CA1168152A - Process for preparing human plasma fractions containing immune globulin (igg) - Google Patents
Process for preparing human plasma fractions containing immune globulin (igg)Info
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- CA1168152A CA1168152A CA000388332A CA388332A CA1168152A CA 1168152 A CA1168152 A CA 1168152A CA 000388332 A CA000388332 A CA 000388332A CA 388332 A CA388332 A CA 388332A CA 1168152 A CA1168152 A CA 1168152A
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- igg
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/34—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood group antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hematology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
TITLE: AN IMPROVED PROCESS FOR PREPARING HUMAN PLASMA FRAC-TIONS CONTAINING IMMUNE GLOBULIN (IgG) ABSTRACT
An improved process for preparing human plasma frac-tions containing immune globulin (IgG) which is suitable for intravenous use comprises the use of a dilute solution con-taining IgG as starting material, preferably obtained as an eluate from the chromatographic separation of plasma on a DEAE-Sephadex or a QAE-Sephadex column. The dilute solution containing IgG is treated with a mixture of sodium chloride and glycine and the dilute solution thus obtained is subjected to ultrafiltration to provide a concentrated solution contain-ing IgG. The latter solution may, if desired, be freeze-dried to provide a solid composition of matter containing IgG.
An improved process for preparing human plasma frac-tions containing immune globulin (IgG) which is suitable for intravenous use comprises the use of a dilute solution con-taining IgG as starting material, preferably obtained as an eluate from the chromatographic separation of plasma on a DEAE-Sephadex or a QAE-Sephadex column. The dilute solution containing IgG is treated with a mixture of sodium chloride and glycine and the dilute solution thus obtained is subjected to ultrafiltration to provide a concentrated solution contain-ing IgG. The latter solution may, if desired, be freeze-dried to provide a solid composition of matter containing IgG.
Description
This invention relates to an improved process for the manufacture of human plasma fractions containing immune glob-ulin (IgG). The fractions may be obtained in the form of a concentrated aqueous solution or as a solid composition of matter containing IgG and these fractions are useful for in-travenous injection.
The human plasma to be used in the process of this invention may be normal human plasma to provide standard or normal human immune serum globulin (HSIG or NSIG) or it may be plasma which has a high-titer of particular antibodies such as Rh factor antibodies, rabies antibodies, tetanus an~
tibodies or zoster antibodies.
The process of this invention is particularly suit-able for the preparation of a concentrated solution contain-ing Rh immune globulin (Rh IgG) or a solid composition ofmatter containing Rh IgG, the solution and the solid composi-tion being useful for intravenous injection.
The prevention of Rh isoimmunization by the passive administration of anti-D is known and originally, high-titer anti-D plasma was given intravenously to the Rh-negative ex-pectant mother at risk. Two problems associated with the use of raw plasma are low Rh-antibody content and the risk of transmitting hepatitis. These problems were resolved by the introduction of an Rh IgG prepared by the Cohn cold ethanol fractionation of plasma with a high anti-D content. Unfor-tunately, IgG prepared by the Cohn method tends to form ag-gregates and thus precludes its use by intravenous admini-stration. Aggregates of IgG fi.x and activate complement in the same way as do antigen-antibody complexes. They could, if injected intravenously, produce severe adverse reactions.
An IgG which can be administered intravenously has a parti-cular advantage in the case of Rh prevention. Intravenous injection results in a much more rapid appearance of the Rh antibody in the circulation as ~ell as a higher maximum level.
In addition, intravenous injection causes much less discomfort than the intramuscular route.
We have now ~ound, and herein lies our inventio~, that an IgG preparation which is suitable for intravenous use can be prepared by treating a dilute aqueous solution containing IgG with a mixture of sodium.chloride and glycine and then concentrating the solution by means of ultrafiltration. The concentrated solution thus obtainea may be used as such for the preparation of intravenous solutions containing IgG or it may be freeze-dried to provide a solid composition of matter which can also be used to prepare intravenous solutions con-taining IgG.
~he invention as claimed herein is a process for the manufacture of an immune globulin (IgG) preparation w.hich is suitable for intravenous use which comprises treating a dilute solution containing IgG with a mixture of sodium chloride and glycine and subjecting said dilute solution to concentration by means of ultrafiltration to provide a concentrated solution containing IgG.
The dilute solution containing IgG used as starting material may be a solution obtained as an eluate from a chro-matographic separa~ion procedure using a column containing an ion exchange resin, for example a DEAE-Sephadex A-50 or a QAE-Sephadex A-50 ion exchange-resin. Such a dilute solution containing IgG, and particularly one containing Rh IgG, is useful as starting material in that the final product con-taining IgG, preferably Rh IgG, obtained therefrom is rela-tively pure and in high yield from.the original plasma. Thefinal product has a relatively.low content of aggregates and relatively low anticomplementary activity and is suitable for . .
~ * r~rade ~ark - 2 -intravenous use.
The process of this invention will be described fur-ther with particular reference to the manufacture of Rh immune globulin (Rh IgG) according to the following procedure:
Mat~iaIs. Membrane filters and ultrafilter membranes were purchased from Millipore Ltd. (Toronto, Canada). DEAE-Sephadex A-50 was purchased from Pharmacia (Canada) Ltd.
(Montreal). All other chemicals were of ACS reagent grade.
DEAE-Sephade~ ~o~umn preparation of R~ immune gIobuZin.
