IE44048B1 - Process for separating precipitated proteins from albumin-containing suspensions - Google Patents

Abstract

The production of albumin for non-therapeutic applications from a suspension obtained from blood plasma is carried out by flotation filtration of the suspension on a fabric filter element. The non-albumin-containing constituents of the emulsion are collected on the fabric filter element and a clear albumin solution is obtained as the filtrate. The process enables the separation of heat-precipitated proteins from the suspension with a substantially smaller outlay compared with known filtration processes, within a shorter filtration time and avoiding a blockage of the filter inserts.

Description

This invention relates to a process for separating precipitated proteins from albumin-containing suspensions obtained from the recovery of albumin from blood plasma.

In the recovery of albumin from blood, blood products, albumin-containing body fluids and the like, an albumin-contai suspension is obtained which, in addition to the dissolved albumin, contains precipitated proteinaceous materials, particularly globulins. In the Specification of Patent Specification No. 42460 (to which this present Applica10 tion is an Addition), there is described and claimed a process of isolating albumin from a medium comprising blood, blood products, other fluids and tissue extracts, including the stej of: (a) separating plasma from the solid constituents of the said medium. (b) precipitating the globulins by heating the separatee plasma in the presence of more than a 4% concentration, of an alcohol having the formula CHg-(CHg)^-OH, where n is Ο, 1 or , and in the presence of one or more albumin stabilisers, such ε sodium caprylate, to a temperature of from 60 to 75°C. and separating off the fluid from globulins; and (c) recovering the albumin contained in the residual fit The separation of the precipitated globulins and of othe proteins which may be precipitated by thermal precipitation ma be effected in this process at a pH of about 4.4 by continuous centrifuging. The separated proteins are collected in the rotors of the centrifuge, whereas albumin remains in the supernatant liquid.

In a conventional process of separation, it is considered to be unsatisfactory that a relatively large amount of albumin is left in the separated concentrate such that a plurality of washing and centrifuging operations is required to recover the valuable residual albumin. Furthermore, the centrifuging step is extremely time consuming and accompanied by high expenditure of work. In addition, the noise of the centrifuges is a nuisance. In spite of these drawbacks, a different mode of operation has not yet been considered because it is customary and necessary in the Cohn method which has been practised for many years, and which is referred to in Patent Specification No. 42460 to separate, by a’ centrifuging step, the suspension comprising precipitated globulins and dissolved albumin. Tests have shown, that the suspensions obtained by the Cohn method can substantially not be filtered under normal requirements.

In view of the fact that the substances recovered in accordance with the process of Patent Specification No. 42460 and the conventional Cohn method involve substances which have been obtained from the same starting material and which show substantially the same consistency in the form as an intermediate product and as the final product, it has heretofore not been throught possible that the substances obtained by the more recent process could be filtered at all. Various filtration techniques have been tried to remove precipitated proteins by cold ethanol fractionation procedures. Following experience, these precipitates cannot be economically removed by filtration techniques, most probably because of their intrinsic consistency. Nevertheless different filtration procedures were investigated, as it 4 0 Ί 8 had been noticed that the consistency of heat-ethanol precipitate: globulins differs considerably from those globulins precipitated with cold ethanol.

Contrary to experience and expectation, it was surprisingly discovered that the thermally precipitated proteins could be separated from the suspension with a substantially low expenditure by alluvial filtration of the suspension.

Accordingly, the present invention provides a process for isolating albumin from a medium comprising blood, blood products, other body fluids and/or tissue extracts, including the steps of: (a) separating plasma from the solid constituents of the said medium; (b) precipitating the globulins by heating the separated plasma in the presence of more than a 4% concentration of an alcohol having the formula CH^-(CH^J^-OH, where n is 0, 1 or 2, and in the presence of one or more albumin stabilisers to a temperature of from 60 to 75°C, and separating off the fluid from the globulins; and (c) recovering the albumin contained in the residual fluid by subjecting the fluid to an alluvial filtration through an openwork filter element of fabric or metal, whereby the non-albumin constituents are collected on the filter element and a clear albumin-solution is obtained as a filtered product.

Alluvial filtration is known per se. Normally, alluvial filtration is performed in centrifugal purification filters compri ing essentially a closed pressure vessel in which circular filter elements are disposed on a centrally rotating hollow shaft, which filter elements are positioned either horizontally or vertically in parallel with each other. Customarily, the filter elements are fitted with a twisted metallic wire or braid fabric. 4 0 4 8 - 5 The substance to be filtered is mixed with a filtering aid, normally kieselguhr or diatomite, which is added to the substance in a metered quantity in accordance with the type or turbidity and with the character of the residues.

