WO1997003092A1

WO1997003092A1 - A process for removal of polyethylene glycol from a protein or peptide solution

Info

Publication number: WO1997003092A1
Application number: PCT/DK1996/000314
Authority: WO
Inventors: Per Kaersgaard; Søren Knud CARLSEN
Original assignee: Hemasure A/S
Priority date: 1995-07-13
Filing date: 1996-07-10
Publication date: 1997-01-30

Abstract

Polyethylene is used for fractional precipitation of proteins and peptides. Protein and peptide fractions obtained by the PEG fractionation methods generally contains residual PEG. The invention relates to a process for removing contaminating PEG from a solution of proteins or peptides, which process comprises adsorption of PEG in the protein or peptide solution to activated carbon.

Description

TITLE

A process for removal of polyethylene glycol from a protein or peptide solution.

FIELD OF THIS INVENTION

The present invention relates to a method for the removal of polyethylene glycol (hereinafter designated PEG) from a protein or peptide solution.

BACKGROUND OF THIS INVENTION PEG for fractional precipitation of proteins was introduced in 1964 by Poison et al. (Biochim.Biophys.Acta 82, 463-475 (1964)). Rapid and simple methods for the preparation of γ-globulin and fibrinogen with aid of PEG are described. The products were homogeneous on electrophoresis and centrifugation and retained satisfactory biological properties.

Haskό et al. (Haematologia 14 (2), 199-206 (1981)) describes a method for fractionation of plasma proteins with PEG-4000 and examine the effects of pH, ionic strenght and temperature.

A method of protein fractionation suitable for large scale production is described by Bjόrling (Vox Sang. 49, 240-243 (1985)). The method take advantage of the high protein- binding capacity of ion exchangers under special conditions in the presence of PEG.

Optimization of the method for selective fractionation of bovine blood plasma proteins using PEG was described by Young-Zoon Lee et al. (J.Agric.Food Chem. 35, 958-962 (1987)).

In US patent specification No. 3,652,530 is described a method for obtaining a high purity Factor VLU concentrate by further treating a lower purity preparation of Factor VIII with PEG, preferably of a molecular weight of 4000, in two steps, the first at about 6 percent and the second at about 12 percent.

Danish patent application No. 646/84 describes a method for manufacturing of a high purity concentrate of Factor VLU by fractionation with PEG in at least two steps, in which one of the steps is a precipitation with PEG in the presence of a salting-in agent.

US patent specification No. 4,164,495 describes a method for recovering IgG in a pure and anticomplementary activity free condition by fractionated precipitation of blood plasma with PEG in the presence of caprylic acid.

European patent application having publication number 123,375 describes manufacturing of a dry γ-globulin preparation capable of intravenous injection by fractionating human plasma with PEG. The method provides a γ-globulin preparation with improved water solubility and stability against increase of anticomplementary activity and decrease of antibody titer.

Ail-though the toxicity of PEG is very low, and the tolerability in humans normally are considered to be good, adverse effects of PEG has been described (Herold et al., Toxicol. Appl.Pharmacol. 65, 329-335 (1985)). The protein fractions obtained by the PEG fractionation methods generally contains residual PEG and it may, uierefore, be desirable to remove or reduce the content of PEG, especially if the purified protein is used as a drug in mammals.

The removal of contaminating PEG from a protein fraction may be carried out according to known methods of protein fractionation such as precipitation of the proteins with another precipitation agent or adsorbtion of the proteins on a chromatography column. Precipitation with another precipitation agent (ethanol, ammonium sulfate a.o.), however, introduces other substances that subsequently has to be removed, and adsorbtion of the proteins to a chromatography column may give rise to high concentrations of elution substances after elution of the proteins from the chromatography material.

Both Busby et al. (Vox Sang. 39, 93-100 (1980)) and Lou (Acta Chem.Scand.B 37, 241- 265)) descibe a method for removal of PEG from proteins by salt-induced phase separation, but the method is only applicable with relative high starting concentrations of PEG and the residual concentration of PEG is still high.

European patent application having publication number 123,375 describes a method for removal of PEG from a solution of γ-globulin by treatment with a nonpolar styrene divinylbenzene copolymer. The adsorbtion treatment is carried out, batchwise or by a column method, by contacting an aqueous solution of γ-globulin contaminated with PEG to remove the PEG from the aqueous solution by adsorbtion of PEG to the synthetic copolymer.

