SE521954C2 - Continuous purification and concentration of leukocytes from blood, useful for production of interferons - Google Patents

Abstract

A method and apparatus for continuous purification and concentration of leukocytes from blood are provided. Continuous purification and concentration of leukocytes from blood comprises: (a) separating plasma from the blood by filtration to achieve a filtered buffy coat fraction; (b) adding an aqueous solution which is hypotonic in relation to plasma, to the buffy coat fraction from step (a) to achieve lysation of erythrocytes contained in buffy coat fraction; (c) mixing the buffy coat fraction and the aqueous hypotonic solution in a mixing device; (d) leading the mixture from (c) through a retention vessel; (e) leading the mixture from (d) through a centrifuge to separate the leukocytes; and (f) collecting the separated leukocytes from (e). An Independent claim is also included for the apparatus for continuous purification and concentration of leukocytes from blood comprising: (i) a membrane filter means for separating plasma from the blood by filtration to achieve a filtered buffy coat fraction; (ii) a static mixer for mixing the buffy coat fraction and an aqueous hypotonic solution to achieve lysation of erythrocytes contained in the buffy coat fraction; (iii) a retention vessel means; and (iv) a centrifuge means for separating leukocytes.

Description

25 30 521 954 2 (Cantell, K., Hirvonen, S, Kauppinen, HL. And Myllyla, G., Production of interferon in human leukocytes from normal donors with the use of Sendai virus, in Methods in Enzymology, vol 78, p .

S. and Koistinen, V., 29-38, and Cantell, K., Hirvonen, Partial purification of human leukocyte interferon on a large scale, in Methods in Enzymology, vol 78, p- 499-505). The process described by Cantell can be summarized as follows: pooled buffy coats from healthy blood donors are suspended in cold 0.83% NH 4 Cl and centrifuged. In this step, the leukocytes are purified and separated from other blood cells. About 30% of leukocytes disappear. The leukocytes are collected and incubated in modified "Eagle's minimum essential medium" (MEM). The suspension is then pretreated with a small amount of interferon (priming) and then inoculated with Sendai virus in order to initiate the production of interferon. The harvested raw interferon is then pooled and the interferon is precipitated and further purified.

The processing steps of the leukocytes are in most cases performed batchwise using glass flasks and other suitable manual equipment. Upscaling has so far been achieved by using more flasks and centrifuges and, of course, more staff to handle these flasks. The production of interferon according to the prior art thus suffers from disadvantages typical of labor-intensive processes; high staff costs, low reproducibility, variations in exchange, etc. The improvement of today's processes has thus mostly focused on how to make the best use of available laboratory equipment and methodology. The following invention helps to overcome these disadvantages and to enable higher yields, better reproducibility and lower personnel costs. In addition, the invention enables simpler scaling up and a process that is more adapted to "Good Manufacturing Practice" (GMP).

Summary of the Invention The present invention provides a solution to the above-mentioned problems and disadvantages of a conventional process by introducing a process according to the appended claims. The invented process has, among other things, the advantages of being suitable for automation, which in turn provides better reproducibility, reduction of personnel costs.

Furthermore, the process according to the invention is easy to scale up and to adapt to large production volumes.

Brief Description of the Drawings The invention will now be described in more detail below with reference to the accompanying drawings, of which - Fig. 1 shows a block diagram which in turn shows the various process steps according to the present invention.

-Pig. 2 schematically shows an example of an automated leukocyte purification process according to the present invention.

Description of the Invention The inventor of the present invention has surprisingly shown that a continuous and automated process can be used in the purification and concentration of leukocytes.

Said process comprises the following steps: - Plasma is removed from the leukocyte suspension by filtration - NH 4 Cl is added to the filtered leukocyte concentrate - The mixture of leukocyte concentrate and EEMCI is passed through a static mixer 10 - The mixture of leukocyte concentrate and NH through a retention vessel - The mixture of leukocyte concentrate and NH 4 Cl is passed through a centrifuge - The harvested cells are collected and further processed According to an embodiment of the invention, the filtration step for separating plasma and leukocytes is performed using "hollow fibers". , which are suitable for use in the process according to the invention and as part of the apparatus or system according to the invention. The filtration parameters are balanced to produce on the one hand a densely concentrated leukocyte fraction and on the other hand a pure plasma fraction with as little discoloration as possible. The plasma fraction can be used as a starting material in other processes because it is pure and it contains no additives.

