WO1994009123A1 - Recycling of enzymes used to convert sub-type a, b and ab erythrocytes - Google Patents
Recycling of enzymes used to convert sub-type a, b and ab erythrocytes Download PDFInfo
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- WO1994009123A1 WO1994009123A1 PCT/US1993/010066 US9310066W WO9409123A1 WO 1994009123 A1 WO1994009123 A1 WO 1994009123A1 US 9310066 W US9310066 W US 9310066W WO 9409123 A1 WO9409123 A1 WO 9409123A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/18—Erythrocytes
Definitions
- This invention is directed to a method of recycling enzymes, including the enzyme ⁇ -galactosidase, used to remove antigens from the surface of cells in blood products.
- the enzymes are recycled from enzyme-treated blood products.
- This invention is further directed to enzymes recycled by the method of this invention.
- blood product includes whole blood and cellular components derived from blood, including red cells and platelets.
- blood group systems there are more than 30 blood group systems, one of the most important of which is the ABO system.
- This system is based on the presence or absence of antigens A and/or B.
- Blood of group A contains antigen A on its erythrocytes.
- blood of group B contains antigen B on its erythrocytes.
- Blood of group AB contains both antigens, and blood of group O contains neither antigen.
- erythrocytes which are red blood cells containing hemoglobin, the principal function of which is the transport of oxygen.
- Blood of group A contains antibodies to antigen B. Conversely, blood of group B contains antibodies to antigen A. Blood of group AB has neither antibody, and blood of group 0 has both. A person whose blood
- the blood transfusion recipient's antibodies coat the red blood cells of the transfused incompatible group and cause the transfused red blood cells to agglutinate, or stick together. Transfusion reactions and/or hemolysis (the destruction of red blood cells) may result therefrom.
- transfusion blood type is cross-matched against the blood type of the transfusion recipient.
- a blood type A recipient can be safely transfused with type A blood which contains compatible antigens.
- type O blood contains no antigens, it can be transfused into any recipient with any blood type, i.e., recipients with blood type A, B, AB or O.
- type O blood is considered "universal", and may be used for all transfusions.
- the enzyme purified in these patents is the enzyme ⁇ -galactosidase, and it is purified from coffee beans.
- This invention is directed to a method of recycling enzymes, including the enzyme ⁇ -galactosidase, said recycled enzymes being capable of removing antigens from the surface of cells in blood products.
- the method of this invention comprises treating enzyme-treated blood products to obtain an enzyme-containing solution free of red blood cells, cell membrane fragments, small molecules, cell metabolites and hemoglobin, concentrating the solution, depyrogenating the concentrated solution and adjusting the solution to the desired final concentration.
- This invention is further directed to enzymes recycled by the method of this invention, said recycled enzymes being capable of removing antigens from the surface of cells in blood products.
- Figure 1 represents SDS-polyacrylamide gel analysis of the recycled ⁇ -galactosidase enzyme of this invention, wherein the enzyme is substantially pure, and appears as a single band.
- Lane 1 shows ⁇ -galactosidase (8 ⁇ g) as a single band, and Lane 2 shows standard marker proteins (6 ⁇ g) as several
- Figure 2 represents SDS-polyacrylamide gel analysis of unrecycled ⁇ -galactosidase.
- Lane 1 shows ⁇ -galactosidase (8 ⁇ g) and Lane 2 shows standard marker proteins (6 ⁇ g) by Coomassie Blue Staining.
- This invention is directed to a method of recycling enzyme capable of removing antigens from the surface of cells in blood products.
- this invention is directed to the recycling of the enzyme ⁇ -galactosidase purified from coffee beans.
- This invention is further directed to enzymes recycled by the methods of this invention, said recycled enzymes being essentially as pure as and having a specific activity similar to that of freshly purified enzymes, said recycled enzymes also being capable of removing antigens from the surface of cells in blood products.
- blood products are treated with enzymes in order to convert the blood products to type 0.
- the enzymes are associated with the cells in the blood products.
- the blood products containing the red blood cells are washed with a buffer to obtain an enzyme-containing wash solution.
- the buffer used to wash the enzyme-treated blood products must be isotonic, and must have near
- red blood cells can be maintained.
- Any isotonic buffer which has a near physiological pH may be used.
- the enzyme-treated blood products may be washed on any appropriate automatic cell washer. Of course, any washing procedure used must result in structural and functional maintenance of the red blood cells.
- red blood cells and membrane fragments may be removed by low speed centrifugation (centrifugation at 2,000 to 4,000 times gravity (g)). Following centrifugation, a 5.0 micron filter disc may be used to free the enzyme-containing solution from small membrane fragments.
- Another method of removing red blood cells and membrane fragments is the use of specific antibodies attached to a solid support, for example beads or filters. Any other method suitable for removing red blood cells and cell membrane fragments may be used.
- red blood cells and membrane fragments are removed from the enzyme-containing wash solution, small molecules, such as cell metabolites, must be removed. This can be performed by chromatography, filtration or dialysis of the enzyme-containing wash solution through a membrane, filter, or resin with a 10,000 MW cut-off. Any method suitable for removing small molecules may be used.
- a cation exchange resin e.g., carboxymethyl (CM) cellulose, CM52, CM Sephadex, CM Sepharose and
- sulphophosphorous (SP) Sephadex columns may be used either alone or in conjunction with an anion exchange resin (e.g., DE53, DEAE Sepharose, DEAE Sepharose columns). It is preferable to use both a CM52 column and a DE53 column, as the use of both columns provides for a more purified preparation. The order of use of these two columns is not critical.