Rh immune globulin was prepared by the fractionation of high-titer Rh human source plasma on columns of DEAE-Sephadex by the following method:
DEAE-Sephadex A-50 (380 g) was treated sequentially with 1 M HCl, 1 M NaOH, and 0.25 M potassium phosphate buffer, pH 7.5, in a 70-liter Bel-art tabletop Buchner funnel. Be-tween each treatment the DEAE-Sephadex was washed with large volumes of pyrogen-free distilled water and finally suspended in 0.025 M phosphate buffer, pH 7.5, for autoclaving (121C, 15 psi, for 30 min.). After cooling the DEAE-Sephadex was packed into a 15 x 50-cm glass column and equilibrated with 0.025 M phosphate buffer, pH 7.5.
A 4-liter pool of Rh plasma was diluted with an equal volume of pyrogen-free water and was applied to the DEAE-Sephadex column at 500 ml/h. When all the plasma had ~een applied the column was eluted with equilibrating buffer (0.025 M phosphate, pH 7.5) at the same flow rate. The column ef-fluent absorption at 280 nm was monitored hy a Uvicord I
LKB-Production AB (Bromma, Sweden) and the 280-nm absorbing peak was collected in one vessel, sodium chloride and glycine were added to this effluent (Rh IgG solution) to a concentra-tion of 0.15 M and 0.1 M, respectively, in the effluent. The dilute Rh IgG solution (about 14 liters) was concentra-ted to ~8~
1000 ml by ultrafiltration with a Millipore Pelicon System using a PTGC 000 05 cassette membrane (NMWL of 10~000/5 ft ) (Millipore Ontario Canada). Column fractionation and efflu-ent concentration were carried out at 4C in a clean room. A11 equipment in contact at any stage of the production process was sterilized by autoclaving, ethylene oxide, or heating to 270C for 1 h. Pyrogen-free water prepared by glass distil-lation of reverse osmosis water was used for all washings and for the preparation of buffers.
The concentrated Rh IgG was centrifuged at 5000 rpm for 30 min. in a Sorvall RC-3 and sterilized via serial fil~
tration through 0.8-, 0.45- and 0.22-~m pore size membrane in a 142-mm stainless-steel membrane holder.
The anti-D concentration of the filtered concentrate was measured by an Auto-Analyzer technique. The concentrate was then filled into 5-ml serum vials in either one or the other of two concentrations and then lyophilized. The volume filled into each vial was sufficient to allow removal of in excess of either 120 or 240 ~g anti-D; the first concentration being for postpartum administration, the second for antenatal administration. All the equipment in contact with th~ product at any stage was sterilized by oven, steam, or ethylene oxide gas.
The purity, anticomplementary activity, safety (ste-rility and toxicity), hepatitis B surface antigen test, resi-dual moisture, quantitative determination of IgG subclasses and anti-A, -B, -C and -E levels in the lyophilized product were determined according to established procedures before the product (*"WinRho") was used in clinical trials, Results:
Preparation of Rh immune gZo~u~in, ~"Win~ho~'. Plasma was diluted with an equal volume of pyrogen-free distilled water prior to fractionation to reduce its ionic strength to * Trade Mark that o:E the eluting buffer (0.025 M phosphate, pH 7.5). The fractionation of the diluted plasma on the column of DEAE-Sephadex A-50 resulted in the adsorption of all the plasma proteins except Rh IgG which was eluted in a very dilute solu-tion (about 0.2 g%). Ultrafiltration of the Rh IgG in thephosphate buffer alone resulted in poor solubility after lyo-philization. However, the addition of sodium chloride and glycine to the column effluent significantly reduced the amount of precipitate formed during concentration. The sodium chlo-ride and ylycine stabilized the dilute Rh IgG during ultra-filtration and subsequent freeze-drying resulted in a readily soluble lyophilized Rh IgG. The Millipore cross flow ultra-filtration system appeared to produce minimum damage to the shear-sensitive IgG molecule in the presence of sodium chlo-ride and glycine.
Recoveries of anti-D (as measured by an ~utoanalyzer) in the final concentrated Rh IgG solution following filtra-tion averaged 91%. This high yield was not, however, an in-dication of the recovery of all of the IgG from the column since only 65 to 75% of all the IgG in the starting plasma was eluted frorn the DEAE-Sephadex.
S~ety. The rigid compliance to the asceptic pro-tocol set out for the production of "WinRho" has resulted in all lots being sterile, nonpyrogenic, and nontoxic according to the test methods required by the Health Protection Branch, Department of Health and Welfare, Canada.
Although the usual human dose of "WinRho" would be the content of one 120- or 240-~g vial, certain clinical in-dications may re~uire administration of several vials of "WinRho". The safety of "WinRho" was investigated by carry~
ing out an I,D50 (half the lethal dose) experiment. Vials of "WinRho" were reconstituted with 0.3 ml of 0.9% NaCl instead of the recommended 2.0 ml and several mice were injected in-travenously with varying doses of up to 1.25 ml per mouse.
The usual safety test dose is 0.5 ml of "WinRho" dissolved in
The human plasma to be used in the process of this invention may be normal human plasma to provide standard or normal human immune serum globulin (HSIG or NSIG) or it may be plasma which has a high-titer of particular antibodies such as Rh factor antibodies, rabies antibodies, tetanus an~
tibodies or zoster antibodies.
The process of this invention is particularly suit-able for the preparation of a concentrated solution contain-ing Rh immune globulin (Rh IgG) or a solid composition ofmatter containing Rh IgG, the solution and the solid composi-tion being useful for intravenous injection.