In order to achieve lysis of the red blood cells in the leukocyte suspension, the concentrated suspension is mixed with ammonium chloride solution (0.8% w / v).

The flow of ammonium chloride is preferably twice the flow of leukocytes. The mixture of leukocyte concentrate and ammonium chloride solution is passed through a static mixer in order to obtain an effective mixture and then through a retention vessel. The retention vessel is designed so that a retention time of about 10 minutes is achieved (depending on the flow / volume ratio) and so that the solution is homogeneous in the entire vessel.

The mixture of leukocyte concentrate and ammonium chloride solution is then passed to a continuous centrifuge.

The centrifuge can be a continuous or semi-continuous centrifuge in sanitary design and preferably a centrifuge adapted for sanitization without disassembly, "Sanitation-In-Place (SIP)" and in addition so-called "Clean- In-Place (CIP) ".

Subsequent process steps may include a second red blood cell lysis step. Finally, the purified and concentrated leukocytes are transferred to an incubation vessel, where interferon production is induced by the addition of Sendai virus. Interferon production preferably takes place in a bioreactor. The advantages of using a bioreactor as an incubation vessel are the possibility to achieve better control of the incubation step, simpler scaling up and simplified SIP and CIP. Together with the other process steps described earlier in this text, this is a more complex process that is better adapted to the industrial production of interferon.

Example Example 1. Test with Doolade human buffv coats on a pilot scale.

Buffy coats were taken from a regular production batch (pooled buffy coats from human donors) and emptied separately. Half of the amount of blood was processed through the invented experimental process and the other half according to the ordinary production process. In the experimental process, the plasma was separated by filtration with hollow fibers. The filtered plasma was fractionated and the highest absorbance fractions at 280 nm (A280) were stored for later functional testing.

After separation of plasma, the blood fraction is pumped directly into a mixing chamber, a static mixer, together with a stream of an aqueous ammonium chloride solution (0.8% w / v). The flow of ammonium chloride is preferably twice the blood flow, but it may vary in the range from 1.5 to 5 times said flow. The mixed solution then flows continuously through the retention vessel. The retention vessel is designed with regard to flow / volume conditions in such a way that the retention time is approximately 10 minutes and ensures that the contents of the vessel are mixed to homogeneity. This is the time required for lysis according to ordinary production. The lysate coming out of the retention vessel is led directly into the semi-continuous centrifuge. The centrifuge is then harvested intermittently. The harvested cells are then lysed once more and then suspended in conventional medium to form a cell suspension with a high cell concentration. The cell suspension is then transferred to a bioreactor containing a suitable medium and then incubated for approx. 17 hours.

After approx. one and a half hours of the 17 hours, the Sendai virus is added to the bioreactor and the cells are induced to produce interferon-alpha. Interferon-alpha secreted into the medium is recovered after incubation.