The filtrate collected on the filter elements is floated away by rotation of the filter elements and countercurrent flushing The residues are discharged as a so-called slurry.

In the present instance, the alluvial filtration offers surprising advantages, since other types of filters and other filtration methods require such long filtering periods that no advantages are obtained at all over the time-consuming centrifuging method. In this connection, the following filtering methods have been tested; purifying filtration by means of carbon and asbestos plate filters; filter layers on the basis of cellulose? fibre15 glass filters; and sintered glass filters. The filtering methods employed resulted either in excessive filtering periods, in clogging of the filter elements, or in turbid filtered liquids. It is only the use of an alluvial filter that provides clear filtrates which do not require any additional clarification or purification filtration and which, thus, permit optimum performance of the operations in the recovery of albumin to be obtained. .

In particular, it is preferred that the filtration be performed within a centrifugal purification filter comprising a closed pressure vessel, using filter elements with a mesh size of 20 to 200 microns, preferably 70 to 90 microns. This mesh size provides an optimum between the throughflow that can be obtained and complete filtering out, in which case a single filtering step only is required to be carried out in each operation.

In order to adequately prepare the filter surface for the subsequent filtration, it is expedient in some instances initially to perform a primary precoating with a neutral liquid whereby the filter element is precoated with a layer of a filtering aid having a thickness of about 0.5 centimetres. The filtering aid may be any of the customary, commercially available kieselguhrs sold under the Trade Marks Hyflo-Super-Cel and Celite 545, or Perlite. Additionally, conventional cellulose filtering aids may be used, although with lesser filtering efficiency. These filter· ing aids likewise enable a product to be obtained which is satisfactory for many applications. It is desirable that the filtering aid be subjected to pre-swelling or pre-soaking.

Favourable filtering effects are obtained with a horizontal: disposed filter element of twisted metallic wire or braid fabric having a mesh size of 80 microns.

A preferred composition comprises a suspension of from 4 to 6% of plasma protein, to which suspension there are added from 20 to 70 grains of kieselguhr filtering aid per litre. Thereafter, this suspension is filtered. Lower concentrations of protein may require lesser quantities of filtering aids under certain circumstances .

In order to recover the albumin present within the slurry or turbid liquid, it is proposed that this albumin is likewise isolated by means of a residual filtering device comprising a small portion of the total filter area, after a cleaning by rotation and countercurrent flushing or a cleaning of the filterir apparatus in a recirculation system has been performed.

In order to increase the yield, the process may be carried out at a pressure of 4.0 + 2.5 bar (atmospheres) without sacrifice to the filtering capacity.

The present invention will now be explained in greater detail by means of the following Examples. The two Examples start with two different suspensions. Example 1 starts with a suspension prepared in accordance with the process of Patent Specification No. 42460.

Example I The solid constituents (blood cells and blood platelets) are separated from human blood, and the coagulation factors are removed. The starting solution contains from 5 to 6% of plasma protein. Ir. accordance with the process disclosed in Patent Specification No. 42460 the clotting factor VIII and the fibrinogen are removed by cryo ethanol sedimentation. The prouhrobin complex is removed by absorption. The original plasma is hepatitis-activity negative, it has normal transaminase values, and it does not contain any visible haemoglobin. Sodium caprylate is added to the original plasma until a concentration of 0.004 moles is obtained. The mixture, containing about 9% of ethanol, is heated at a pH of 6.5, the pH value being adjusted by means cf 0.5N HCl. The temperature is increased to 68°C. within a period of about 3 hours, with uniform heating and the liquid is then cooled to 10°C. The suspension is further cooled and processed at a temperature of 10°C. The solution subjected to further processing contains from 2 to 2.5% of albumin.

The accompanying drawing illustrates in schematic form the processing of the starting solution. In the process a modified rotary discharge filter type ZHF-S (Schenk, upright model) for effecting the separation of a 600 1. plasma batch within a maximum of 5 hours filtering time was used. The central device of the complete system is a filter vessel 20, double walled to make both heating and cooling possible, having a capacity of about 220 1. and a filtration surface area of about 3 m2. The filter has circular filter discs 22 sealed to a central rotary column 21 in such a manner that the filtrate, once having passed through a filter medium coating each disc, flows into the central column and thence to an outlet through a line 23 and valve 8. Removal of the residue (filter aid and precipitated globulins) between filtration cycles is accomplished by centrifugal washing and a backwash using 1% sodium chloride, so that the filter is readily prepared for the next cycle. The filter elements are covered with a twisted metallic wire or braid fabric.