- Appropriate buffer solution for these columns has a salt concentration of 50-125 mM and a pH of 5.0 to 6.0 (such as acetate, citrate, citrate-phosphate, Tris-HCl).
- the solution is concentrated and equilibrated into the appropriate buffer for the next step.
- An Amicon Ultrafiltration Cell using a 10,000 MW cut-off membrane may be used to concentrate and equilibrate the enzyme-containing wash solution.
- the concentrated solution may be passed through a column which has specific affinity for the enzyme being recycled in order to remove any remaining impurities.
- a column which has specific affinity for the enzyme being recycled in order to remove any remaining impurities.
- a divinyl-sulfone galactose column is utilized for this step. The use of this column step may not be critical, depending on the purity of the enzyme being recycled.
- the concentrated solution is then depyrogenated, for example, by passing the concentrated solution
- This column may be a gel filtration column, such as Sephadex G-100, or any other method known in the art for depyrogenation.
- This column may be a gel filtration column, such as Sephadex G-100, or any other method known in the art for depyrogenation.
- Sephadex G-100 any other method known in the art for depyrogenation.
- commercially available depyrogenating columns or other depyrogenating methods known in the art may be used.
- the depyrogenated column effluent is sterile filtered by passing it through a non-pyrogenic, sterile, low protein binding filtration unit.
- a 0.22 micron filter unit may be used.
- the unit is rinsed with sterile buffer, such as phosphate citrate buffer.
- a volume of 2 liters of enzyme-containing wash solution treated by the method of this invention allows for at least 95% recovery of the ⁇ -galactosidase enzyme.
- Two liters represent buffer wash solution after 4 washes. However, the number of washes used for enzyme recovery may be reduced. At least 90% of the
- ⁇ -galactosidase enzyme activity is seen in the first 2 washes (1 liter of wash volume).
- the amount of buffer wash solution utilized may depend on the enzyme being recycled and whether it has some association with the cells being treated. Buffer used for the washing may be stored overnight and processed for recovery of enzyme the next day. If the enzyme to be recycled is ⁇ -galactosidase, it is likely that washes from multiple red cell units may be pooled, since ⁇ -galactosidase is a stable enzyme.
- the enzymes recycled by the method of this invention have as high a level of specific activity as freshly purified enzymes.
- the recycled enzymes are also substantially pure.
- the recycled enzyme is ⁇ -galactosidase purified from coffee beans, it appears as a single band on SDS-acrylamide gel analysis. See Figure 1.
- Stable enzymes recycled by the method of this invention may be recycled several times after treating blood products.
- ⁇ -galactosidase enzyme recycled at least 6 times by the method of this invention is still capable of removing antigens from the surface of cells in blood products.
- the method of this invention may be used to recycle any enzymes used to remove antigens from the surface of cells which maintain activity under conditions of use, recovery and repurification, as well as storage.
- recycled enzyme which may contain virus may be virus-inactivated by the methods discussed in U.S. Patent No. 4,764,369 issued to Neurath et al., dated August 16, 1988, entitled "Undenatured Virus-Free Biologically Active Protein Derivatives", which is incorporated herein by reference, and then the enzymes recycled by the method of this invention used to remove antigens from the surface of cells in blood products without the risk of virus contamination with blood-borne lipid-enveloped viruses.
- Alternative virus inactivation methods known in the art may also be utilized, in combination with or independent of the Neurath, et al. method.
- PBS phosphate buffered saline
- Red cells and cell membrane fragments were removed from the enzyme-containing wash solution using filtration through a 5.0 micron filter disc and low speed centrifugation (centrifugation at 2,000 to 4,000 times gravity (g)).
- Concentrated phosphate-citrate buffer titrate 50 mM citric acid with 100 mM dibasic sodium phosphate to pH 3.7
- the columns were equilibrated with 50 mM sodium acetate at pH 5.5 and the enzyme-containing wash solution was passed through and the columns were washed with 50 mM sodium acetate pH 5.5, collecting the
- phosphate citrate buffer is made by titrating 50 mM citric acid with 100 mM dibasic sodium phosphate to a pH of 3.7; when diluted 1:50 with H 2 O the pH becomes 4.0.
- a divinyl-sulfone-galactose column which binds the ⁇ -galactosidase enzyme, was equilibrated with a 1:50 dilution of phosphate citrate buffer pH 4.0, and the enzyme-containing solution applied to the column to remove any remaining impurities.
- PCS buffer for this step can have a pH range of 5.0 to 7.0.
- the elution buffer used for this affinity column step should have a higher molarity of salt and a higher pH than the buffer used to load the enzyme on the column.
- the enzyme-containing solution was concentrated and equilibrated in buffer (PCS at pH 5.6) in an Amicon Ultrafiltration Cell using a 10,000 MW cut-off membrane.
- the equilibration buffer used for this and the following steps should be isotonic and with a pH suitable for use in removing antigens from the surface of cells in blood products (pH 5.5-7.5).
- the enzyme-containing solution was concentrated to approximately 25 ml.
- the concentrated enzyme-containing solution was then depyrogenated by passing the solution through a pyrogen-free Sephadex G-100 gel filtration column. Approximately 250 ml of depyrogenated enzyme solution was recovered after passing the solution through the Sephadex G-100 column.