PEG may also be removed by diafiltration, as described in european patent application having publication number 270,025, but some proteins tend to aggregate at the ultrafiltration membrane, building up a secondary and denser membrane, that reduces the permeability of PEG. The diafiltration time will, therefore, be very long, especially if the PEG is of a high molecular weight, such as PEG-3000 or PEG-4000.

BRIEF DESCRD7TION OF THIS INVENTION

It has, surprisingly, been found that PEG can be removed from a protein or peptide solution, which process comprises adsorbtion of PEG to activated carbon.

This absorbtion can be done by filtration of the protein or peptide solution through a filter containing activated carbon. PEG may also be removed by adding activated carbon to the PEG containing protein or peptide solution, and then removing the activated carbon by centrifugation or filtration, or by leading the solution through a column with activated carbon.

DETAILED DESCRD7TION OF THIS INVENTION

This invention relates to a process for removing PEG from a protein or peptide solution. The PEG containing solution may have been obtained by a process by which PEG is used in a provisional purification of the protein or peptide in question. PEG is removed from this protein or peptide solution by adsorbing the PEG to activated carbon.

Polyethylene glycol or poly(ethylene oxide) or polyoxyethylene or simply PEG is a water soluble polymer with the general chemical formula: HOCH₂CH₂(CH₂CH₂O)_nCH₂CH₂OH, wherein n is an integer.

Some polyethylene glycols have designations containing a figure, which figure is the approximate average molecular weight. For example, PEG-3000 or Macrogol 3000 signify heterogeneous mixtures having nominal average molecular weights of 3000. PEG 400 and PEG 4000 signify heterogeneous mixtures having nominal average molecular weights of 400 and 4000, respectively.

PEG is a well-known agent for fractional precipitation which stems primarily from its benign chemical properties. Unlike ethanol and other organic precipitating agents, PEG has little tendency to denature or otherwise interact with proteins even when present at high concentrations and elevated temperamres. In principle, the process of this invention is performed by contacting the PEG containing protein or peptide solution with activated carbon and, thereafter, removing the activated carbon. Generally, the removal of the activated carbon is to be performed in such a way that the resulting protein or peptide solution has a substantially lower content of PEG, compared with the content of protein or peptide, than the content of PEG in the parent protein solution. This can, for example be obtained by contacting the parent protein or peptide solution with the activated carbon in a sufficient period of time to allow a substantial amount of PEG to adsorb to the activated carbon whereafter the PEG enriched activated carbon may be removed from the protein or peptide solution.

The protein or peptide solution from which PEG is to be removed, is preferably an aqueuos protein or peptide solution containing PEG.

Activated carbon or activated charcoal is commercial available as a non-soluble porous powder or granular with grains of different sizes or mesh. Activated carbon is videly used in the chemical and pharmaceutical industry for decolorisation or organic contaminant retention. Activated carbon filters are commercial available as filter sheets, filter pads, or filter cartrigdes in which the finely pulverised activated carbon is bonded to a non-migrating fibre matrix made by e.g. cellulose.

The protein or peptide to be purified may be any protein or peptide obtained from plants, animals, or microbes including proteins or peptides obtained by recombinant technology. The protein is especially a plasma protein selected from the group consisting of albumin, immunoglobulins, coagulation factors, antithrombin HI, protein C, protein S, complement factors, transferrin, haptoglobin, α₂-macroglobulin, and α,-antitrypsin. Preferably the protein is selected from immuglobulins and albumin.

In one aspect of this invention, the activated carbon is added to the PEG-containing protein or peptide solution batchwise, and after stirring for a period of time that is sufficient for adsorbing the PEG, the activated carbon is separated from the solution by methods known per se such as centrifugation, sedimitation, or filtration. The removed activated carbon may subsequently be washed and the washing solution may be added to the purified, more protein or peptide containing solution, to increase the recovery of, for example, a valuable protein or peptide in the purified solution. In another aspect of this invention, granular activated carbon is packed into a column. The PEG-containing protein or peptide solution is then led through the column and the effluent is collected. The column may subsequently be washed and the washing solution may be added to the purified more protein or peptide containing solution, to increase the recovery of, for example, a valuable protein or peptide in the purified solution.

