~ ~I. Mean sieving coefficients and plasma volumes processed at varying temperatures; in vitro filtration test of ~uraray ~val 4A (1.0 M
surface area) using fanlilial hypercholestero-l~mic plasma.

Processed M n Sieving Coefficients Temp Volume l'otal H~L LDL
(C) (ml) Chol Chol Chol Alb _ . . .
4 365 0.10 0.5~ O.U7 0.72 1135 0.11 0.76 0.10 0.84 37 1780 0.12 0.~7 0.~6 0.81 42 2150 0.16 0.72 0.08 0.91 ~7 2350 0.14 0.79 0.0~ 0.9q ~.;2g3~

Figure 3 denlonstrates that cholesterol removal differs at varying temperatures. A~ 37 to 42C, the removed LnL cholesterol amollnt is largest (4.5~ module), while IIDL cholesterol is below O.lg. Removal amount per module was limited by the maxinluln Pt~ 0~ mln tlg) permitted.

Conclusion: Th~rnlofiltration is highly effective in selectively removing large quantities of LDL cholesterol from plasma while retaining lar~e quantities of useful HDL cholesterol and albumin undér in vitro conditions. The in vitro membrane filtration of FtlC plasma with the EVAL 4A filter permits near complete rejection of LDL cholesterol with high sieving or retainment of HDL cholesterol and albumin. The sieving coefficients of HDL cholesterol ~nd albumin increased with increasing temperature, while the sieving coefficient of LDL cholesterol was near comple~e rejection at all temperatures. Thus, membrane filtration near or above physiologic temperatures, i.e., thermofiltration, improves the selectivity of LDL
cholesterol removal over that of HDL cholesterol and albumin.
Moreover, thermo~iltration also permits higher plaslna volumes to be processed. This is a result of the reduction of cryogel formation and less removal of solutes not intended to be removed at the elevated temperatures. Also, higher volumes of plasma are processed and lar~er quantitics of cholesterol are removed per unit module.

IX~MPI~

In vitro module ~iltration ~ests were carricd out wi~h normal human plasma (NHP) and sclerosin~
cholangitis plasma (SCP) usin~ the Eval 4A (copolymer of ethylene and vinyl alcohol; surface area 2~0 ~2) at 4, 25, 37, 42, 47 and 52~C, respectively, according to the extracorporeal circuit demonstrated in Figure 1 and as described above. The NHP was procured by filtration at 37C of outdated citrated plasma using the Toray PS-05 plasma separator (polymethylmethacrylate;
surface area O.5m2; Toray Industries, Japan). The SCY
was procured by membrane plasma exchange. The SCP
differed from the NH~ in that the SCP had 1.5 fold higher fibrinogen and four fold higher LDL cilolesterol concentrations with similar levels of albumin and sntithrombin III when compared to NHP.
All filtration tests were carried out with a plasola flow of 30 ml/min. Changes in inlet pressure were monitored as a function of the transmembrane pressure and reflect membrane plugging.
Samples obtained pre and post filtra~ion were analyzed for various biochemical solutes including albu~in (Alb), fibrinogen (Fib), total cholesterol (T
Chol), LDL cholesterol (LDL Chol), HD~ Cholesterol (HDL
Chol), antithrombin III (AT III) and heparin. Alb was measured with an autoanalyzer (SMA-II, Tech~icon Instrument Co., Tarrytown, NY) by the bromocresol green method. Fib was measured by the Fibrosystem (B~L, Ockeysville, MD). T chol and triglycerides were measured with an autoanalyzer (AA II, Technicon Inst.
Co.) using the cholesterol oxidase-peroxidase enzymatic - l s-method. L~L chol was culculated as: 1' chol - ~IDL chol -1/5 triglycerides. II~L chol was oleasLIIe(l by th~
dextran-sll1fato-Mg2~ precipitation metilo~l.
Antithrombin III and heparin were measured by the Protopath antithrombin III and heparin synthetic substrate assay (American Dade, Miami, FL).