The prevention of Rh isoimmunization by the passive administration of anti-D is known and originally, high-titer anti-D plasma was given intravenously to the Rh-negative ex-pectant mother at risk. Two problems associated with the use of raw plasma are low Rh-antibody content and the risk of transmitting hepatitis. These problems were resolved by the introduction of an Rh IgG prepared by the Cohn cold ethanol fractionation of plasma with a high anti-D content. Unfor-tunately, IgG prepared by the Cohn method tends to form ag-gregates and thus precludes its use by intravenous admini-stration. Aggregates of IgG fi.x and activate complement in the same way as do antigen-antibody complexes. They could, if injected intravenously, produce severe adverse reactions.
An IgG which can be administered intravenously has a parti-cular advantage in the case of Rh prevention. Intravenous injection results in a much more rapid appearance of the Rh antibody in the circulation as ~ell as a higher maximum level.
In addition, intravenous injection causes much less discomfort than the intramuscular route.
We have now ~ound, and herein lies our inventio~, that an IgG preparation which is suitable for intravenous use can be prepared by treating a dilute aqueous solution containing IgG with a mixture of sodium.chloride and glycine and then concentrating the solution by means of ultrafiltration. The concentrated solution thus obtainea may be used as such for the preparation of intravenous solutions containing IgG or it may be freeze-dried to provide a solid composition of matter which can also be used to prepare intravenous solutions con-taining IgG.
~he invention as claimed herein is a process for the manufacture of an immune globulin (IgG) preparation w.hich is suitable for intravenous use which comprises treating a dilute solution containing IgG with a mixture of sodium chloride and glycine and subjecting said dilute solution to concentration by means of ultrafiltration to provide a concentrated solution containing IgG.
The dilute solution containing IgG used as starting material may be a solution obtained as an eluate from a chro-matographic separa~ion procedure using a column containing an ion exchange resin, for example a DEAE-Sephadex A-50 or a QAE-Sephadex A-50 ion exchange-resin. Such a dilute solution containing IgG, and particularly one containing Rh IgG, is useful as starting material in that the final product con-taining IgG, preferably Rh IgG, obtained therefrom is rela-tively pure and in high yield from.the original plasma. Thefinal product has a relatively.low content of aggregates and relatively low anticomplementary activity and is suitable for . .
~ * r~rade ~ark - 2 -intravenous use.
The process of this invention will be described fur-ther with particular reference to the manufacture of Rh immune globulin (Rh IgG) according to the following procedure:
Mat~iaIs. Membrane filters and ultrafilter membranes were purchased from Millipore Ltd. (Toronto, Canada). DEAE-Sephadex A-50 was purchased from Pharmacia (Canada) Ltd.
(Montreal). All other chemicals were of ACS reagent grade.
DEAE-Sephade~ ~o~umn preparation of R~ immune gIobuZin.
Rh immune globulin was prepared by the fractionation of high-titer Rh human source plasma on columns of DEAE-Sephadex by the following method:
DEAE-Sephadex A-50 (380 g) was treated sequentially with 1 M HCl, 1 M NaOH, and 0.25 M potassium phosphate buffer, pH 7.5, in a 70-liter Bel-art tabletop Buchner funnel. Be-tween each treatment the DEAE-Sephadex was washed with large volumes of pyrogen-free distilled water and finally suspended in 0.025 M phosphate buffer, pH 7.5, for autoclaving (121C, 15 psi, for 30 min.). After cooling the DEAE-Sephadex was packed into a 15 x 50-cm glass column and equilibrated with 0.025 M phosphate buffer, pH 7.5.
A 4-liter pool of Rh plasma was diluted with an equal volume of pyrogen-free water and was applied to the DEAE-Sephadex column at 500 ml/h. When all the plasma had ~een applied the column was eluted with equilibrating buffer (0.025 M phosphate, pH 7.5) at the same flow rate. The column ef-fluent absorption at 280 nm was monitored hy a Uvicord I
LKB-Production AB (Bromma, Sweden) and the 280-nm absorbing peak was collected in one vessel, sodium chloride and glycine were added to this effluent (Rh IgG solution) to a concentra-tion of 0.15 M and 0.1 M, respectively, in the effluent. The dilute Rh IgG solution (about 14 liters) was concentra-ted to ~8~
1000 ml by ultrafiltration with a Millipore Pelicon System using a PTGC 000 05 cassette membrane (NMWL of 10~000/5 ft ) (Millipore Ontario Canada). Column fractionation and efflu-ent concentration were carried out at 4C in a clean room. A11 equipment in contact at any stage of the production process was sterilized by autoclaving, ethylene oxide, or heating to 270C for 1 h. Pyrogen-free water prepared by glass distil-lation of reverse osmosis water was used for all washings and for the preparation of buffers.
The concentrated Rh IgG was centrifuged at 5000 rpm for 30 min. in a Sorvall RC-3 and sterilized via serial fil~
tration through 0.8-, 0.45- and 0.22-~m pore size membrane in a 142-mm stainless-steel membrane holder.
The anti-D concentration of the filtered concentrate was measured by an Auto-Analyzer technique. The concentrate was then filled into 5-ml serum vials in either one or the other of two concentrations and then lyophilized. The volume filled into each vial was sufficient to allow removal of in excess of either 120 or 240 ~g anti-D; the first concentration being for postpartum administration, the second for antenatal administration. All the equipment in contact with th~ product at any stage was sterilized by oven, steam, or ethylene oxide gas.
