CA1201063A - Process for preparing purified immune globulin (igg) - Google Patents
Process for preparing purified immune globulin (igg)Info
- Publication number
- CA1201063A CA1201063A CA000407649A CA407649A CA1201063A CA 1201063 A CA1201063 A CA 1201063A CA 000407649 A CA000407649 A CA 000407649A CA 407649 A CA407649 A CA 407649A CA 1201063 A CA1201063 A CA 1201063A
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- Prior art keywords
- igg
- deae
- rich fraction
- buffer
- eluate
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
ABSTRACT
An improved process for preparing animal plasma frac-tions, particularly human plasma fractions, containing puri-fied immune globulin (IgG) which is suitable for intravenous use comprises the use of a dilute solution containing IgG as starting material. This solution is subjected to a two stage process of separation, firstly on an anion exchange column containing an agarose cross-linked anionic exchange resin and secondly, on an anion exchange column containing a different anionic exchange resin. The eluate thus obtained contains pure IgG an may, if desired, be freeze-dried to provide a solid composition of matter containing IgG.
An improved process for preparing animal plasma frac-tions, particularly human plasma fractions, containing puri-fied immune globulin (IgG) which is suitable for intravenous use comprises the use of a dilute solution containing IgG as starting material. This solution is subjected to a two stage process of separation, firstly on an anion exchange column containing an agarose cross-linked anionic exchange resin and secondly, on an anion exchange column containing a different anionic exchange resin. The eluate thus obtained contains pure IgG an 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 ~n;m~l plasma fractions, particularly human plasma fractions, containing immune globulin (IgG). The fractions may be obtained in the form of a concentrated aqueous solution or as a solid composition of matter contain-ing pure IgG. Such solution or solid compositions are use-ful for intravenous injection.
The animal, particularly human, plasma to be used in the process of this invention may be, for example, 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 antibodies or v~r;c~ -zQster ~nt;hn~
The process of this invention is particularly suit-able for the preparation of a concentrated solution contain-ing normal human immune serum globulin (HSIG) or a solid composition of matter containing HSIG, said solution and said solid composition being useful for intravenous injection.
We have now found, and herein lies our invention, that an IgG preparation which is suitable for intravenous use can be prepared by subjecting a plasma fraction in the form of a dilute aqueous solution containing IgG to a two stage separation process using two different anionic exchange resins. The dilute aqueous solution thus obtained r devoid of unwanted proteins and containing essentially IgG, may be stabilized, if desired, and concentra-ted. The concentrated solution thus p~epared may be used as such for the prepara-tion of intravenous solutions containing IgG. Alternatively, it may be freeze-dried to provide a solid composition of matter which may also be used to prepare intravenous solu-tions containing IgG.
The invention as claimed herein is an improved pro-........ ... .
cess for the preparation of purified immune globulin (IgG) which comprises (a) contacting an aqueous animal, preferably human, plasma fraction containing IgG with a first anionic exchanger to produce an IgG-rich fraction and then (b) con-tacting said IgG-rich fraction with a second different an-ionic exchanger to produce a purified IgG-rich fraction.
The invention as claimed herein is furthermore an improved process for the preparation of purified i~nune glo-bulin (IgG) which comprises (a) passing an aqueous animal, preferably human, plasma fraction cont~ining IgG through a first chromatographic separation column cont~;n;~g a first anionic exchanger to produce an IgG-rich fraction and then (b) passing said IgG-rich fraction through a second chroma-tographic separation column containing a second different anionic exchanger to produce a purified IgG-rich fraction.