- the depyrogenated column effluent was passed through a sterile non-pyrogenic low protein binding 0.22 micron filter unit, which was rinsed with sterile, pyrogen-free phosphate citrate saline buffer at a pH of 5.6, to yield a final enzyme concentration of 1500-2000 units ⁇ -galactosidase per ml.
- the recovered ⁇ -galactosidase was analyzed by SDS-polyacrylamide gel filtration, and appeared as a single band. See Figure 1.
- Table II shows the recovery and rechromatography of ⁇ -galactosidase after various treatment steps. As shown in Table II, a high percentage of enzyme was recovered after each step of the recycling procedure was performed. In addition, Table II shows that the specific activity of the recycled ⁇ -galactosidase enzyme was 27-30 units/mg protein, which is comparable to the specific activity of freshly purified
- the recycled -galactosidase meets the same lot release specifications as the freshly purified enzyme.
- Table III represents the lot release specifications of recycled ⁇ -galactosidase. Again, a specific activity of 28 units/mg protein is indicated at 26°C, at a pH of 6.5.
- the recycled ⁇ -galactosidase appears as a single band on SDS-polyacrylamide gel analysis. The protease and neuraminidase activities were within background range. Both the apyrogenicity and sterility of the recycled ⁇ -galactosidase meet FDA requirements.
- Protease Within background range (20 units -galactosidase, 6 hrs at 37°C,
- Neuraminidase Within background range (10 units -galactosidase, 16 hrs at 37°C,
- Alpha-galactosidase enzyme isolated from coffee beans was used to treat type B blood cells eight times without repurification or recycling in between cell treatments. For each treatment, 0.5 mLs of type B red cells were treated for 1 hour at 26°C (pH 5.7). The cells were pelleted by centrifugation, and supernatant containing the enzyme was taken off and added to the next aliquot of B cells. After each successive treatment, no reactivity was seen with human polyclonal anti-B serum. This demonstrates the stability of the enzyme and maintenance of activity on reuse. To demonstrate the use of recovered and repurified enzyme, ⁇ -galactosidase enzyme was recovered and reused for six successive cycles. In four of these six cycles, recycled enzyme was used for ex vivo treatment of B cells for transfusion without complications. No loss in enzyme activity was observed, demonstrating successive reuse of enzyme after multiple cycles of recovery, repurification and reuse.
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Abstract
This invention is directed to a method of recycling enzymes, including the enzyme α-galactosidase, used to remove antigens from the surface of cells in blood products. The enzymes are recycled from enzyme-treated blood products. This invention is also directed to recycled enzymes.
Description
RECYCLING OF ENZYMES USED TO
CONVERT SUB-TYPE A, B AND AB ERYTHROCYTES
Statement of Government Interest
This invention was made with government support under NMRDC grant No. N00014-90-J-1638. As such, the government has certain rights in this invention.
FIELD OF THE INVENTION
This invention is directed to a method of recycling enzymes, including the enzyme α-galactosidase, used to remove antigens from the surface of cells in blood products. The enzymes are recycled from enzyme-treated blood products. This invention is further directed to enzymes recycled by the method of this invention.
BACKGROUND OF THE INVENTION
As used herein, the term "blood product" includes whole blood and cellular components derived from blood, including red cells and platelets.
There are more than 30 blood group systems, one of the most important of which is the ABO system. This system is based on the presence or absence of antigens A and/or B. Blood of group A contains antigen A on its erythrocytes. Similarly, blood of group B contains antigen B on its erythrocytes. Blood of group AB
contains both antigens, and blood of group O contains neither antigen. These antigens are found on the surface of erythrocytes, which are red blood cells containing hemoglobin, the principal function of which is the transport of oxygen.
Blood of group A contains antibodies to antigen B. Conversely, blood of group B contains antibodies to antigen A. Blood of group AB has neither antibody, and blood of group 0 has both. A person whose blood
contains either (or both) of the anti-A or anti-B antibodies cannot receive a transfusion of blood containing the corresponding incompatible antigen(s). If a person receives a transfusion of blood of an incompatible group, the blood transfusion recipient's antibodies coat the red blood cells of the transfused incompatible group and cause the transfused red blood cells to agglutinate, or stick together. Transfusion reactions and/or hemolysis (the destruction of red blood cells) may result therefrom.
In order to avoid red blood cell agglutination, transfusion reactions and hemolysis, transfusion blood type is cross-matched against the blood type of the transfusion recipient. For example, a blood type A recipient can be safely transfused with type A blood which contains compatible antigens. Because type O blood contains no antigens, it can be transfused into any recipient with any blood type, i.e., recipients with
blood type A, B, AB or O. Thus, type O blood is considered "universal", and may be used for all transfusions. Hence, it is desirable for blood banks to maintain large quantities of type O blood. However, there is a paucity of blood type O donors. Therefore, it is would be useful to convert types A, B and AB blood to type O blood in order to maintain large quantities of universal products.
In an attempt to increase the supply of type O blood, methods have been developed for converting certain subtypes of A and AB and B type blood to type O blood. For example, enzymes have been used to convert blood products to type 0. Several methods of purifying enzymes capable of converting blood products from types A, B, or AB to type O blood products have been developed. For example, U.S. Patent Nos. 4,427,777 and 4,330,619 are directed to enzymatic conversion of red blood cells for transfusion, wherein methods of purifying the enzyme used for such conversion are
described. The enzyme purified in these patents is the enzyme α-galactosidase, and it is purified from coffee beans.