~SULTS
_ Pigures 4A and 4B show the volumes processed and the sieving coefficisllts for the filtration of NHP
and SCP over the temperature range of 4 to 52C, In both plasmas hi8her volumes were processed as the temperature was increased from 4 to 52C. ~owever, the volume processed did not increase at temperatures over 42C and dropped significantly at 52C. Sievings for Alb ~ncreased with increasing temperature from 4 to 42C and thereafter also dropped. A similar tendency can be seen in ~ibrinogen. Significant increases of IIDL
chol sieving were observed in the temperature range of 4 to 42C where no major changes in LDL chol were noted. HDL chol sieving also dropped at 52C. Total removal of LDL chol and removal raeios of HDL chol and Alb to LDL chol were liste(l in Table III. Maximum removal amounts of LDI. chol and minimal removal of HDL
chol an~ Alb versus LDL chol removal were obtained at 42C, 3~;~8 TABLE III: Total removal an~ ratios of ~IDI./LDL and albumin/LDL choles~erol removal using NHP an(l SCP
patients plasma at varying temperatures.

Plasma Temp ~ 4 25 37 42 47 52 NHP LDL Chol (mg) 130 347 932 1854 1549 198 .. _ HDL chol 0.95 0.76 0,45 0.190.23 0.48 LDL chol Alb 31.1 18.2 10 5 5.66.5 14~1 _ _ ~ __ .
LDL chol . . _ . . . _ SCP LDL chol (mg) 887 1458 2368 3130 2790 1216 _ _ _ _ _ _ _ _ _ HDL chol 0.0160.021 0.008 0.001 0.007O.OS
LDL ohol Alb 6.2 5.6 ~,~ 2.2 3.24.8 LDL chol . . . HP - normal llulllan plasma SCP ~ sclerosing chol~ngitis plasma Conclusion: These results suggest that operation at near physiologic tempeIature is promising for prevention ~35~

of heparin induced aggregation which occurs below physiologic temperature and filter p~ugging caused by these deposits when smaller pore size membranes are used to separate molecules. As shown in Figure ~A and 4B, filtration above physiologic temperature ~up to 47C) produces higher volumes processed and a higher passage of albumin and }IDL chol. These results indicate tha~
plasma filtration near or above 37C, thermofiltration, is promising for clinical use in the separation of plasma solutes based on size differences (i.e., LDL
selective separation vs. HDL sieving).

II. Ex Vivo Filtration Ex vivo filtration is the continuous on-line filtering of plasma with living organisms. The ex vivo filtration tests were carried out at 37C according to the extracorporeal circuit demonstrated in Figure 2.
Referring to Figure 2, blood is drawn from the artery of a living organism illtO line 36 and fed into a pump 38 from which it is pumped into a line 40, passing by pressure gauge 42 and into plasma separator 44, where the plasma and blood cellular elements are separated.
The concentrated blood cellular elements are fed into line 46, while the plasma is fed into line 48. From line 48, the plasma flows through pump S0 to line 52 where it enters water bath 54 controlled by thermo-regulator 56. Within water bath 54, the plasma passes through heat exchanger 58 and by pressure gauge 60 into filter 62, where the LDL cholesterol is subs~antially retained and the HDL cholesterol, albumill, and other low molecular weight macromolecules subs~antially pass through.

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Prom ~ilteI 62, the ~iltere~! plaslna which is essentially deficient in LDL cholesterol flows through line 64 an~ is in~ermixe(l witll the blood cellular elements of line 46. The mixture tllen is either cooled to body temperature in heat exchanger 66, or passed into line 68 where it passes pressure gauge 70 and is fed into line 72 and returned to the vein of a living organism in a continuous process.
Ihe following specific examples further illustrate the practice of the present invention.