The purity, anticomplementary activity, safety (ste-rility and toxicity), hepatitis B surface antigen test, resi-dual moisture, quantitative determination of IgG subclasses and anti-A, -B, -C and -E levels in the lyophilized product were determined according to established procedures before the product (*"WinRho") was used in clinical trials, Results:
Preparation of Rh immune gZo~u~in, ~"Win~ho~'. Plasma was diluted with an equal volume of pyrogen-free distilled water prior to fractionation to reduce its ionic strength to * Trade Mark that o:E the eluting buffer (0.025 M phosphate, pH 7.5). The fractionation of the diluted plasma on the column of DEAE-Sephadex A-50 resulted in the adsorption of all the plasma proteins except Rh IgG which was eluted in a very dilute solu-tion (about 0.2 g%). Ultrafiltration of the Rh IgG in thephosphate buffer alone resulted in poor solubility after lyo-philization. However, the addition of sodium chloride and glycine to the column effluent significantly reduced the amount of precipitate formed during concentration. The sodium chlo-ride and ylycine stabilized the dilute Rh IgG during ultra-filtration and subsequent freeze-drying resulted in a readily soluble lyophilized Rh IgG. The Millipore cross flow ultra-filtration system appeared to produce minimum damage to the shear-sensitive IgG molecule in the presence of sodium chlo-ride and glycine.
Recoveries of anti-D (as measured by an ~utoanalyzer) in the final concentrated Rh IgG solution following filtra-tion averaged 91%. This high yield was not, however, an in-dication of the recovery of all of the IgG from the column since only 65 to 75% of all the IgG in the starting plasma was eluted frorn the DEAE-Sephadex.
S~ety. The rigid compliance to the asceptic pro-tocol set out for the production of "WinRho" has resulted in all lots being sterile, nonpyrogenic, and nontoxic according to the test methods required by the Health Protection Branch, Department of Health and Welfare, Canada.
Although the usual human dose of "WinRho" would be the content of one 120- or 240-~g vial, certain clinical in-dications may re~uire administration of several vials of "WinRho". The safety of "WinRho" was investigated by carry~
ing out an I,D50 (half the lethal dose) experiment. Vials of "WinRho" were reconstituted with 0.3 ml of 0.9% NaCl instead of the recommended 2.0 ml and several mice were injected in-travenously with varying doses of up to 1.25 ml per mouse.
The usual safety test dose is 0.5 ml of "WinRho" dissolved in
2.0 ml with observation of the mice for 7 days. A11 mice had normal weight gains and only -the l.0- and 1.25-ml dose caused convulsions which lasted 2 and 5 min., respec*ively. The 1.25-ml dose was the equivalent of the injection of eight vials (lO00 ~g anti-D) of "WinRho" to a 20 g mouse. The LD50(lethal dose) of "WinRho" was not determined since no mice died. This test indicated that very large doses are safe for human use.
; "WinRho" has also been shown to be safe in regard to the transmission of hepatitis B. All Rh source plasma frac-tionated into "WinRho" was obtained by the plasmapheresis of naturally immunized Rh-negative female volunteers by the Winnipeg Rh Institute under the direct supervision oi -the Medical Director of the Institute. The medical history of each donor is known and in each case there has been no clini-cal evidence of hepatitis. Each bag of Rh plasma is negative for ~IB3Ag when tested by the Abbott(Ausria II) RIA test as is ~ 20 each lot of final lyophilized Rh immune qlobulin.
; Stabi~ity. The relative Rh antibody to total protein ratio for "WinRho" is about 1.2%. In order to fill the vials with volumes of 1.0 to 2.0 ml the protein concentration of the Rh immune globulin solution containing 120 and 240 ~g would be about l and 2 g~. This protein concentration is too low to allow storage of "WinRho" as a liquid and retain sta-bility of anti-D activity for as long as l year. Lyophili-zation immediately after production of the Rh IgG provided a solid composition of matter. With the maintenance of resi-dual moisture levels below 3% the anti-D activity of "WinRho"
remained stable and fragmentation and aggregation remained below 3% for at least 18 months at ~4C., and at least 18 months at room temperature.
CZini~aZ t~iaZs. A detailed report of the preclinical and clinical trial of "WinRho" has been published. The com-bination of low protein, low anticomplementary activity and very low contamination with IgA make "WinRho" suitable for in-travenous as well as intramuscular administration. Our studies showed that intravenous administration of "WinRho" resulted in the appearance of maximum levels of anti-D in the serum of !~ volunteers within 1 h after administration compared with 2 to
; "WinRho" has also been shown to be safe in regard to the transmission of hepatitis B. All Rh source plasma frac-tionated into "WinRho" was obtained by the plasmapheresis of naturally immunized Rh-negative female volunteers by the Winnipeg Rh Institute under the direct supervision oi -the Medical Director of the Institute. The medical history of each donor is known and in each case there has been no clini-cal evidence of hepatitis. Each bag of Rh plasma is negative for ~IB3Ag when tested by the Abbott(Ausria II) RIA test as is ~ 20 each lot of final lyophilized Rh immune qlobulin.