The unfiltered starting solution is maintained in suspensior within a slurry tank 24 with constant agitation by means of an agitator 3. The starting substance is introduced into the filter vessel 20 via a slurry feed valve 10 through a pair of inlet valves 11 and 12 by means of a pump 2. The vessel may be pressurized with compressed air by means of compressed air valve 5.

Initially, the filter elements are precoated with filter and suspended in distilled water in a suspension of about 1 kilopond of ‘'Celite545 in 500 litres of water, to form a primary precoat having a thickness of about 0.2 centimetre at atmospheric pressure. Thereafter, 500 litres of albumin-containing suspension are mixed with 25 kiloponds of “Celite545 and stirred. The mixture is introduced into the filter vessel from the slurry tank, and urged through the filter surfaces of the discs 22 at a positiv pressure of 2 bars. Prior to the actual dispensing of the liquid to the clear tank, the substance is repeatedly passed through the filter discs in the so-called recirculation mode of using a recirculation valve 6, and the pump 2, until the filtrate appears to be sufficiently clear when viewed in a sight glass 25. Thereafter, the filter is discharged to the clear tank through a valve As the clarified albumin-containing solution leaves the filter, a 1% sodium chloride wash solution is simultaneously pumped through the filter, the volume of wash solution used being equal to half the volume of the original batch. The filtration rate is 150 l./h. and the protein concentration of the filtrate at the beginning of the process is about 2.5%, while the end concentration of non-albumin constituents is only about 0.05%.

The resultant solution is clear with a protein concentration of about 1.5% albumin and an osmolality of approximately 1.000 mosm.

This clear filtrate is then diafiltered using the Pellicon 2 Cassette system (filtration surface area 9.0 m . Millipore GmbH. Heu-Isenburg, Germany). Diafiltration means, that the solution is filtered through a dialysis membrane. The flow rate at the beginning of the process is 5 to 6 litres filtrate/minute.

Operating on an 8-hour shift per day, the entire fractionation procedure takes 3 days. day 1: heat-ethanol precipitation, subsequent cooling and adjustment of pH to 4.4 (5 hours). day 2: alluvial filtration (4 hours) and diafiltration (flow rate so regulated that process is finished at beginning of 3rd working day). The material is kept at a temperature between + 6°C. and +8°C by cooling the double walled vessels in both procedures. day 3: final adjustment of pH to 7.0 protein concentration (5%, 20%). osmolality (300 mosm): clear filtration: sterile filtration (membrane 0.2μ.): bottling, pasteurizing (10 hours, 60°C).

While the yield of albumin using the alluvial filtration/ diafiltration procedure is 93% of the albumin present in the original starting material (average from 60 batches) and the quality of the final product is the same as that achieved with previous methods, the greatest advantage of this method lies in the fact that the entire process, with the exception of bottling, may be performed by one person.

Cleaning of the filters is effected by rotating the filter elements by means of a drive motor 1 and by discharging the slurry through a discharge valve 9. Through a valve 4, the discharged substance may be suspended and filtered again.

After its passage through the filter, the liquid no longer shows any Tyndall effect, and is as clear as water. The liquid is free of accompanying substances, such as lipids or foreign proteins. The liquid may be used directly for albumin saturatio and subjected to a subsequent filtration process.

In principle, the following steps occur in the process: PIASMA 600 kg: 33,6 kg. PROTEIN 18.48 kg. ALBUMIN ( = 55%) 1. + 9% ETHANOL + 0.004 M SODIUM CARPRYIATE + HCl to pH 6.5 2. HEAT PRECIPITATION: 30 min. 68°C. 3. + HCl to pH 4.4 4. ALLUVIAL FILTRATION FILTRATE . DIAFILTRATION .1 CONCENTRATION .2 DIALYSIS 6. + NaOH to pH 7.0 7. CLEAR FILTRATION ALBUMIN SOLUTION 17.2 kg. ALBUMIN ( = 93% YIELD) Example 2 A placenta extract is initially treated in a manner similai to that described in Example 1, the haemoglobin contained in the placenta extract being initially removed with the aid of a solver such as, for instance, trichloroacetic acid, chloroform or diethj ether. Sodium caprylate is added to the original plasma until a concentration of 0.004 moles is obtained. Then, the protein-containing supernatant liquid containing about 8.5% of ethanol is heated to a temperature of 68°C. at a pH of 6.5. Upon cooling and adjusting the pH to a value of 4.4 with the addition of 0.5N HCI, 100 litres of the thus obtained liquid are mixed with 3 kiloponds of Hyflo-Super-Cel filtering aid and stirred.

At a temperature of IS°C. and a pressure of 4 bar, the liquid is urged through an alluvial filter of the above-specified type and having a mesh size of 70 microns, the operation being performed without a primary precoat. The liquid is recircuh-ted until it appears in the sight glass to be clear as water and free of the Tyndall effect. Thereupon, the albumin contained in the liquid is adjusted to the desired concentration by conventional methods.