Example 3 As a result of the si~ilarity of lipoproteins and its suitable body size for on-line filtration, a hypercholestrol dog was the model chosen for ex vivo filtration. The thyroidectomized/diet canine model is well established and has been studied extensively.
Using this model, three different choles~erol level ran~es up to 600mg/dl (normal, 120 mg/dl) were evaluated.
Arterio-venous (AV) fistulae were constructed in two healthy male mongrel dogs weighing from 24 to 30 kg. As a control, dogs were maintained with a normal diet (lab Canine Diet 5006; Lab Chow, St. Louis, M0).
As a middle cholesterol concentration model, the same dog was maintàined with the same diet a~ter surgical thyroidectomy. As a high cholesterol concentration model, thè dog was maintained with a special diet that consisted of the normàl meal with 4% hydrogenated coconut oil and 0.75% cholic acid added (TD 75337, Taklad, Madison, WI) after thyroidectomy.
Ex vivo filtration tests were carried out in the dogs at different cholesterol levels under general 12~392B

_19_ anesthesia with nitrous oxide gas and nelnbutal injection ~Nembutal sodium sollltion, Abbott Lab, I~.) An AV
fistula was used for blood access and Z00 unit/kg heparin was injected as an anticoagulant. Plasma was separate~ in the on^line extracorporeal system using a membrane plasma separa~or ~Mitsubishi 60TW, polyethelene, Mitsubishi Rayon Co , Japan). The separated plasma was filtered using the same method as in vitro filtration (at 37C) and the filtered plasma was then recombined and returned to the dog (Fig. 3).
Blood and plasma flows employed were 60 and 15 ml/min, respectively. One calculated plasma volume was filtered. ~ight hundred to 1000 ml of lactated Ringer's solution was infused intr~venously during the extracorporeal circulation.
Samples were drawn simultaneously from the arterial and plasma lines of the filter inlet and outlet when one half and one volumc were processed, and pre-and post-treatment. At one hour and 1, 3, 7, 14 and 21 days post treat~ent, bloo~ samples were taken following 14 eo 16 hours fasting. Dogs were fed the same diet as before filtration ad libitum. Biochemical measurements included cholesterol and tri~lyceride (automatic analysis AA II, Technicon Instrument Co., NY), HDL
cholesterol (Dextran Sulfate MgCl~ precipit~tion procedure), in addition to routine serum multiple analysis (SMA-II system, Technicon Instrument Co.) and hematological analysis (autolnated cell counter, Coulter Electronics Inc., FL).
The LDL cholesterol concentration of huulan plasma was calculated using the equation: total cholesterol - IIDL cholesterol - l/S triglyceride. The LDL-VLDL choles~erol concentration in dogs was calculated as follows: total cholesterol HDL

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~20-cholesterol. 'rhe lipoprotei~ fractioIls wcre prepared ~or analysis by preparutive ultracentlifuge (human LDI.;
1.006'd ~1.063, human HI)L; 1.063 'd ~1.21, canine LDL-VLDL; d ~1.06~, canine }IDL; 1.087' d <1.21~, where d ~ density. Lipoprotein particle sizes were measured using negative straining electron ~icrographs. These fractions oE canine lipoproteins are no~ homogeneous but are comparable ~o the fractions obtained by precipitation me~hods.

R}.SULT
Table IV outlines choles~erol concen~rations at the various stages of the dog model. Total cholesterol, particularly LDL-VLD~ cholesterol, was increased and the ratio of L~L-VLDL cholesterol to ~IDL cholesterol was increased over 10 times. Albumin showed no significant change. On-line plasma filtration was carried out at each cholesterol level. Transmembrane pressure (Ptm) of the plasma separator was stable throughout the procedure. Sieving coefficients of albumin and total cholesterol and other macromolecules were over 95~. ~he Ptm of the macromolecule ~ilter increased gradually during the perfusion. The Pt~ values at one plasma volume processed ranged from 10 to 256 mmHg.
Significant differences in Ptm changes were not dependent on cholesterol concentrations.

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ABLE IV. Cholesterol an~l albumin levels on canine hypercholesterolemic model;

CH0LESTEROL_LEVEL (~ /dl) Albumin level TOTAL l~l)L LDL-VLDL

1; 137 ~ 2~115 ~ 15 21 ~ 9 3.00 ~ 6 (~3~

II; 395 ~ 30181 ~ 13 214 ~ 17 3.25 ~ 0.07 (n=2) III; 600 + 14 219 ~ 30 382 16 3.25 ~ 0.21 (n~2) . . .
I; Normal dog with normal diet II; Thyroidectomized dog with normal diet III; Thyroidectomized dog with 4~ hydrogenated coconut oil and 0.75~ cholic acid addition on norJnal diet Table V outlines mean sieving coefficients at the varying cholesterol level. LDL-VLDL cholesterol was highly rejected by the plas~a filter, whereas albumin and HDL cholesterol showed high sieving. The sc of the LDL-VLDL cholesterol ~ecreased with increasing cholesterol.