The first anionic exchanger, which removes a substan-tial proportion of unwanted proteins, may ben an agarose cross-linked support anionic exchanger, such as DEAE-Sepha-rose CL6B or DEAE-Biogel. The DEAE-Sepharose CL6B may be used in conjunction with a buffex which may be from about 0.06M to about 0.08M sodium acetate~ preferably about 0.07M
SOaium acetate, at a pH within the range of from about 4.
to about 5.6, preferably at a pH of about 5.2. The DEAE-Biogel anionic exchanger may be used in conjunc~ion with a buffer which may be from about 0.015 to about 0.025M sodium acetate, prefexably about 0.02M sodium acetate, at a pH
within the range of from about 5.6 to about 6.4, preferably at a pH of about 6.0~
The second anionic exchanger which is used to remove the re -i ni ng minor ~nounts of unwanted proteins from the IgG-rich fraction may be any appropriate anionic exchanger known to be effective ~n adsorbing proteins from aqueous solution. Thus the second anionic exchanger may be, for ex-J~ ~
The animal, particularly human, plasma to be used in the process of this invention may be, for example, 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 antibodies or v~r;c~ -zQster ~nt;hn~
The process of this invention is particularly suit-able for the preparation of a concentrated solution contain-ing normal human immune serum globulin (HSIG) or a solid composition of matter containing HSIG, said solution and said solid composition being useful for intravenous injection.
We have now found, and herein lies our invention, that an IgG preparation which is suitable for intravenous use can be prepared by subjecting a plasma fraction in the form of a dilute aqueous solution containing IgG to a two stage separation process using two different anionic exchange resins. The dilute aqueous solution thus obtained r devoid of unwanted proteins and containing essentially IgG, may be stabilized, if desired, and concentra-ted. The concentrated solution thus p~epared may be used as such for the prepara-tion of intravenous solutions containing IgG. Alternatively, it may be freeze-dried to provide a solid composition of matter which may also be used to prepare intravenous solu-tions containing IgG.
The invention as claimed herein is an improved pro-........ ... .
cess for the preparation of purified immune globulin (IgG) which comprises (a) contacting an aqueous animal, preferably human, plasma fraction containing IgG with a first anionic exchanger to produce an IgG-rich fraction and then (b) con-tacting said IgG-rich fraction with a second different an-ionic exchanger to produce a purified IgG-rich fraction.
The invention as claimed herein is furthermore an improved process for the preparation of purified i~nune glo-bulin (IgG) which comprises (a) passing an aqueous animal, preferably human, plasma fraction cont~ining IgG through a first chromatographic separation column cont~;n;~g a first anionic exchanger to produce an IgG-rich fraction and then (b) passing said IgG-rich fraction through a second chroma-tographic separation column containing a second different anionic exchanger to produce a purified IgG-rich fraction.
The first anionic exchanger, which removes a substan-tial proportion of unwanted proteins, may ben an agarose cross-linked support anionic exchanger, such as DEAE-Sepha-rose CL6B or DEAE-Biogel. The DEAE-Sepharose CL6B may be used in conjunction with a buffex which may be from about 0.06M to about 0.08M sodium acetate~ preferably about 0.07M
SOaium acetate, at a pH within the range of from about 4.
to about 5.6, preferably at a pH of about 5.2. The DEAE-Biogel anionic exchanger may be used in conjunc~ion with a buffer which may be from about 0.015 to about 0.025M sodium acetate, prefexably about 0.02M sodium acetate, at a pH
within the range of from about 5.6 to about 6.4, preferably at a pH of about 6.0~
The second anionic exchanger which is used to remove the re -i ni ng minor ~nounts of unwanted proteins from the IgG-rich fraction may be any appropriate anionic exchanger known to be effective ~n adsorbing proteins from aqueous solution. Thus the second anionic exchanger may be, for ex-J~ ~
- 2 -* Trade Mark ample, selected from DEAE-Sepharose CL6B, DEAE-Sephadex A-50, DEAE-Biogel, DEAE-Trisacryl (a cross-linked acrylamide), a DEAE-cellulose (a granular form of cellulose support) and DEAE-Sephacel (a beaded form of cellulose support).
The IgG-rich fraction in the form of a dilute aqueous solution from the first separation column is preferably con-centrated, for example by ultrafiltration, and then equili-brated, for example by diafiltration, to the buffer which is to be used in the second anion exchange column~
The two anion exchange column process of this inven-tion may be carried out as follows:-1. Plasma, be it simply normal non-immunized plasma from a human source, or hyper-immune plasma such as tetanus, rabies, v~r;c~ -zoster or Rh plasma from a hu~an source, is mn~;f;~d, by diafiltration or column gel permeation, to the ionic strength and pH of the initial buffer to be used in the elu-tion of the first anion exchange column. This modification provides conditioned or equilibrated plasma to be used as starting material for the first anion exchange column.