In "Affinity Purification of α-Galactosidase A From Human Spleen, Placenta And Plasma With Elimination of Pyrogen Contamination", by Bishop et al. in The Journal of Biological Chemistry, Vol. 286, No. 3, pages 1307-1316 (February 10, 1981), the enzyme
α-galactosidase is purified from human spleen, placenta and plasma. Further, in "Single-Unit Transfusions Of RBC Enzymatically Converted From Group B To Group O To A And O Normal Volunteers", by Lenny et al., in Blood. Vol. 77, No. 6, pages 1383-1388 (March 15, 1991), the enzyme α-galactosidase is purified from coffee beans.
The methods described in the patents and articles discussed above to purify the α-galactosidase enzyme are all extremely complicated and time consuming procedures. In order to avoid the need to continually purify enzymes for use in the removal of antigens from the surface of cells in blood products, a need has arisen to develop a method of recycling enzymes already used for such removal, wherein the recycled enzymes are as pure as original unrecycled enzyme, have a high specific activity and are capable of removing antigens from the surface of cells in blood products with the same efficiency and specific activity as freshly
purified, nonrecycled enzymes.
To date, no method has been developed which allows for the recycling of the enzyme α-galactosidase or any other enzyme used to remove antigens from the surface of cells ex vivo, thereby enabling the enzyme to be used many times for blood product type conversion. Hence, it is desirable to develop a method of recycling enzymes, including α-galactosidase, so that the
enzymes may be used many times for the removal of antigens from the surface of cells and conversion of such cells to more useful, transfusable cellular products. Enzyme recycling eliminates the need to continually purify enzymes from various sources utilizing the time consuming, complicated and expensive methods of the prior art.
It is therefore and object of this invention to provide a method of recycling enzymes capable of
removing antigens from the surface of cells in blood products.
It is another object of this invention to provide a method of recycling the enzyme α-galactosidase after said enzyme is used for removal of antigens from the surface of cells or in blood type conversion of blood products.
It is a further object of this invention to provide a method of obtaining a purified enzyme by recycling an enzyme after the enzyme has been used to remove antigens from the surface of cells in blood products.
It is yet another object of this invention to provide a recycled enzyme capable of removing antigens from the surface of cells in blood products.
It is a still further object of this invention to provide a recycled enzyme which has a level of specific activity similar to that of freshly purified
enzyme, said recycled enzyme being capable of removing antigens from the surface of cells in blood products.
It is another object of this invention to provide a recycled enzyme which is essentially as pure as freshly purified enzyme, said recycled enzyme being capable of removing antigens from the surface of cells in blood products.
It is another object of the present invention to provide a recycled enzyme which is virus inactivated and free of blood-borne viruses.
SUMMARY OF THE INVENTION
This invention is directed to a method of recycling enzymes, including the enzyme α-galactosidase, said recycled enzymes being capable of removing antigens from the surface of cells in blood products. The method of this invention comprises treating enzyme-treated blood products to obtain an enzyme-containing solution free of red blood cells, cell membrane fragments, small molecules, cell metabolites and hemoglobin, concentrating the solution, depyrogenating the concentrated solution and adjusting the solution to the desired final concentration. This invention is further directed to enzymes recycled by the method of this invention, said recycled enzymes being capable of removing antigens from the surface of cells in blood products.
BRIEF DESCRIPTION OF THE DRAWING
The above brief description, as well as further objects and features of the present invention, will be more fully understood by references to the following detailed description of the presently preferred albeit illustrative embodiment of the present invention when taken in conjunction with the accompanying drawing wherein:
Figure 1 represents SDS-polyacrylamide gel analysis of the recycled α-galactosidase enzyme of this invention, wherein the enzyme is substantially pure, and appears as a single band. Lane 1 shows α-galactosidase (8 μg) as a single band, and Lane 2 shows standard marker proteins (6 μg) as several
bands, by Coomassie Blue Staining.
Figure 2 represents SDS-polyacrylamide gel analysis of unrecycled α-galactosidase. Lane 1 shows α-galactosidase (8 μg) and Lane 2 shows standard marker proteins (6 μg) by Coomassie Blue Staining.
DETAILED DESCRIPTION OF THE INVENTION
This invention is directed to a method of recycling enzyme capable of removing antigens from the surface of cells in blood products. In a preferred embodiment, this invention is directed to the recycling of the enzyme α-galactosidase purified from coffee beans. This invention is further directed to enzymes recycled by the methods of this invention, said recycled
enzymes being essentially as pure as and having a specific activity similar to that of freshly purified enzymes, said recycled enzymes also being capable of removing antigens from the surface of cells in blood products.
Typically, blood products are treated with enzymes in order to convert the blood products to type 0. At the end of the enzyme treatment process, the enzymes are associated with the cells in the blood products. In order to recycle these enzymes, the blood products containing the red blood cells are washed with a buffer to obtain an enzyme-containing wash solution. The buffer used to wash the enzyme-treated blood products must be isotonic, and must have near
physiological pH so that the red blood cells can be maintained. Any isotonic buffer which has a near physiological pH may be used. The enzyme-treated blood products may be washed on any appropriate automatic cell washer. Of course, any washing procedure used must result in structural and functional maintenance of the red blood cells.