In a preferred aspect of this invention, the PEG-containing protein or peptide solution is filtered through an activated carbon filter with a flow rate that permits the adsorbtion of the PEG to the activated carbon in the filter. The removal of the PEG by filtration may be combined with the removal of other contaminating substances, with a decolorization, or with a clarification of the solution by the activated carbon filter. The filtration may be performed by techniques known per se and with known equipments usually obtainable from the manufacturer of the filters. The filter may subsequently be washed and the washing solution may be added to the purified more protein or peptide containing solution, to increase the recovery of e.g. a valuable protein or peptide in the treated solution.

Another aspect of this invention is a process for purifying a PEG containing solution of a protein or peptide, which process comprises precipitating the protein or peptide with PEG, resolubilizing the precipitated protein or peptide and treating the resolubilised protein or peptide solution with activated carbon.

The desired protein or peptide is extracted from the purified protein or peptide solution in a manner known per se, for example, by lyophilization.

This invention is explained more in detail in the below examples, which illustrates the invention. It is not to be considered as limiting the scope of the invention being defined by the appended claims.

EXPERIMENTAL PART

Example 1.

A vial of Immunoglubulin G, Nordimmun^® (5 g) (Novo Nordisk A/S, Denmark) was dissolved in 200 ml of destilled water and to the solution 1 g of PEG-3000 was added. The solution with PEG was then divided into eight parts of 25 ml and different amounts of activated carbon (Charcoal activated, 2186, Merck, Germany) was added (the amount added are stated in table 1, below). After mixing for 15 minutes on a hematology mixer, the samples were centrifuged at 4700 G for 15 minutes. The supernatants were filtered through a 5 μm filter (Millex-SV, Millipore, USA). The content of PEG and IgG in the filtrates were measured giving the results stated in table 1.

5 The PEG content was determined by means of spectrophotometric measurements of the extinction value at 535 nm after formation of a complex of with barium iodide: 1000 μl of 0.5 M perchloric acid was added to 200 μl of test solution, in duplo standard solutions (10 - 75 μg/ml), and blind sample (destilled water) in centrifuge glasses and mixed thoroughly. After at least 15 minutes, the solutions were centrifuged for 15 minutes at 3000 φm. To 10 800 μl of the supernatants were added 200 μl of a 5 % barium chloride solution. For each 30 seconds 100 μl of iodine-R (0.1 M) was added to all test tubes and mixed thoroughly. After exactly 20 minutes the extinction value at 535 nm were measured towards the blind sample.

IgG was measured by radial immuno diffusion towards rabbit anti-human IgG (A-424, 15 DAKO, Denmark).

Table 1. Concentration of PEG-3000 and IgG ind samples after treatment with different amounts of activated carbon.

Amount of activated carbon PEG, mg/ml IgG, mg/ml added, g/25 ml

0.0 5.7 24.0

0.2 3.1 23.7

0.4 1.8 24.0

0.6 < 0.1 23.9

0.8 < 0.1 24.0

1.0 < 0.1 23.7

1.5 < 0.1 24.0

2.0 < 0.1 23.9

20 After addition of 0.6 g of activated carbon to a 25 ml sample with 5.7 mg/ml PEG-3000 and removal of the activated carbon no PEG could be detected. This corresponds to adsorption of more than 233 mg PEG-3000 per g of activated carbon. No adsorption of IgG was found.

Example 2

A similar experiment as that described in example 1 was performed, but this time with

PEG-6000 in stead of PEG-3000. The results obtained appear from table 2, below.

Table 2. Concentration of PEG-6000 and IgG ind samples after treatment with different amounts of activated carbon.

Amount of activated carbon PEG, mg/ml IgG, mg/ml added, g 25 ml

0.0 5.4 27.2

0.2 3.6 27.2

0.4 2.0 27.2

0.6 0.8 27.9

0.8 0.2 27.4

1.0 < 0.1 27.2

1.5 < 0.1 27.0

2.0 < 0.1 27.2

After addition of 1.0 g of activated carbon to a 25 ml sample with 5.4 mg/ml PEG-6000 and removal of the activated carbon no PEG could be detected. This corresponds to adsorption of more than 132 mg PEG-6000 per g of activated carbon. No adsorption of IgG was found.