2. The initial column fractionation procedure con-sists of applying this conditioned or equilibrated plasma to an anion exchange column which may contain an agarose cross-linked anionic exchange resin, for example, DEAE-Sepharose CL6B or DEAE-Biogel.
DEAE-Sepharose CL6B is used as the anionic exchanger in the first anion exchange column, the column is preferably pre-equilibratecl with a buffer which is within the range of from about 0.06M to about 0.08M, preferably 0.07M, sodium aceta-te at a pH
of from 4.8 to 5.6, preferably 5.2. This is the same buffer which would ear]ier have been used ror conditioning or equili-brating the plasma prior to applying such plasma to the column. Following the application of the equilibrated plasma to the column, an IgG-rich solution is obtained in the form of an eluate by elution of the column with the equi-librating buffer, i.e., the same sodium acetate ~uffer which is preferably 0.07M sodium acetate at a pH of 5.2.
Alternatively, if DEAE-Biogel is to be used as the anionic exchanger in the first anion exchange column, the buffer to be used for initially conditioning or equilibrating the plasma, for pre-equilibrating the column and for eluting the column to provide an IgG-xich solution in the form of an eluate maybe within the range of from about 0.015M to about 0.025M, preferably be 0.02M sodium acetate at a pH of from about 5.6 to about 6.4, preferably about 6Ø
The IgG-rich fraction in the form of a dilute aqueous solution from the first separation column is preferably con-centrated, for example by ultrafiltration, and then equili-brated, for example by diafiltration, to the buffer which is to be used in the second anion exchange column~
The two anion exchange column process of this inven-tion may be carried out as follows:-1. Plasma, be it simply normal non-immunized plasma from a human source, or hyper-immune plasma such as tetanus, rabies, v~r;c~ -zoster or Rh plasma from a hu~an source, is mn~;f;~d, by diafiltration or column gel permeation, to the ionic strength and pH of the initial buffer to be used in the elu-tion of the first anion exchange column. This modification provides conditioned or equilibrated plasma to be used as starting material for the first anion exchange column.
2. The initial column fractionation procedure con-sists of applying this conditioned or equilibrated plasma to an anion exchange column which may contain an agarose cross-linked anionic exchange resin, for example, DEAE-Sepharose CL6B or DEAE-Biogel.
DEAE-Sepharose CL6B is used as the anionic exchanger in the first anion exchange column, the column is preferably pre-equilibratecl with a buffer which is within the range of from about 0.06M to about 0.08M, preferably 0.07M, sodium aceta-te at a pH
of from 4.8 to 5.6, preferably 5.2. This is the same buffer which would ear]ier have been used ror conditioning or equili-brating the plasma prior to applying such plasma to the column. Following the application of the equilibrated plasma to the column, an IgG-rich solution is obtained in the form of an eluate by elution of the column with the equi-librating buffer, i.e., the same sodium acetate ~uffer which is preferably 0.07M sodium acetate at a pH of 5.2.
Alternatively, if DEAE-Biogel is to be used as the anionic exchanger in the first anion exchange column, the buffer to be used for initially conditioning or equilibrating the plasma, for pre-equilibrating the column and for eluting the column to provide an IgG-xich solution in the form of an eluate maybe within the range of from about 0.015M to about 0.025M, preferably be 0.02M sodium acetate at a pH of from about 5.6 to about 6.4, preferably about 6Ø
3. The IgG-rich dilute solution obt~ined as eluate from the first anionic ~chAnge column is concentrated by ultrafiltration to about O.Sg~ to l.Og% protein concentra-tion followed by diafiltration against 2 to 3 volumes of the eluting buffer which is to bP used in the secona anion exchange column.