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ABLE V. Mean sievin~ coefficients of macrolllolecule filter; ex vivo on~ e fiitration test (37C) of Kuraray Eval 4A on differer1t chol~st~rol levels.

CHOLESTE~OL
TO~AL tlDL LDL-YLDL AL~UMIN

I; 0.60~0.lO 0.63~0.09 0,3~0.07 0.~8~0.07 ~n~3) II; 0.42~0,08 0.61*0.ll 0,32~0.0l 0.95~0.06 (n=2) III; 0.34~0.05 0.59~0.09 0.l9~0.04 0.93~0.04 (n=2) Mean + Standard Deviation -Pigure 5 shows the particle size distribution of the lipoprotein for the FHC plas~a and the dog plasmas with various cholesterol levels. The size difference between the HDL and LD~ of FllC plasma was grea~er than that of ~he dog plasmas. The particle diameters and ~eviation of LDL-VLDL in do~ plasmas also increased, but not in as great de8ree as with the hu~an plasma. The HDL size was not significantly different among the groups.
Figure 5 shows the post treatment recovery of HDL-VLDL and HDL cholesterols. Recovery of LDL-VLDL
cholesterol was prolonued in th~ hi~her cholesterol level groups. For Groups ~1 and III, it took about 2 weeks to return to pre-treatment values. IIDL
cholesterol recovery was constant and returned to pre-v~lues within 7 days for all groups. Pi~ure 6 shows ~9~3~2~

the changes in the LDL-Vl,DI. cholesterol/l-lDL cholesterol ratios. The ratio decre~sed during the post filtration periods and was maintained a~ a lower level for 14 days. The tendency for a hi~her reduced ratio for longer periods in comparison to the pre or post treatment values was greater in Croup III which had the highest cholesterol.

Conclusion: The data indicates that lipoprotein particle size and sieving coeficients are highly cholesterol concentration dependent. As the cholesterol concentration increases, lipoprotein particle size (particularly LDL-VLDL) increases, sieYing decreases and the total cholesterol (LDL-VLDL cholesterol) removal increases.
A comparison of the sieving coef~icients indica~es that canine LDL-VLDL sieving is greater than that of human LDL. This correla~ion is explàined by the particle size study which indicates larger deviations and overlaps between canine LDL-VLDL and HDL, ~aking it more difficult to separate the lipoproteins in canines than in humans. These results indicate that thermofiltration would be quite effective in humans in regard to the selective re00val of lipoproteins.
Moreover, the data indicates that there is a more prolonged recovery of the LDL-VLDL cholesterol in the more hypercholesterolenlic stages, while HDL
cholesterol recovery remains relatively normal. The reduction of LDL choles~erol with preservation of HDL
cholesterol by thermofiltra~ion and the prolonged recovery of LDL-VLDL cholesterol with the maintenance of lower LDL/~IDL ratio is higllly sugKestive oE an effective method of treating lipoprotein abnormalities.

~3~8 ~,, Exall!ple IV
Initial clinical thermofiltration procedures were perormed on a secon(lary hypercholesterolemia patient. The patient selected for the trial was a 39 year old man who ha~ a high concentration (210-450 mg/dl) of cholesterol and a very high LDL/HDL
cholesterol ratio (8-30) due to the cholestasis of sclerosing cholangitis.
The thermofiltr~tion tests were carried out according to the on-line system exhibited in Figure 2.
The blood flow was set at 100 ml/min and the plasma flow at 30 ml/min. The Toray PS-05 (Toray Industries, Tokyo, Japan), and Asahi Plasmaflo~(AP 0511: Asahi Medical Co..
Tokyo, Japan) modules were used as the plasma separator, and the ~VAL 4A 2.0 m2 (~uraray Co.. Osaka~ Japan) was used as the plasma ~ilter. The filter and a warmer plate were wrapped with an electric heatin~ pad to maintain the temperatule at 37C in the cryochamber of the Cryomax ~Cryomax 360; Parker Biomedical, Irvine, CA, U.S.A.). For anticoagulation, 5000 U of heparin was injected as a bolus prior to initiation of the extracorporeal circulation. The processed plaslna volume and transmembrane pressure were monitored continuously throughout the procedure. The Eiltration was carried out until the transmenlbrane pressure (Ptm) o~ the plasma filter reaches 500 mm Hg. Samples were drawn simultaneously from ~he plasma inlet and outlet lines of the ~ilter when the Ptm reached 150 and 300 ~m tl8 to calculate the sieving coefficients (sc). Solute sieving was calculated as concentration in the ~iltrate divided by the concentration in the plasma inlet to the plasma ~ilter. Biochemical measurements included cholesterol and triglyceride (automatic analysis AAIII Technicon ~a~le m~r~