Example 3. A 20 % solution of human serum albumin (HSA) (ALBUMIN Novo Nordisk, Gentofte, Denmark) was diluted until a concentration of 3% HSA and 2.55 g/litre of PEG-3000 was added. The pH value was adjusted to 5.0. 830 ml of this solution was then filtered through an AKS-4 activated carbon filter from Seitz (Seitz-Filter-Werke, Bad Kreuznach, Germany). The filter area was 143 cm². Samples of 100 ml of the filtered solution was collected and PEG was determined similarly as described in example 1. The data are given in table 3.

Before filtration, the concentration of PEG was determined to 2600 μg/ml. After filtration of 300 ml of the PEG-containing HSA-solution, more than 90 % of PEG was removed, corresponding to an adsorbtion of about 5 mg PEG/cm² of filter-area. No adsoφtion of HSA to the filter was observed.

PEG was measured similarly as described in example 1 and HSA was measured by rocket immuno electrophoresis towards rabbit anti-Human Albumin (A001 , DAKO, Denmark).

Table 3. Removal of PEG-3000 from a solution of human serum albumin by filtration through AKS-4.

Filtered volume, ml Concentration of PEG in % of PEG before filtration

100 1

200 3

300 12

400 35

500 53

600 79

700 83

800 86

Example 4. Resolubilized crude-IgG was obtained from the routine production of Nordimmun^®. The crude-IgG was obtained by precipitation with PEG-3000, and the precipitate, therefore contained residual amounts of PEG-3000. Crude-IgG consist of the human plasma proteins IgG, IgA, IgM, albumin, haptoglobin, and α₂-macroglobulin.

Samples of resolubilized crude-IgG were then filtered through an In line 90 filter housing (filter area 64 cm²) from CUNO equiped with either Zeta plus activated carbon R 33 SLP (CUNO), or Zeta plus activated carbon R 53 SLP (CUNO). The filters were of pharmaceutical grade.

The filters were flushed with 100 ml phosphate buffer (conductivity - 800 μS/cm, pH = 6.5) both before and after filtration. A sample of resolubilized crude-IgG was centrifugated and filtered through one of the filters. 50 ml samples of filtrate were collected and PEG and IgG were determined analogous as described in example 1 (table 4). The concentration of PEG and IgG were 3.91 mg/ml and 86.6 mg/ml before filtration, respectively.

For both filters, 200 - 300 ml of resolubilized crude-IgG could be filtered before d e concentration of PEG increased dramatically. The recovery of IgG was calculated to be 94 % for R 33 SLP and 99 % for R 53 SLP. The recovery seemed only to depend on the efficiency of the washing after filtration.

Table 4. Filtration of 535 ml and 440 ml resolubilized crude-IgG through activated carbon filters R 33 SLP and R 53 SLP, respectively. Concentration of PEG and IgG in filtered samples.

R 33 SLP R 53 SLP Filtered vol. PEG IgG Filtered vol. PEG IgG ml mg/ml mg ml ml mg/mlmg/ml

50 0.01 39.2 50 0.01 45.7

100 0.02 75.4 100 0.02 79.3

150 0.03 81.9 150 0.02 83.9

200 0.08 77.2 200 0.03 87.0

250 0.31 83.7 250 0.32 84.8

300 0.77 83.0 300 0.74 84.8

350 1.68 81.2 350 1.67 83.9

400 2.45 81.9 400 2.52 83.2

450 3.08 76.3 450 + 3.17 77.3 10 ml wash

500 3.63 76.2 50 ml wash 2.08 37.2

535 + 3.67 73.9 40 ml wash 0.53 7.9 15 ml wash

50 ml wash 1.81 36.8

35 ml wash 0.56 7.8

Example 5

In the routine production of Nordimmun^®, resolubilized crude-IgG was filtered through a Zetaplus filter housing (filter area 2.0 or 2.8 m²) from CUNO equiped with Zeta plus activated carbon R 33 SLP (CUNO). After filtration through the activated carbon filter, the solution was filtered through a 20" Sartopure GF prefilter (Sartorius) and a 30" Durapore sterile filter (Millipore). The filters were flushed with phosphate buffer (conductivity = 800 μS/cm, pH = 6.5) both before and after filtration.