After obtaining the enzyme-containing wash solution, it is necessary to remove any red blood cells remaining in the wash solution, including red blood cell membrane fragments. Removal of red blood cells and membrane fragments from the enzyme-containing wash solution may be performed by low speed centrifugation
(centrifugation at 2,000 to 4,000 times gravity (g)). Following centrifugation, a 5.0 micron filter disc may be used to free the enzyme-containing solution from small membrane fragments. Another method of removing red blood cells and membrane fragments is the use of specific antibodies attached to a solid support, for example beads or filters. Any other method suitable for removing red blood cells and cell membrane fragments may be used.
After red blood cells and membrane fragments are removed from the enzyme-containing wash solution, small molecules, such as cell metabolites, must be removed. This can be performed by chromatography, filtration or dialysis of the enzyme-containing wash solution through a membrane, filter, or resin with a 10,000 MW cut-off. Any method suitable for removing small molecules may be used.
Next, various forms of hemoglobin remaining in the enzyme-containing wash solution must be removed.
Any suitable method which selectively removes hemoglobin, but does not remove or have affinity for the enzyme being recycled, may be used. For example, a cation exchange resin (e.g., carboxymethyl (CM) cellulose, CM52, CM Sephadex, CM Sepharose and
sulphophosphorous (SP) Sephadex columns) may be used either alone or in conjunction with an anion exchange resin (e.g., DE53, DEAE Sepharose, DEAE Sepharose
columns). It is preferable to use both a CM52 column and a DE53 column, as the use of both columns provides for a more purified preparation. The order of use of these two columns is not critical. Appropriate buffer solution for these columns has a salt concentration of 50-125 mM and a pH of 5.0 to 6.0 (such as acetate, citrate, citrate-phosphate, Tris-HCl).
After red blood cells, small molecules and hemoglobin have been removed from the enzyme-containing wash solution, the solution is concentrated and equilibrated into the appropriate buffer for the next step. An Amicon Ultrafiltration Cell using a 10,000 MW cut-off membrane may be used to concentrate and equilibrate the enzyme-containing wash solution. After concentration and equilibration, the concentrated solution may be passed through a column which has specific affinity for the enzyme being recycled in order to remove any remaining impurities. For example, for α-galactosidase enzyme, a divinyl-sulfone galactose column is utilized for this step. The use of this column step may not be critical, depending on the purity of the enzyme being recycled.
The concentrated solution is then depyrogenated, for example, by passing the concentrated solution
through a column. This column may be a gel filtration column, such as Sephadex G-100, or any other method known in the art for depyrogenation. For example,
commercially available depyrogenating columns or other depyrogenating methods known in the art may be used.
After depyrogenation, the depyrogenated column effluent is sterile filtered by passing it through a non-pyrogenic, sterile, low protein binding filtration unit. A 0.22 micron filter unit may be used. After sterile filtration the unit is rinsed with sterile buffer, such as phosphate citrate buffer.
A volume of 2 liters of enzyme-containing wash solution treated by the method of this invention allows for at least 95% recovery of the α-galactosidase enzyme. Two liters represent buffer wash solution after 4 washes. However, the number of washes used for enzyme recovery may be reduced. At least 90% of the
α-galactosidase enzyme activity is seen in the first 2 washes (1 liter of wash volume). The amount of buffer wash solution utilized may depend on the enzyme being recycled and whether it has some association with the cells being treated. Buffer used for the washing may be stored overnight and processed for recovery of enzyme the next day. If the enzyme to be recycled is α-galactosidase, it is likely that washes from multiple red cell units may be pooled, since α-galactosidase is a stable enzyme. The method
outlined herein may be used for batches of at least 40,000 units of enzyme, with the process scaled up for larger batches.
The enzymes recycled by the method of this invention have as high a level of specific activity as freshly purified enzymes. In addition, provided that the enzymes used to treat the blood products are pure, the recycled enzymes are also substantially pure. Where the recycled enzyme is α-galactosidase purified from coffee beans, it appears as a single band on SDS-acrylamide gel analysis. See Figure 1.
Stable enzymes recycled by the method of this invention may be recycled several times after treating blood products. α-galactosidase enzyme recycled at least 6 times by the method of this invention is still capable of removing antigens from the surface of cells in blood products. In addition, the method of this invention may be used to recycle any enzymes used to remove antigens from the surface of cells which maintain activity under conditions of use, recovery and repurification, as well as storage.
Further, recycled enzyme which may contain virus may be virus-inactivated by the methods discussed in U.S. Patent No. 4,764,369 issued to Neurath et al., dated August 16, 1988, entitled "Undenatured Virus-Free Biologically Active Protein Derivatives", which is incorporated herein by reference, and then the enzymes recycled by the method of this invention used to remove antigens from the surface of cells in blood products without the risk of virus contamination with blood-borne
lipid-enveloped viruses. Alternative virus inactivation methods known in the art may also be utilized, in combination with or independent of the Neurath, et al. method.
Example
Blood treated with the enzyme α-galactosidase purified from coffee beans was washed several times with phosphate buffered saline (PBS) buffer (150 πiM sodium chloride, 150 mM sodium phosphate) in a COBE cell washer to obtain an enzyme-containing wash solution. Red cells and cell membrane fragments were removed from the enzyme-containing wash solution using filtration through a 5.0 micron filter disc and low speed centrifugation (centrifugation at 2,000 to 4,000 times gravity (g)).