In Example 1, the cell suspensions are then added to 100 ml incubation flasks or to 3 liter laboratory bioreactors in order to compare cells from the ordinary production process with cells from the experimental process. The interferon from the bioreactors and the 100 ml bottles are then analyzed for interferon content using an ELISA method specific for quantitative measurements of leukocyte interferon-alpha. 521 954 7 Table 1. Yields of interferon from the experiments Amount of Cell ELISA Yield Yield Tot. till- conc. / cell byte / cell delayed byte (x 107 (IU / (10-3 (ml) pcs / ml) ml) IU / (%) (%) cell) 961009 Exp.- 1.5 0.8 32000 4 100 100 100% cells Ref.- 1.5 0.8 32000 4 cells 961023 Exp.- 1.1 1 41000 4.1 cells Exp.- 1.1 1 47000 4.7 85 90 155% cells Ref.- 2 1 52000 5.2 cells Ref.- 2 1.1 51000 4.6 cells 961030 Exp.- 1 0.8 42000 5.3 cells Exp.- 1 0.8 43000 5.4 cells Ref.- 1 0.8 46000 5.8 cells Ref.- 1 0.8 46000 5.8 cells Exp.- cells Exp.- cells Ref.- cells Ref.- cells 961114 Exp.- cells Exp.- cells Exp.- cells Exp.- cells Ref.- cells Ref.- cells Ref.- cells Ref.- cells 521 954 61000 54000 65000 64000 38000 37000 56000 56000 42000 46000 70000 64000 5.8 91 84 91 82 91 84 521 954 9 (Table 1 cont.) Quantity Cell - ELISA Exchange Exchange- Exchange Tot. till- conc. / cell byte / cell delayed byte (x 107 (IU / (10-3 (ml) pcs / ml) ml) IU / (%) (%) cell) 961121 Exp.- 1.5 1.1 58000 5 , 3 cells EXp.- 1.5 l, l 53000 4.8 cells EXp.- 2 1.5 73000 4.9 cells Exp.- 2 1.5 66000 4.4 139 94 196% cells Ref.- 2 0 , 7 29000 4.1 cells Ref.- 2 0.7 46000 6.9 cells Ref.- 3 l, l 56000 5.1 cells Ref.- 3 l, l 51000 4.6 cells 961127 Exp.- 1.1 1.1 41000 3.7 cells Exp.- 1.1 1.1 47000 4.3 cells EXp.- 1.5 1.7 69000 4.1 cells Exp.- cells Ref.- cells Ref.- cells Ref. - cells Ref.- cells Labferm .Exp Labferm .Ref 961204 Exp.- cells Exp.- cells Exp.- cells Exp.- cells Ref.- cells Ref.- cells Ref.- cells Ref.- cells Labferm 45 84 65 521 954 10 69000 32000 36000 53000 56000 46000 40000 30000 31000 48000 50000 37000 35000 53000 56000 50000 4.1 128 87 92 96 193% 87% 521 954 ll .Exp Labferm 65 1.1 55000 5 .Ref 961206 Exp.- 1.5 1 51000 5.1 cells Exp.- 1.5 1 48000 4.8 cells Exp.- 2.25 1.5 83000 5.5 cells Exp.- 2.25 1.5 79000 5.3 116 116 78% cells Ref.- 1 l 40000 4 cells Ref.- 1 l 50000 5 cells Ref.- 1.5 1.5 70000 4.7 cells Ref.- 1.5 1.5 62000 4.1 cells (Table 1 cont. ) Amount Cell- ELISA Yield Yield Yield Tot. till- conc. / cell byte / cell delayed byte (ml) (X 107 (IU / (10-3 pcs / ml) ml) IU / (%) (%) cell) 961211 Exp.- 1.5 1.1 66000 6 cells Exp.- cells Exp.- cells Exp.- cells Ref.- cells Ref.- cells Ref.- cells Ref.- cells Labferm .Exp Labferm .Ref 521 954 12 61000 88000 86000 61000 67000 79000 74000 68000 64000 5.5 6.1 106 89 6.8 7.4 6.2 106% AVERAGE CARE 104 94 121 STAND.

DEVIATE SE. 20 10 47 REL.

STAND.

DEVIATE SE. 19% 10% NUMBER.

The results show a better yield with the invented experimental line. It is possible to obtain more cells from the experimental line compared to the conventional production line and thus a higher total yield of interferon since the yield per cell is approximately equal.

The function tests of the plasma separated by means of "hollow fibers" were performed by adding this plasma instead of ordinary plasma to the medium when incubating leukocytes on a small scale, 100 ml bottles. The interferon produced in these vials was analyzed by an ELISA method and compared with ELISA results from vials incubated with regular plasma. 521 954 14 Table 2. Experimental yields plasma quality.

Plasma A 280 Amount of Media Cell- ELISA Out- Out- Out- prep. till- till- conc. byte byte byte sat. true. / cell / cell (ml) (ml) (X (IU / (10-3 (%) (%) 107pcs ml) IU / / cell) ml) M-5296, 25.6 1.6 40 1.2 63000 5.3 100 100 Ref.

M-5296, 25.6 1.6 40 1.2 62000 5.2 100 100 Ref. 961014 18.4 2.23 39.4 1.2 71000 5.9 ll5 ll5 961014 18.4 2.23 39.4 1.2 73000 6.1 961028 17.3 2.38 39.2 1.2 64000 5.3 109 109 961028 17.3 2.38 39.2 l, 2 72000 6.0 961030 18.8 2.25 39.4 1.2 65000 5.4 102 102 961030 18.8 2.25 39, 4 1.2 62000 5.2 961114 14.6 2.81 38.8 1.2 66000 5.5 102 102 961114 14.6 2.81 38.8 1.2 62000 5.2 961202 12.3 3, 28 38.3 1.2 64000 5.3 106 106 961202 12.3 3.28 38.3 1.2 68000 5.7 AVERAGE 107 107 VALUE 521 954 15 STAND. 6 DEVIATION.