Instrument Co., Tarrytowl-l, NY, U.S.A.), all~ HU~
cholesterol (dextran sulEclte MUcl2 precipitation procedure), in addition to routine serum lnul~iple analysis (SMA-II, Technicon Instrument Co.). The filter was remove~ f 1`01!1 the circuit following plugKing, and the plasmapheresis procedure was changed to plasma exchange, using 53 albumin solution as a substitution fluid.
Plasma exchange was continued until one calculated plasma volume (2893 ml) was processed by plasma exchange alone.
In vitro filtration studies were done using the same filter, the same temperature, and plasma from the same patient as described in Example I.

~ESULTS
The procedure was well tolerated by the patient and no adverse reac~ions were noted. No substitution fluid was used during the thermofiltration phase. To a Ptm Of 500 mul ~Ig. 1355 + 275 ml (1160 and 1540) ml of plasma were filtered and 1117 ~ 183 ml (980 and 1253 ml) were filtered to a YtD, of 300 mm Hg. The course o Ptm versus the filtered volume was comparable to in vitro studies with this patient's plas~a, as was the filtered volume (1060 ml processed to 300 m~ Hg of Pt~
in vitro).
There was near complete rejection for LDL +
VLDL cholesterol (sc ~ 0.02) and high passage of albumin (sc ~ 0.75) and HDL cholesterol sc~0.7B). Fibrirlogell sievin~ was low (0.07). These results were comparable to the in vitro filtration results. (Table Vl below).

~2~3~3Z~3 -2~-_ABLF. VI
Concentration nnd SieVill~ coefficients (sc) of solute (mean ~ SD) Clinlcal (Ex Yivo) n Concentration sc .
Total protein (g/dl) 6.8 ~ 1.00.64 ~ 0.09 Albumin (g/dl) 3.2 ~ 0.20.75 + 0.0~
Total cholesterol (mg/dl) 181 ~ 47 0.09 ~ 0,00 HDL cholesterol (mg/dl) 17 ~ 7 0.78 ~ 0.09 LDL cholesterol (m~/dl) 140 ~ 40 0.01 LDL-YLDL cholesterol (mg/dl) 1~3 ~ 41 0.02 _ 0.01 Fibrinogen (mg/dl) 286 ~ 84a. 07 ~ 0.03 In vitro *~
Concentration sc Total protein (g/dl) 6.2 0.3 0.72 0.02 Albumin (g/dl) 3.6 ~ 0.10,82 ~ 0.03 Total cholesterol (mg/dl) 274 ~ 2 0.06 ~ 0.01 HDL cholesterol (m~/dl) 11 ~ 3 0.84 1 0.08 LDL cholesterol (mg/dl) 228 ~ 120.02 ~ 0.02 LDL-VLDL cholestercl tm~/dl) 263 ~ 50.03 ~ 0,02 Fibrinogen (mg/dl) 298 ~ 28 0.13 ~ 0.03 HDL: hi8h density lipoproteins; LDL: low denslty lipoproteins; VLDL: very low density lipoproteins;
Ptm: transmelnbrane pressure.
* Mean value of four samples, taken at Pt~n of 150 m Hg and 300 ~Inlltg froln each of two treatments.
~ Mean value o~ three filtration tests at the same conditions.