Next, small molecules, including cell metabolites, were removed by concentration and washing with 50 mM sodium acetate buffer at pH 5.5 to a final volume of approximately 50 ml, using a 10,000 MW cut-off ultrafiltration membrane. Table I shows the
chromatography steps for recycling of the α-galactosidase enzyme. In order to remove various forms of hemoglobin, a combination of 2 small ion exchange columns was used. The 2 small ion exchange columns used were DE53 and CM52. Two columns were used so that any contaminants or impurities, including hemoglobin, remaining in the enzyme-containing wash solution would be removed.
TABLE I
Chromatography steps for Recycling of -galactosidase [40,000 U] a recovered from treatment of 1 unit RBC
Col. size Wash Elution
Support Diam. x Length Equil. Buffer Buffer Buffer Vol. Applied Vol. Eluted
I
DE 53 2 × 20 cm 50 mM 50 ml
Na acetate
CM 52 2 × 20 cm pH 5.5 200 ml
II
Divinyl Phosphate PCS+
Sulfone 10 × 20 cm Citrate Buffer pH 5.6 200 ml 500 ml Galactose 1:50 dil.
pH 4.0
*III
Seph G100 5 × 100 cm PCS+ buffer
pH 5.6 25 ml 250 ml
*This column was run at 4°C; the other two were run at room temperature.
Concentrated phosphate-citrate buffer: titrate 50 mM citric acid with 100 mM dibasic sodium phosphate to pH 3.7
+PCS buffer pH 5.6 = 58 mM dibasic sodium phosphate
21 mM citric acid
77 mM sodium chloride
a 1 unit of enzyme liberates 1 micromole -galactose per minute at 26°C and pH 6.5
The columns were equilibrated with 50 mM sodium acetate at pH 5.5 and the enzyme-containing wash solution was passed through and the columns were washed with 50 mM sodium acetate pH 5.5, collecting the
solution from the columns until all the enzyme washed through. This resulted in approximately 200 ml of pass-through solution. The 200 ml of enzyme solution was then concentrated and equilibrated in a 1:50 dilution of phosphate citrate buffer, pH 4.0
(Concentrated phosphate citrate buffer is made by titrating 50 mM citric acid with 100 mM dibasic sodium phosphate to a pH of 3.7; when diluted 1:50 with H2O the pH becomes 4.0.) A divinyl-sulfone-galactose column, which binds the α-galactosidase enzyme, was equilibrated with a 1:50 dilution of phosphate citrate buffer pH 4.0, and the enzyme-containing solution applied to the column to remove any remaining impurities. Approximately 500 ml of enzyme-containing solution was eluted from the divinyl-sulfone-galactose column using phosphate citrate saline (PCS) buffer (58 mM dibasic sodium phosphate, 21 mM citric acid, 77 mM sodium chloride) at pH 5.6 (PCS buffer for this step can have a pH range of 5.0 to 7.0). The elution buffer used for this affinity column step should have a higher molarity of salt and a higher pH than the buffer used to load the enzyme on the column.
Next, the enzyme-containing solution was concentrated and equilibrated in buffer (PCS at pH 5.6) in an Amicon Ultrafiltration Cell using a 10,000 MW cut-off membrane. The equilibration buffer used for this and the following steps should be isotonic and with a pH suitable for use in removing antigens from the surface of cells in blood products (pH 5.5-7.5). The enzyme-containing solution was concentrated to approximately 25 ml. The concentrated enzyme-containing solution was then depyrogenated by passing the solution through a pyrogen-free Sephadex G-100 gel filtration column. Approximately 250 ml of depyrogenated enzyme solution was recovered after passing the solution through the Sephadex G-100 column.
Finally, after depyrogenation, the depyrogenated column effluent was passed through a sterile non-pyrogenic low protein binding 0.22 micron filter unit, which was rinsed with sterile, pyrogen-free phosphate citrate saline buffer at a pH of 5.6, to yield a final enzyme concentration of 1500-2000 units α-galactosidase per ml. The recovered α-galactosidase was analyzed by SDS-polyacrylamide gel filtration, and appeared as a single band. See Figure 1.
Table II shows the recovery and rechromatography of α-galactosidase after various treatment steps. As shown in Table II, a high percentage of enzyme was recovered after each step of the recycling procedure was
performed. In addition, Table II shows that the specific activity of the recycled α-galactosidase enzyme was 27-30 units/mg protein, which is comparable to the specific activity of freshly purified
α-galactosidase enzyme.
TABLE II
Recovery and Rechromatography of -Galactosidase After Red Cell Treatment
STEP SPEC. ACTIVITY
STEP RECOVERY YIELD U/mg PROTEIN
Red Cell
Washes > 90% 100% *N. D.
Removal of Red
Cell fragments > 90% 99% *N. D.
10,000 MW
Ultrafiltration 90% 98% 27-30
Sepharose Divinylsulfone Galactose 81% 90% 27-30
Sephadex G 100 73% 90% 27-30
*N. D. = Not determined
The recycled -galactosidase meets the same lot release specifications as the freshly purified enzyme.