; Stabi~ity. The relative Rh antibody to total protein ratio for "WinRho" is about 1.2%. In order to fill the vials with volumes of 1.0 to 2.0 ml the protein concentration of the Rh immune globulin solution containing 120 and 240 ~g would be about l and 2 g~. This protein concentration is too low to allow storage of "WinRho" as a liquid and retain sta-bility of anti-D activity for as long as l year. Lyophili-zation immediately after production of the Rh IgG provided a solid composition of matter. With the maintenance of resi-dual moisture levels below 3% the anti-D activity of "WinRho"
remained stable and fragmentation and aggregation remained below 3% for at least 18 months at ~4C., and at least 18 months at room temperature.
CZini~aZ t~iaZs. A detailed report of the preclinical and clinical trial of "WinRho" has been published. The com-bination of low protein, low anticomplementary activity and very low contamination with IgA make "WinRho" suitable for in-travenous as well as intramuscular administration. Our studies showed that intravenous administration of "WinRho" resulted in the appearance of maximum levels of anti-D in the serum of !~ volunteers within 1 h after administration compared with 2 to
3 days following the intramuscular injection of the same dose.
Peak anti-D levels were twice as high as when the same dose was given intramuscularly.
The clinical trial consisted of an intravenous injec-tion of "WinRho" at 28 weeks gestation to every unimmunized Rh-negative woman whose husband was Rh-positive or Rh unknown, followed by a second injection within 72 h after delivery of an Rh-positive baby. Our studies showed that a 240-~ anti-D
dose resulted in a demonstrable level of Rh antibody for at least 12 weeks and that a 120-~g dose was sufficient for pro-tection after delivery of an Rh-positive baby. The clinical trial showed "WinRho" to be a safe and effective means of prevention of Rh immunization.
The DEAE-Sephadex A-50 or QAE-Sephadex A-50 column method is very suitable for the production of the dilute aqueous solution of IgG used as starting material in the pro-cess of this invention. The procedure is relatively simple and the column technique lends itself to small scale produc-tion of hyperimmune globulin. The only pretreatment of the Rh plasma required before application to the column is reduc-tion in ionic strength to that of the eluting buffer by dilu-tion with pyrogen-free distilled water. Dilution of the plasma results in additional dilution of the Rh IgG eluted from the DEAE-Sephadex or OAE-Sephadex column. We have found that the tendency for pxecipitation oE Rh IgG during ultrafil-tration concentration of the dilute solution is significantly reduced by stabilization of the dilute Rh IgG by addition of sodium chloride and glycine to the column effluent to a con-centration of 0.15 M and 0.1 ~ respectively in the effluent.
The use of sodium chloride and glycine has been found by us to be a very effective and improved process for the stabilization of the dilute IgG as well as for increasing the solubility of lyophilized IgG preparations.
It will be understood that the degree of concentration of the dilute IgG solution by ultrafiltration may vary accord-ing to the type of immune globulin product being prepared.
In the case of Rh IgG, the concentration by ultrafiltration may be of the order of from about 1/10 to about 1/30, prefer-ably from about 1/15 to about 1/20. In the case of anti-rabies or anti-Zoster immune globulin, the concentration may be of the order of from about 1/10 to about 1/100.
The use of an enclosed sterile column and sterile plastic tubing completely isolates the plasma and effluent from the outside contaminated environment. This together with the sterilization procedures and elimination of pyrogens sig nificantly reduces the hazard of bacterial or pyrogen conta-mination of the final product.
Characterization of the product produced by the ion-exchange column shows that the Rh IgG is highly purified and is suitable for intravenous administration. Both immunoelec-trophoresis and double-diffusion analysis indicate a pure IgG
with no evidence of other plasma proteins. IgA, which is a frequent low-level con-taminate of Cohn ethanol-produced IgG
fractions, could not be found in "WinRho" when assayed by the low-level RID technique. Thus the concentration of IgA in "WinRho" must be 0.01 g/liter or less which is less than 10%
of the lowest concentration of IgA (0.1 g/liter) found in Cohn prepared IgGs produced by 13 different manufacturers.
Sedimentation velocity experiments demonstrated the homogeneity of the IgG produced by the ion-exchange method.
A single symmetrical peak was observed with a sedimentation ~ coefficient in the 6.0 S range. The purity and recovery of ;~ IgG from the DEAE-Sephadex or QAE-Sephadex column depends upon the ionic strength and pH of the eluting buffer. The use of 0.025 M phosphate buEfex, pH 7.5, results in a high-purity IgG but a low recovery of IgG from the plasma (70-80%). How-ever, the yield of Rh antibody in the preparation was consis-tently above 90%. The high yield of Rh activity relative to total IgG is not unexpected since IgG consists of a hetero-geneous group of proteins classified into four subclasses according to their types of heavy chains. Studies of the IgG
subclasses of anti-D indicate that it is restricted to the IgGl and IgG3 subclasses. Our observation that IgG produced by our method contains a higher percentage of IgGl and IgG3 and no IgG4 compared with normal plasma is in agreement with these studies. The loss of IgG during column fractionation appears to be due to the loss of the components with lower isoelectric point subclasses IgG2 and IgG4, which contain only a small percentage of anti-D.
soth precipitation of IgG and exposure to ethanol have been implicated as factors in the development of aggre-gates and anticomplementary activity in Cohn prepared IgG.