~3~

(`_NCI.US ION
Sieving coefficients of LDL cholegterol (0.~2), HDL choles~erol (~ 78) and albumin (0.75) demonstrate che selectivity of thermoÇiltration. These results are comparable to in vitro filtrations tests using the plasma of the same patient. The advanta~e of this system compared to plasnla exchange is the maintenance o ~IDL (antiathero~enic lipoprotein) and other essential plasma solutes that would be discarded in plasma exchange. Thermofiltration is more selective than membrane schelnes without temperature control and simpler to apply, as it does not require other plasma treatment steps or the addition of potentially harmful chemical additives. Moreover, abnormal concentra~ions of various proteins (such as immulloglobulins) can also be effectively removed by thermofiltration.

While there haYe been described herein what are at present considered to be the preferred embodiments of this invention, it will be apparent to those skilled in the art that various changes and modifications may be made therein without departing from the invention, and it is, therefore, intended in the appended clai~s to cover all such changes and modifica~ions as fall within the true spirit and scope of the invention.

Claims (45)

1. A method of more selectively removing macromolecules from a plasma solution including the steps of:
a. providing a plasma solution containing the macromolecules to be removed;
b. heating said plasma to a temperature near or above normal body temperature but below the boiling point of said plasma solution; and c. filtering said heated plasma solution while at a temperature near or above normal body temperature but below its boiling point with a membrane filter to selectively remove macromolecules from the plasma solution.

2. The method of claim 1 wherein said heating is carried out at a temperature range from about 35° to about 60°C.

3. The method of claim 2 wherein said heating is carried out at a temperature ranging from about 37°to about 52°C.

4. The method of claim 1 wherein said membrane filter has a porosity less than that of the selective macromolecules to be removed from the plasma solution.

5. The method of claim 1 wherein said macromolecules are separated from said plasma solution based on differences in sieving coefficients at elevated temperatures.

6. The method of claim 1 wherein said macromolecules to be removed are selected from the group consisting of low-density lipoproteins (LDL), pyroglobulins, high molecular weight proteins and mixtures thereof.

7. The method of claim 1 wherein said plasma includes normal; sclerosing cholangitis and type IIa hypercholesterolemia plasma.

8. A method of selectively removing macromolecules from a plasma solution including the steps of:
a. securing a blood flow from a specimen;
b. separating said blood flow into a concentrated cellular element stream and a plasma stream containing macromolecules to be removed;
c. heating said plasma stream containing macromolecules to be removed to a temperature near or about normal body temperature but below its boiling point;
d. filtering said heated plasma stream while at a temperature near or about normal body temperature but below its boiling point with a membrane filter to selectively remove macromolecules from the plasma solution to form a filtered plasma stream; and e. combining said filtered plasma stream and said cellular element stream to form a processed plasma stream.

9. The method of claim 8 wherein said process stream plasma is cooled before it is combined with said cellular element stream.

10. The method of claim 8 wherein the processed stream plasma is returned to the specimen.

11. The method of claim 8 which further includes the step of pumping the blood from the patient before it is formed into a separate stream.

12. The method of claim 8 wherein the separation of the blood flow into a concentrated cellular element stream and a plasma stream is effected by either a membrane filter or by a centrifuge.

13. The method of claim 8 wherein the heating of plasma stream is carried out in the temperature range of 35°C to 60°C.

14. The method of claim 13 wherein said heating is carried out at a temperature ranging from about 37°C to about 52°C.

15. The method of claim 8 wherein said macromolecular membrane filter has a porosity less than that of the selective macromolecules to be removed from the plasma solution.

16. The method of claim 8 wherein said selective macromolecules are separated from said plasma solution based on differences in sieving coefficients at elevated temperature.

17. The method of claim 8 wherein said selective macromolecules to be removed are low density lipoproteins (LDL), pyroglobulins, high molecular weight proteins, or mixtures thereof.

18. The method of claim 8, wherein the process is continuous.

19. An apparatus for removing selective macromole-cules from a plasma solution comprising:
a) a means for dividing a plasma containing solution containing macromolecules into a concentrated cellular element stream and a plasma stream;
b) a means for heating said plasma stream to a temperature above normal body temperature but below its boiling point;
c) a means for filtering said heated plasma stream to selectively remove macromolecules;
d) a means for receiving said filtered plasma stream and said concentrated cellular element stream combining to form a processed stream substantially free of selective macromolecules.