Table III represents the lot release specifications of recycled α-galactosidase. Again, a specific activity of 28 units/mg protein is indicated at 26°C, at a pH of 6.5. The recycled α-galactosidase appears as a single band on SDS-polyacrylamide gel analysis. The protease and neuraminidase activities were within background range. Both the apyrogenicity and sterility of the recycled α-galactosidase meet FDA requirements. Further, there was limited activity for β-N-acetylgalactosaminidase, β-N-acetylglucosaminidase, α-glucosidase, β-glucosidase, β-galactosidase, α-mannosidase and α-fucosidase at 37°C with a pH of 5.6.
TABLE III
LOT RELEASE SPECIFICATIONS OF -GALACTOSIDASE FROM GREEN COFFEE BEANS
Specific activity: Minimum = 28 units/mg protein (26°C; 1.25 mM p-nitrophenyl¬
-D-galactosidase as substrate; pH 6.5)
Protease: Within background range (20 units -galactosidase, 6 hrs at 37°C,
5 mg Azocoll as substrate)
Neuraminidase: Within background range (10 units -galactosidase, 16 hrs at 37°C,
1 mM N-acetylneuraminlactose as substrate)
B-N-acetylgalactosaminidase Maximum = 0.0002%
B-N-acetylglucosaminidase Maximum = 0.0002%
-glucosidase Maximum = 0.0002%
B-glucosidase Maximum = 0.0002%
B-galactosidase Maximum = 0.0002%
-mannosidase Maximum = 0.0002%
-fucosidase Maximum = 0.0002%
(37°C; 2 mM p-nitrophenyl glycosidase as substrate, pH 5.6)
SDS-Page analysis: Single band
Load = 8 ug -galactosidase
(Coomassie Blue staining)
Apyrogenicity: Meets FDA requirements
(Limulus Amebocyte Lysate gel clot procedure)
Sterility: Meets FDA requirements
Alpha-galactosidase enzyme (isolated from coffee beans) was used to treat type B blood cells eight times without repurification or recycling in between cell treatments. For each treatment, 0.5 mLs of type B red cells were treated for 1 hour at 26°C (pH 5.7). The cells were pelleted by centrifugation, and supernatant containing the enzyme was taken off and added to the next aliquot of B cells. After each successive treatment, no reactivity was seen with human polyclonal anti-B serum. This demonstrates the stability of the enzyme and maintenance of activity on reuse. To demonstrate the use of recovered and repurified enzyme, α-galactosidase enzyme was recovered and reused for six successive cycles. In four of these six cycles, recycled enzyme was used for ex vivo treatment of B cells for transfusion without complications. No loss in enzyme activity was observed, demonstrating successive reuse of enzyme after multiple cycles of recovery, repurification and reuse.
In order to show the applicability of the enzyme recycling process of this invention to other enzymes, a mix of recovered α-N-acetylgalactosaminidase (7.55 units) and α-galactosidase (23.8 units) was loaded onto DE53 and CM52 columns in 100 mM sodium acetate buffer at a pH of 5.5. Contaminating hemoglobin bound to the columns, while both the α-galactosidase and α-N-acetyl-galactosaminidase enzymes passed through
and were collected in the column wash. As shown in Table IV below, ninety-five percent of the α-galactosidase and 80% of the α-N-acetyl-galactosaminidase were recovered in the column wash solution.
TABLE IV
Recycle a mixture of NGal + Gal using a combination col CM 52 + DE 53
CM 52 = 1.5 ml )
DE 53 = 1.5 ml ) equil in 100 mM Acetate pH 5.5
Apply 0.5 ml of used NGal + Gal equil in 100 mM Na Acetate pH 5.5
1:20 dil of appl. En2(+ ALb)
Gal = 10 5'37° 405 = 0.880 = 47.7 U/ml
NGal = 10 10'37° 0.664 = 15.1 U/ml
Etritest with wash buffer 100 mM Na Acetate
Bzyme: 9 ml 10 5'37° 405 = 0.900 = 21.95 units total
3 ml 10 10'37° = 0.15 = 0.60 "
22.55 units total applied = 23.8 units
recovered = 22.5 units = i 95%
NGal: 9 ml 10 10'37° 405 = 0.580 = 5.95 units total
3 ml 10 10'37° 0.085 = 0.15 "
6.10 units total applied = 7.55 units
recovered = 6.10 units = 80%
Although this invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of various aspects of the invention. Thus, it is to be understood that numerous modifications may be made in the illustrative embodiments and other arrangements may be devised without departing from the spirit and scope of the invention.
Claims
1. A method of recycling enzyme from enzyme-treated blood product comprising:
(a) washing enzyme-treated blood product to
obtain an enzyme containing solution;
(b) removing red blood cells, including cell membrane fragments from the enzyme-containing solution;
(c) removing small molecules, including cell metabolites from the enzyme-containing solution;
(d) removing hemoglobin from the enzyme- containing solution;
(e) concentrating and equilibrating the solution;
(f) depyrogenating the concentrated solution; and
(g) adjusting the concentration of the depyrogenated solution such that the remaining solution contains enzyme at a concentration for use in the removal of antigens from the surface of cells in blood products.
2. The method of Claim 1 which further comprises passing the concentrated solution through a column which has affinity for the enzyme being recycled so as to remove remaining impurities, if any.
3. The method of Claim 2 wherein the column used is a divinyl-sulfone-galactose column and the enzyme is α-galactosidase.