The present method of this invention separates IgG from other plasma proteins without the use of ethanol or any other pre-cipitation procedure. We have found that Rh IgG produced by the ion-exchange method and use of our stabilization process using sodium chloride and glycine, has a very small content g 5;2 of aggregates and very low-anticomplementary activity making it suitable for intravenous use. Our studies have shown that Rh IgG, ("WinRho"), when injected intravenously, not only appears in the circulation more rapidly but peak levels are twice as high as when it is given intramuscularly. Rh IgG, ("WinRho"), in solution is not stable in terms of anti-D con-tent for as long as one year when stored at 4C. but lyophi-lization results in a solid product which is stable for up to 18 months when stored at 4C. and can be dissolved readily when required for intravenous use.
The above process involving the use of a column ion-exchange method using, for example, DEAE-Sephadex A-50 or QAE-Sephadex A-50 as the ion exchange resin, for the preparation of a dilute solution of IgG and subsequent treatment with a mixture of sodium chloride and glycine as a stabilizer follow-ed by concentration by ultrafiltration provides a concentrated solution of purified IgG. The latter may be used to provide suitable preparations for intravenous use or it may be lyo-philized to produce a solid composition of matter containing IgG for intravenous use. This process is clearly the method of choice for the preparation of Rh IgG (anti-D) in the form of the product "WinRho".
It will be understood by one skilled in the art that the process of the present invention may likewise be applied as the process of choice for other immune globulin ~IgG~ pro-ducts for clinical use such as standard or normal human im-mune serum globulin (HSIG or NSIG), anti-tetanus immune serum globulin, anti-rabies immune serum globulin and anti-Zoster immune serum globulin.
Peak anti-D levels were twice as high as when the same dose was given intramuscularly.
The clinical trial consisted of an intravenous injec-tion of "WinRho" at 28 weeks gestation to every unimmunized Rh-negative woman whose husband was Rh-positive or Rh unknown, followed by a second injection within 72 h after delivery of an Rh-positive baby. Our studies showed that a 240-~ anti-D
dose resulted in a demonstrable level of Rh antibody for at least 12 weeks and that a 120-~g dose was sufficient for pro-tection after delivery of an Rh-positive baby. The clinical trial showed "WinRho" to be a safe and effective means of prevention of Rh immunization.
The DEAE-Sephadex A-50 or QAE-Sephadex A-50 column method is very suitable for the production of the dilute aqueous solution of IgG used as starting material in the pro-cess of this invention. The procedure is relatively simple and the column technique lends itself to small scale produc-tion of hyperimmune globulin. The only pretreatment of the Rh plasma required before application to the column is reduc-tion in ionic strength to that of the eluting buffer by dilu-tion with pyrogen-free distilled water. Dilution of the plasma results in additional dilution of the Rh IgG eluted from the DEAE-Sephadex or OAE-Sephadex column. We have found that the tendency for pxecipitation oE Rh IgG during ultrafil-tration concentration of the dilute solution is significantly reduced by stabilization of the dilute Rh IgG by addition of sodium chloride and glycine to the column effluent to a con-centration of 0.15 M and 0.1 ~ respectively in the effluent.
The use of sodium chloride and glycine has been found by us to be a very effective and improved process for the stabilization of the dilute IgG as well as for increasing the solubility of lyophilized IgG preparations.
It will be understood that the degree of concentration of the dilute IgG solution by ultrafiltration may vary accord-ing to the type of immune globulin product being prepared.
In the case of Rh IgG, the concentration by ultrafiltration may be of the order of from about 1/10 to about 1/30, prefer-ably from about 1/15 to about 1/20. In the case of anti-rabies or anti-Zoster immune globulin, the concentration may be of the order of from about 1/10 to about 1/100.
The use of an enclosed sterile column and sterile plastic tubing completely isolates the plasma and effluent from the outside contaminated environment. This together with the sterilization procedures and elimination of pyrogens sig nificantly reduces the hazard of bacterial or pyrogen conta-mination of the final product.
Characterization of the product produced by the ion-exchange column shows that the Rh IgG is highly purified and is suitable for intravenous administration. Both immunoelec-trophoresis and double-diffusion analysis indicate a pure IgG
with no evidence of other plasma proteins. IgA, which is a frequent low-level con-taminate of Cohn ethanol-produced IgG
fractions, could not be found in "WinRho" when assayed by the low-level RID technique. Thus the concentration of IgA in "WinRho" must be 0.01 g/liter or less which is less than 10%
of the lowest concentration of IgA (0.1 g/liter) found in Cohn prepared IgGs produced by 13 different manufacturers.
Sedimentation velocity experiments demonstrated the homogeneity of the IgG produced by the ion-exchange method.
A single symmetrical peak was observed with a sedimentation ~ coefficient in the 6.0 S range. The purity and recovery of ;~ IgG from the DEAE-Sephadex or QAE-Sephadex column depends upon the ionic strength and pH of the eluting buffer. The use of 0.025 M phosphate buEfex, pH 7.5, results in a high-purity IgG but a low recovery of IgG from the plasma (70-80%). How-ever, the yield of Rh antibody in the preparation was consis-tently above 90%. The high yield of Rh activity relative to total IgG is not unexpected since IgG consists of a hetero-geneous group of proteins classified into four subclasses according to their types of heavy chains. Studies of the IgG
subclasses of anti-D indicate that it is restricted to the IgGl and IgG3 subclasses. Our observation that IgG produced by our method contains a higher percentage of IgGl and IgG3 and no IgG4 compared with normal plasma is in agreement with these studies. The loss of IgG during column fractionation appears to be due to the loss of the components with lower isoelectric point subclasses IgG2 and IgG4, which contain only a small percentage of anti-D.
soth precipitation of IgG and exposure to ethanol have been implicated as factors in the development of aggre-gates and anticomplementary activity in Cohn prepared IgG.