PEG and IgG were determined similarly as described in example 1. The results from the assays for PEG before and after filtration and the yield of IgG appears from table 5. The results shows that the invention can be used in a large scale routine production for removing PEG from a solution of proteins.

Table 5. Concentration of PEG before and after filtration and the yield of IgG. Results from the routine production of Nordimmun^®.

Batch Weight of solution, kg PEG cone., mg/ml Yield of IgG, % IgB no. Before After BeforeAfter

10081 75 119 3.70 0.1 92

29088.1 63 131 3.70<0.1 80

20114.1* 68 105 3.69<0.1 82

a) 2.8 m² filter area (2.0 m ² for the other two batches)

Claims

1. A process for purifying a protein or peptide solution containing PEG, characterized in a) contacting the PEG containing protein or peptide solution with activated carbon and b) removing the activated carbon from the protein or peptide solution.

2. A process according to the preceding claim, wherein the solution is an aqueous solution.

3. A process according to the preceding claims, wherein the protein or peptide solution is a solution of a protein or peptide extracted from animal or plant tissue or microbial cultures.

4. A process according to the preceding claims, wherein the solution to be purified is a protein solution.

5. A process according to the preceding claim, wherein the protein solution is a solution of plasma proteins.

6. A process according to the preceding claim, wherein the plasma protein is selected from die group consisting of albumin, immunoglobulins, coagulation factors, antithrombin III, protein C, protein S, complement factors, transferrin, haptoglobin, α₂-macroglobulin, and α,-antitrypsin.

7. A process according to the preceding claim, wherein the plasma protein is selected from albumin and immunoglobulins.

8. A process according to any one of the claims 1 dirough 3, wherein the the solution to be purified is a peptide solution.

9. A process according to the preceding claims, wherein the solution is filtered through a filter containing activated carbon.

10. A process according to the preceding claim, wherein the amount of activated carbon in the filter is 1 - 100 g per g of PEG in the solution.

11. A process according to any one of the preceding claims, wherein the filter is a 5 cellulose filter.

12. A process according to any one of the preceding claims, wherein die activated carbon filter is washed after filtration of the protein or peptide solution and tlie washing solution is added to the protein or peptide solution treated with activated carbon.

10

13. A process according to any one of the preceding claims, wherein the flow across the filter is in the range from about 0.1 to about 1000 ml/cm²/hour.

14. A process according to any one of the claims 1 dirough 8, wherein the solution is 15 applied to a column packed with activated carbon.

15. A process according to the preceding claim, wherein the flow rate through the column is in the range from about 0.1 to about 100 bed volumes per hour.

20 16. A process according to claim 14 or 15, wherein the column with activated carbon is washed after loading me protein or peptide solution to the column and me washing solution is added to die protein or peptide containing flow-through.

17. A process according to any one of the claims 1 through 8, wherein the activated 25 carbon is added batchwise to the protein or peptide solution followed by stirring to adsorb PEG and removal of the activated carbon from the protein or peptide solution.

18. A process according to the preceding claim, wherein the activated carbon is washed after removal from the protein or peptide solution, and tlie washing solution is added

30 to the activated carbon treated solution.

19. A process for purifying a protein or peptide solution, the process comprising

(a) precipitating the protein or peptide with polyethylene glycol,

(b) resolubilizing the precipitated protein or peptide from step (a),

35 (c) treating the resolubilised protein or peptide solution of step (b) wim activated carbon, and (d) removing the activated carbon from the protein or peptide solution.

20. A process according to me preceding claim containing the characteristic feamre of any one of the claim 2 through 18.

5

21. A process according to any one of the preceding claims comprising a further step wherein the protein or peptide is extracted from d e purified protein or peptide solution obtained in any one of diese claims.

10 22. A protein or peptide obtained by die process according to me preceding claim.

23. A process according to any one of me preceding claims wherein me contacting of the PEG containg protein or peptide solution wim activated carbon and die removal of me activated carbon is performed in such a way that me range between me content of 15 PEG in d e parent protein or peptide solution and the content of PEG in the purified protein or peptide per gram of protein is above about 10, preferably about 100, more prefered above about 1000.

20