4. The method of Claim 1 wherein the enzyme is α-galactosidase which was initially purified from coffee beans.
5. The method of Claim 1 wherein the enzyme is α-N-acetylgalactosaminidase.
6. The method of Claim 1 wherein red blood cells and
cell membrane fragments are removed using low speed centrifugation.
7. The method of Claim 1 wherein small molecules and cell metabolites are removed by dialysis with a 10,000 MW cut-off membrane.
8. The method of Claim 1 wherein hemoglobin is removed from the enzyme-containing solution by passing said solution through a CM52 column, a DE53 column or both columns.
9. The method of Claim 1 wherein the
enzyme-containing solution is concentrated by ultrafiltration using a 10,000 MW cut-off membrane.
10. The method of Claim 1 wherein the enzyme-containing solution is depyrogenated using a pyrogen-free Sephadex G-100 gel filtration column.
11. The method of Claim 1 wherein at least 80% of the enzyme is recovered from the enzyme-containing solution.
12. An enzyme recycled by the method of Claim 1.
13. An enzyme recycled from enzyme-treated blood product, said enzyme being substantially pure.
14. The enzyme of Claim 13 wherein said enzyme is α-galactosidase which was initially purified from coffee beans.
15. An enzyme recycled from enzyme-treated blood product, said enzyme being capable of removing antigens from the surface of cells in blood products.
16. The enzyme of Claim 15 wherein said enzyme is α-galactosidase which was initially purified from coffee beans.
17. An enzyme recycled from enzyme-treated blood product, said enzyme having specific activity similar to that of freshly purified enzyme.
18. The enzyme of Claim 16 wherein said enzyme is α-galactosidase which was initially purified from coffee beans.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU53650/94A AU5365094A (en) | 1992-10-22 | 1993-10-21 | Recycling of enzymes used to convert sub-type a, b and ab erythrocytes |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US96472792A | 1992-10-22 | 1992-10-22 | |
US07/964,727 | 1992-10-22 |
Publications (1)
Publication Number | Publication Date |
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WO1994009123A1 true WO1994009123A1 (en) | 1994-04-28 |
Family
ID=25508897
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1993/010066 WO1994009123A1 (en) | 1992-10-22 | 1993-10-21 | Recycling of enzymes used to convert sub-type a, b and ab erythrocytes |
Country Status (2)
Country | Link |
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AU (1) | AU5365094A (en) |
WO (1) | WO1994009123A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0807165A1 (en) * | 1995-01-30 | 1997-11-19 | The New York Blood Center, Inc. | RECOMBINANT $g(a)-GALACTOSIDASE ENZYME |
WO2003027245A3 (en) * | 2001-09-25 | 2003-07-10 | Zymequest Inc | CONVERSION OF RED BLOOD CELLS A, B, AND AB USING a-N-ACETYLGALACTOSAMINIDASES AND a-GALACTOSIDASE |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4427777A (en) * | 1980-08-14 | 1984-01-24 | New York Blood Center, Inc. | Enzymatic conversion of red cells for transfusion |
US4698302A (en) * | 1983-05-12 | 1987-10-06 | Advanced Magnetics, Inc. | Enzymatic reactions using magnetic particles |
-
1993
- 1993-10-21 AU AU53650/94A patent/AU5365094A/en not_active Abandoned
- 1993-10-21 WO PCT/US1993/010066 patent/WO1994009123A1/en active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4427777A (en) * | 1980-08-14 | 1984-01-24 | New York Blood Center, Inc. | Enzymatic conversion of red cells for transfusion |
US4698302A (en) * | 1983-05-12 | 1987-10-06 | Advanced Magnetics, Inc. | Enzymatic reactions using magnetic particles |
Non-Patent Citations (2)
Title |
---|
BIOCHIMICA ET BIOPHYSICA ACTA, Vol. 341, issued 1974, HARPAZ et al., "Purification of Coffee Bean Alpha-Galactosidase by Affinity Chromatography", pages 213-221. * |
JOURNAL OF BIOCHEMISTRY, Volume 90, No. 5, issued 1981, CHINEN et al., "Purification and Properties of Alpha-Galactosidase from Immature Stalks of Saccharum Officinarum (Sugar Cane)", pages 1453-1461. * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0807165A1 (en) * | 1995-01-30 | 1997-11-19 | The New York Blood Center, Inc. | RECOMBINANT $g(a)-GALACTOSIDASE ENZYME |
EP0807165A4 (en) * | 1995-01-30 | 1998-07-15 | New York Blood Center Inc | RECOMBINANT -g(a)-GALACTOSIDASE ENZYME |
WO2003027245A3 (en) * | 2001-09-25 | 2003-07-10 | Zymequest Inc | CONVERSION OF RED BLOOD CELLS A, B, AND AB USING a-N-ACETYLGALACTOSAMINIDASES AND a-GALACTOSIDASE |
US7767415B2 (en) | 2001-09-25 | 2010-08-03 | Velico Medical, Inc. | Compositions and methods for modifying blood cell carbohydrates |
US7993896B2 (en) | 2001-09-25 | 2011-08-09 | Velico Medical, Inc. | Streptomyces griseoplanus α-galactosidases for removing immunodominant α-galactose monosaccharides from blood group B or AB reactive cells |
US8697411B2 (en) | 2001-09-25 | 2014-04-15 | Velico Medical, Inc. | Streptomyces griseoplanus comprising an α-galactosidase for removing immunodominant α-galactose monosaccharides from blood group B or AB reactive cells |
Also Published As
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