REL. 5% STAND.

DEVIATION.

QUANTITY. 5 10 15 20 25 521 954 16 Example 2. Sanitary experiments to determine the result of CIP.

Sanitation of the experimental process line comprising the centrifuge, plasma filter, static mixer and retention vessel was performed by pumping NH4Cl through the system directly after the blood, except through the plasma filter which is filled with PBS (phosphate buffered saline). The flow direction of PBS was the reverse in the plasma filter. The entire system was then drained and then PBS was pumped in.

The system was again drained and then charged with 0.5 M NaOH. The system was allowed to stand in 0.5 M NaOH for approx. one hour and then it was drained and refilled with storage solution 0.1 M NaOH. When the system is used again, it is drained and washed with PBS.

The system was dismantled and visually inspected at critical points to see that no cells or cell fragments etc were left inside the system. Samples were taken from the rinsing solution before each experiment on the rinsing solution which had passed the whole system or as shown in Table 3. The samples were analyzed for bacterial content (according to European Pharmacopeia, 2nd Edition) and endotoxin content (according to European Pharmacopeia, 2nd Edition) .

Table 3. Experimental results: sanitary status Baked. Endotox. (cfu / ml) (EU / ml) 961009 Rinse solution: PBS <0.5 <0.2 961023 521 954 17 Rinse solution: water 0.5 <0.2 961030 Rinse solution: water <0.5 <0.2 961114 Rinse solutionz water, <0.5 <0.2 after hollow fiber Rinse solution: water, 1 after retention vessel 961121 Rinse solution: water, <0.5 ND filtrate Rinse solution: water, <0.5 after retention vessel 961127 Rinse solution: water <0.5 <0.2 (Table 3. Cont. ) Baked. Endotox. (cfu / ml) (EU / ml) 961204 Rinse solution: water <0.5 ND 961206 Rinse solution: water <0.5 ND 961211 Rinse solution: water <0.5 ND 10 521 954 18 No cells or cell fragments could be seen inside the system . Table 3 shows that the invented system can be run under aseptic conditions and is suitable for CIP.

The invention has been described with regard to preferred embodiments, which constitute the best way of carrying out the invention at present according to the inventors.

Of course, certain changes and modifications may be made which are obvious to those skilled in the art without departing from the scope of the invention, as described in the appended claims.

Claims (7)

fl _ “v 10 15 20 25 30 521 954É?“ fš¶§f 19 PATENTKRAV Process for the continuous purification and concentration of leukocytes from human blood, characterized in that said process comprises the following steps: (a) plasma is separated from buffy coats from blood by filtration to obtain a leukocyte concentrate; (b) ammonium chloride is added to the filtered leukocyte concentrate from step (a) in the form of an aqueous ammonium chloride solution, to obtain lysis of red blood cells; (c) mixing leukocyte concentrate and ammonium chloride solution from step (b) in a mixer; (d) passing the mixture from step (c) through a retention vessel; (e) passing the mixture from step (d) through a centrifuge to separate the leukocytes; (f) the separated and harvested leukocytes are collected and further processed. Process according to claim 1, characterized in that the filtration is carried out by passing the buffy coat through a membrane filter with a pore size in the range of 0.1-1.0 μm. Process according to claim 1, characterized in that the retention vessel is designed in such a way that the retention time for the mixture of leukocyte concentrate and ammonium chloride becomes approx. 10 minutes. Process according to claim 1, characterized in that the plasma separated in step (a) is reused. Process according to claim 1, characterized in that the process is automatic and adapted for CIP ("Clean-In-Place") cleaning and sanitation. fc n: 1 v H ...., '= - ^ f- i,' f = = '20 Process according to claim 1, characterized in that the harvested leukocytes are subjected to a second lysis. Process according to claim 1, characterized in that the harvested leukocytes are incubated in a bioreactor.