After the entire liquid has passed through the filter, the slurry or turbid volume retained in the filter is re-washed with distilled water or with 0.9% sodium chloride solution. The resulting liquid is likewise clear as water, and still contains from about 0.5% to 20% of albumin. This liquid may likewise be added directly to the albumin concentration, as is explained e.g. in Patent Specification No. 42460.

Further reference may be made to the fact that the floating filtration may be carried out both with and without a primary percoat. Clearing of the filter elements is effected by rotation and countercurrent flushing, the filter elements being rotated and the residues being discharged as slurry.

If a sufficient quantity of washing liquid is employed (namely distilled water or sodium chloride solution), an increase of the yield of up to about 96% of the originally used quantity of - 12 albumin can be obtained. The quantities of impurities or turbidities (slurries) - caused by variation of the density ratio between solvent and solid substance as well as optimum mechanical distribution - as result in other separation processes such as e.g. by centrifuging, particularly in the elution of the initially separated proteins, are avoided by residual volume filtration, either cleaning the volume of the filtering apparatus by rotation and countercurrent flushing or after a clearing therei in the recirculation process has taken place. Filtration prior IO to the further processing of the diluted albumin solution may be dispensed with in view of the high degree of purity thereof.

The separation of the solids is independent of temperature, the rate of filtration varying only slightly with increasing or decreasing temperature, and such variation may be neglected.

Normally, the heat-treated plasma acidified to a pH of 4.4 is subjected to separation at room temperature within a centrifugal purification filter adapted to be cooled by means of an external jacket. The filtration step may be performed at different tempera tures and experience has shown that the temperature may range of from 4° to + 40°C. Following the primary precoating which is performed almost without pressure, the filtration pressure may be increased to 4.0 + 2.5 bar. The resulting filtration capacity amounts to about 150 litres of filtrate per square metre per hour. As mentioned above, kieselguhrs prove to be of particular useful25 ness as filtering aids.

The quantities specified in the Example are variable depending upon the capacity of the filter. Tests have been performed successfully, both with small volumes (about 10 litres) and with large volumes in large vessels (about 500 litres).

Claims (10)

1. A process for isolating albumin from a medium comprising blood, blood products, other body fluids and/or tissue extracts, including the steps of: (a) separating plasma from the solid constituents of the said medium; (b) precipitating the globulins by heating the separated plasma in the presence of more than a 4% concentration of an alcohol having the formula CH,-(CH,) -CH, wherein n is 0, 1 or 2, and in the presence of one or more albumin stabilisers to a temperature of from 60 to 75°C. and separating off the fluid from the globulins; and (c) recovering the albumin contained in the residual fluid by subjecting the fluid to an alluvial filtration through an openwork filter element of fabric or metal, whereby the non-albumin constituents are collected on the filter element and a clear albumin-solution is obtained as a filtered product.

2. A process as claimed in Claim 1, wherein the filtration is carried out in a centrifugal purification filter comprising a sealed pressure vessel, and wherein the filter elements have a mesh size of from 20 to 2000 microns.

3. A process as claimed in Claim 2, wherein the filter elements have a mesh size of from 70 to 90 microns.

4. A process as claimed in Claim 1 or 2, wherein the filtration is carried out using a horizontally disposed filter element comprising twisted metallic wire or braided fabric having a mesh size of 80 microns.

5. A process as claimed in any one of Claims 1 to 4, wherein a suspension containing from 4 to 6% of plasma protein 440 48 is mixed with from 30 to 70 grams of kieselguhr filtering aid per litre.

6. A process as claimed in any one of Claims 1 to 4, wherein a cellulose filtering aid is employed in a pre-swollen 5 or pre-soaked condition.

7. A process as claimed in Claim 2 or Claim 5 or 6, wherein the filtration is carried out in filtering apparatus in which the filter elements have been provided with a pre-coat of a filtering aid, and wherein the slurry or turbid volume which is contained in IO the apparatus at the end of the filtration is discharged through a residual volume filtering device, either after cleaning by rotation and countercurrent flushing or after clearing of the volume of the filtering apparatus by a recirculation process has taken place. 15 S.A process as claimed in any preceding Claim wherein the process is carried out under a positive filtration pressure of 4.0 + 2.5 bar.

8.

9. A process as claimed in any preceding Claim, wherein the clear albumin-solution is filtered through a dialysis membrane. 20

10. A process for separating proteins from an albumincontaining suspension in accordance with claim 1 and substantially as hereinbefore described in either of the foregoing Examples.