The present method of this invention separates IgG from other plasma proteins without the use of ethanol or any other pre-cipitation procedure. We have found that Rh IgG produced by the ion-exchange method and use of our stabilization process using sodium chloride and glycine, has a very small content g 5;2 of aggregates and very low-anticomplementary activity making it suitable for intravenous use. Our studies have shown that Rh IgG, ("WinRho"), when injected intravenously, not only appears in the circulation more rapidly but peak levels are twice as high as when it is given intramuscularly. Rh IgG, ("WinRho"), in solution is not stable in terms of anti-D con-tent for as long as one year when stored at 4C. but lyophi-lization results in a solid product which is stable for up to 18 months when stored at 4C. and can be dissolved readily when required for intravenous use.
The above process involving the use of a column ion-exchange method using, for example, DEAE-Sephadex A-50 or QAE-Sephadex A-50 as the ion exchange resin, for the preparation of a dilute solution of IgG and subsequent treatment with a mixture of sodium chloride and glycine as a stabilizer follow-ed by concentration by ultrafiltration provides a concentrated solution of purified IgG. The latter may be used to provide suitable preparations for intravenous use or it may be lyo-philized to produce a solid composition of matter containing IgG for intravenous use. This process is clearly the method of choice for the preparation of Rh IgG (anti-D) in the form of the product "WinRho".
It will be understood by one skilled in the art that the process of the present invention may likewise be applied as the process of choice for other immune globulin ~IgG~ pro-ducts for clinical use such as standard or normal human im-mune serum globulin (HSIG or NSIG), anti-tetanus immune serum globulin, anti-rabies immune serum globulin and anti-Zoster immune serum globulin.
Claims (32)
1. A process for the manufacture of an immune globu-lin (IgG) preparation which is suitable for intravenous use which comprises treating a dilute solution containing IgG with a mixture of sodium chloride and glycine and subjecting said dilute solution to concentration by means of ultrafiltration to provide a concentrated solution containing IgG.
2. The process of claim 1 wherein the dilute solution containing IgG used as starting material is an eluate obtained by a chromatographic procedure from a column containing an ion-exchange resin.
3. The process of claim 2 wherein the ion-exchange resin is DEAE-Sephadex A-50 or QAE-Sephadex A-50.
4. The process of claim 3 wherein human plasma which may or may not be prior diluted with water is subjected to chromatographic separation on the ion exchange column and the column is then eluted with an equilibrating buffer of 0.025 M
phosphate buffer at a pH of 7.5.
phosphate buffer at a pH of 7.5.
5. The process of claim 1 wherein the dilute solution of IgG contains a high-titer of antibodies selected from the group consisting of pH antibodies, rabies antibodies, tetanus antibodies and zoster antibodies.
6. The process of claim 1 wherein the dilute solution of IgG is prepared from normal human plasma to provide stan-dard or normal human immune serum globulin (HSIG or NSIG).
7. The process of claim 1 wherein the mixture of so-dium chloride and glycine added to the dilute solution of IgG
is such that the concentration of sodium chloride in said di-lute solution is 0.15 M and the concentration of glycine in said dilute solution is 0.1 M prior to ultrafiltration.
is such that the concentration of sodium chloride in said di-lute solution is 0.15 M and the concentration of glycine in said dilute solution is 0.1 M prior to ultrafiltration.
8. The process of claim 1 wherein the ultrafiltration is carried out by means of a Millipore Pelicon System using a PTGC 000 05 cassette membrane (NMWL of 10,000/5 ft2).
9, The process of claim 1 wherein the ultrafiltration concentrates the dilute solution containing IgG to a volume which is from about 1/10 to about 1/100 of the volume of said dilute solution before said concentration.
10. The process of claim 1 wherein the ultrafiltration concentrates the dilute solution containing IgG to a volume which is from about 1/10 to about 1/30 of the volume of said dilute solution before said concentration.
11. The process of claim 1 wherein the ultrafiltration concentrates the dilute solution containing IgG to a volume which is from about 1/15 to about 1/20 of the volume before said concentration.
12. The process of claim 1 wherein the concentrated solution resulting from ultrafiltration is subsequently freeze-dried to produce a solid composition of matter con-taining IgG.
13. A process for the manufacture of a concentrated solution containing Rh immune globulin (Rh IgG) which is suitable for intravenous use which comprises diluting high-titer Rh human plasma with an equal volume of water, sub-jecting said diluted plasma to chromatographic separation by applying said diluted plasma to a DEAE-Sephadex A-50 or a QAE-Sephadex A-50 column, eluting said column with an equil-ibrating buffer comprising 0.025 M phosphate buffer of pH 7.5 to provide an eluate which is a dilute solution containing Rh IgG, adding sodium chloride and glycine to said dilute so-lution to provide a dilute solution containing Rh IgG having a concentration of 0.15 M sodium chloride and 0.1 M glycine therein, and then subjecting said dilute solution to ultra-filtration to provide a concentrated solution containing Rh IgG.
14. The process of claim 13 wherein the diluted plasma is applied to the column at a rate of 500 ml per hour and the equilibrating buffer is applied to the column at a rate of 500 ml per hour.
15. The process of claim 13 wherein the ultrafiltra-tion provides a concentrated solution containing Rh IgG which is from about 1/10 to about 1/30 of the volume of the dilute solution before concentration.