20. The apparatus of claim 19, further including means for cooling said combination stream to normal body temperature and for returning said fluid to the specimen.

21. The apparatus of claim 19, wherein said means for heating is adapted to warm the plasma stream to a temperature rangeing from about 35°C to about 60°C.

22. A method of selectively removing macromolecules from a plasma solution by thermofiltration including the steps of:
a) providing a plasma solution containing the macromolecules to be removed;
b) heating said plasma solution to a temperature about or above 35°C but below the boiling point of said plasma solution;
c) thermofiltering said heated plasma solution while at a temperature about or above 35°C but below its boiling point with a membrane filter to selectively remove macromolecules from the plasma solution by differences in sieving coefficients of said macromolecules at higher temperatures.

23. The method of claim 22, wherein said plasma solution is a human plasma solution.

24. The method of claim 22, wherein said plasma solution is a canine plasma solution.

25. The method of claim 22, wherein said heating is carried out at a temperature range from about 35°C to about 60°C.

26. The method of claim 22, wherein said heating is carried out at a temperature range from about 37°C to about 52°C.

27. The method of claim 22, wherein said membrane filter has a porosity less than the size of the selective macromolecules to be removed from the plasma solution.

28. The method of claim 22, wherein said macromole-cules are separated from said plasma solution based on differences in sieving coefficients at elevated tempera-tures.

29. The method of claim 22, wherein said macromole-cules to be removed are selected from the group consisting of low density lipoproteins (LDL), pyroglobulins, high molecular weight proteins and mixtures thereof.

30. The method of the claim 22, wherein said plasma includes normal, sclerosing cholangitis and type IIa hypercholesterolemia plasma.

31. A method of selectively removing macromolecules from a plasma solution including the steps of:
a) securing a blood flow from a specimen;
b) separating said blood flow into a concen-trated cellular element stream and a plasma stream con-taining macromolecules to be removed;
c) heating said plasma stream containing macro-molecules to be removed to a temperature about or above 35°C but below its boiling point;
d) filtering said heated plasma stream while at a temperature about or above 35°C but below its boiling point with a membrane filter to selectively remove macro-molecules from the plasma solution to form a filtered plasma stream; and e) combining said filtered plasma stream and said cellular stream to form a processed plasma stream.

32. The method of claim 31, wherein said process stream plasma is cooled before it is combined with said cellular element stream.

33. The method of claim 31, wherein said plasma solution is a human plasma solution.

34. The method of claim 31, wherein said plasma solution is a canine plasma solution.

35. The method of claim 31, wherein said heating is carried out at a temperature range from about 35°C to about 60°C.

36. The method of claim 31, wherein said heating is carried out at a temperature range from about 37°C to about 52°C.

37. The method of claim 31, wherein said membrane filter has a porosity less than the size of the selective macromolecules to be removed from the plasma solution.

38. The method of claim 31, wherein said macromole-cules are separated from said plasma solution based on differences in sieving coefficients at elevated tempera-tures.

39. The method of claim 31, wherein said macromole-cules to be removed are selected from the group consisting of low density lipoproteins (LDL), pyroglobulins, high molecular weight proteins and mixtures thereof.

40. The method of claim 31, wherein said plasma includes normal, sclerosing cholangitis and type IIa hypercholesterolemia plasma.

41. The method of claim 32, 33 or 34, wherein said heating is carried out at a temperature range from about 37°C to about 52°C.

42. The method of claim 32, 33 or 34, wherein said membrane filter has a porosity less than the size of the selective macromolecules to be removed from the plasma solution.

43. The method of claim 32,33 or 34, wherein said macromolecules are separated from said plasma solution based on differences in sieving coefficients at elevated temperatures.

44. The method of claim 32,33 or 34, wherein said macromolecules to be removed are selected from the group consisting of low density lipoproteins (LDL), pyro-globulins, high molecular weight proteins and mixtures thereof.

45. The method of claim 32,33 or 34, wherein said plasma includes normal, sclerosing cholangitis and type IIa hypercholesterolemia plasma.