16. The process of claim 13 wherein the ultrafiltra-tion provides a concentrated solution containing Rh IgG which is from about 1/15 to about 1/20 of the volume of the dilute solution before concentration.
17. A process for the manufacture of a solid composi-tion of matter containing Rh IgG which is suitable for intra-venous use which comprises subjecting the concentrated solu-tion prepared as defined in claim 13 to freeze drying to pro-vide a solid composition of matter containing Rh IgG.
18. A process for the manufacture of a solid composi-tion of matter containing Rh IgG which is suitable for intra-venous use which comprises subjecting the concentrated solu-tion prepared as defined in claim 14 to freeze drying to pro-vide a solid composition of matter containing Rh IgG.
19. A process for the manufacture of a solid composi-tion of matter containing Rh IgG which is suitable for intra-venous use which comprises subjecting the concentrated solu-tion prepared as defined in claim 15 to freeze drying to pro-vide a solid composition of matter containing Rh IgG.
20. A process for the manufacture of a solid composi-tion of matter containing Rh IgG which is suitable for intra-venous use which comprises subjecting the concentrated solu-tion prepared as defined in claim 16 to freeze drying to pro-vide a solid composition of matter containing Rh IgG.
21. A concentrated solution containing IgG when pre-pared by the process of claim 1.
22. A concentrated solution containing IgG when pre-pared by the process of claim 2, 3 or 4.
23. A concentrated solution containing IgG when pre-pared by the process of claim 5, 6 or 7.
24. A concentrated solution containing IgG when pre-pared by the process of claim 8, 9 or 10.
25. A concentrated solution containing IgG when pre-pared by the process of claim 11.
26. A solid composition of matter containing IgG when prepared by the process of claim 12.
27. A concentrated solution containing Rh IgG when prepared by the process of claim 13.
28. A concentrated solution containing Rh IgG when prepared by the process of claim 14, 15 or 16.
29. A solid composition of matter containing Rh IgG
when prepared by the process of claim 17.
when prepared by the process of claim 17.
30. A solid composition of matter containing Rh IgG
when prepared by the process of claim 18.
when prepared by the process of claim 18.
31. A solid composition of matter containing Rh IgG
when prepared by the process of claim 19.
when prepared by the process of claim 19.
32. A solid composition of matter containing Rh IgG
when prepared by the process of claim 20.
when prepared by the process of claim 20.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000388332A CA1168152A (en) | 1981-10-20 | 1981-10-20 | Process for preparing human plasma fractions containing immune globulin (igg) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000388332A CA1168152A (en) | 1981-10-20 | 1981-10-20 | Process for preparing human plasma fractions containing immune globulin (igg) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1168152A true CA1168152A (en) | 1984-05-29 |
Family
ID=4121212
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000388332A Expired CA1168152A (en) | 1981-10-20 | 1981-10-20 | Process for preparing human plasma fractions containing immune globulin (igg) |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1168152A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0102731A2 (en) * | 1982-07-26 | 1984-03-14 | Ortho Diagnostic Systems Inc. | Methods for preparation of hepatitis B surface antigen free gamma globulins and products |
| EP0173999A2 (en) * | 1984-09-06 | 1986-03-12 | Biotest Pharma GmbH | Process for preparing a solution of milk and/or colostrum immunoglobins |
| EP0186360A2 (en) * | 1984-12-10 | 1986-07-02 | Ortho Diagnostic Systems Inc. | Methods for preparation of highly purified, gamma globulins free of hepatitis-B-virus infectivity |
| US5087695A (en) * | 1988-07-29 | 1992-02-11 | Mcy Laboratories Canada, Inc. | Immunoglobulin extraction utilizing properties of colloidal solutions |
| WO1995005196A1 (en) | 1993-08-13 | 1995-02-23 | Univax Biologics, Inc. | Antibody-based treatment of hiv infection |
-
1981
- 1981-10-20 CA CA000388332A patent/CA1168152A/en not_active Expired
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0102731A2 (en) * | 1982-07-26 | 1984-03-14 | Ortho Diagnostic Systems Inc. | Methods for preparation of hepatitis B surface antigen free gamma globulins and products |
| EP0102731A3 (en) * | 1982-07-26 | 1986-03-26 | Ortho Diagnostic Systems Inc. | Methods for preparation of hepatitis b surface antigen free gamma globulins and products |
| EP0173999A2 (en) * | 1984-09-06 | 1986-03-12 | Biotest Pharma GmbH | Process for preparing a solution of milk and/or colostrum immunoglobins |
| EP0173999A3 (en) * | 1984-09-06 | 1986-12-30 | Biotest Pharma Gmbh | Process for preparing a solution of milk and/or colostrum immunoglobins and its use |
| EP0186360A2 (en) * | 1984-12-10 | 1986-07-02 | Ortho Diagnostic Systems Inc. | Methods for preparation of highly purified, gamma globulins free of hepatitis-B-virus infectivity |
| EP0186360A3 (en) * | 1984-12-10 | 1986-12-03 | Ortho Diagnostic Systems Inc. | Methods for preparation of highly purified, gamma clobulins free of hepatitis-b-virus infectivity |
| US5087695A (en) * | 1988-07-29 | 1992-02-11 | Mcy Laboratories Canada, Inc. | Immunoglobulin extraction utilizing properties of colloidal solutions |
| WO1995005196A1 (en) | 1993-08-13 | 1995-02-23 | Univax Biologics, Inc. | Antibody-based treatment of hiv infection |
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