4. The concentrated and diafiltered IgG-rich solu-tion is then applied to an equilibrated second anion ex-change column for further purification. If DEAE-Sepharose CL6B has been used in the first anion exchange column, the secor.d anion exchange column preferably contains either * *
DEAE-Biogel or DEAE-Sephadex A-50 as the second anionic exchanger. When DEAE-Biogel is used in the secon~ exchange column, the equilibrating and eluting buffer may be from about 0.015M 1:o about 0.025M sodium acetate, preferably 0.02M sodium acetate, at a pH of about 5.6 to ab~ut 6.4, preferably at about 6Ø When DEAE-Sephaaex A-50 is used in the second anion exchange column, the equilibrating and eluting buffer may be from about 0.02M to about 0.03~ potas-sium phosphate, preferably 0.025M potassium phosphate, at ,~.
* Trade Mark ,3 a pH of about 7.l to about 7.9, preferably at about 7.5.
Following application of the IgG-rich solution to the second anion exchange column, elution may be performed with the appropriate equilibrating buffer. The eluate provides a dilute solution of pure IgG. This dilute solution of IgG
may be stabilized by the addition of sodium chloride, for example to a concentration of 0.15~ sodium chloride, and glycine, for example to a concentration of O.lM glycine. The stabilized solution thus obtained may then be concentrated by ultrafiltration.
When DEAE-Biogel is used as the anionic exchanger in the first anion exchange column, it is preferable to use either DEAE-Sephadex A 50 or DEAE-Sepharose C16B as the an-ionic exchanger in the second anion exchange purification column. The procedure for applica-tion of the crude IgG
solution to the column, elution of the column, and concentra-tion of the dilute pure IgG, which may or may not be stab-ilized, is similar to the procedure outlined in paragraph 4 ahove. The equilibrating and eluting buffer to be used with DEAE-Sephadex A-50 in the second anion exchange column may be from about 0.02M to about 0.03M potassium phosphate, preferably 0.025M potassium phosphate, at a pH of about 7.l to about 7.9, preferably at about 7.5. Alternatively, the equilibrating and eluting buffer to be used with DEAE-Sepharose CL6B in the second anion exchange column may be from about 0.025M to about 0.035M sodium acetate, preferably 0.03M sodium acetate, at a pH of from about 4.8 to about 5.6, preferably at about 5.2.
DEAE-Biogel or DEAE-Sephadex A-50 as the second anionic exchanger. When DEAE-Biogel is used in the secon~ exchange column, the equilibrating and eluting buffer may be from about 0.015M 1:o about 0.025M sodium acetate, preferably 0.02M sodium acetate, at a pH of about 5.6 to ab~ut 6.4, preferably at about 6Ø When DEAE-Sephaaex A-50 is used in the second anion exchange column, the equilibrating and eluting buffer may be from about 0.02M to about 0.03~ potas-sium phosphate, preferably 0.025M potassium phosphate, at ,~.
* Trade Mark ,3 a pH of about 7.l to about 7.9, preferably at about 7.5.
Following application of the IgG-rich solution to the second anion exchange column, elution may be performed with the appropriate equilibrating buffer. The eluate provides a dilute solution of pure IgG. This dilute solution of IgG
may be stabilized by the addition of sodium chloride, for example to a concentration of 0.15~ sodium chloride, and glycine, for example to a concentration of O.lM glycine. The stabilized solution thus obtained may then be concentrated by ultrafiltration.
When DEAE-Biogel is used as the anionic exchanger in the first anion exchange column, it is preferable to use either DEAE-Sephadex A 50 or DEAE-Sepharose C16B as the an-ionic exchanger in the second anion exchange purification column. The procedure for applica-tion of the crude IgG
solution to the column, elution of the column, and concentra-tion of the dilute pure IgG, which may or may not be stab-ilized, is similar to the procedure outlined in paragraph 4 ahove. The equilibrating and eluting buffer to be used with DEAE-Sephadex A-50 in the second anion exchange column may be from about 0.02M to about 0.03M potassium phosphate, preferably 0.025M potassium phosphate, at a pH of about 7.l to about 7.9, preferably at about 7.5. Alternatively, the equilibrating and eluting buffer to be used with DEAE-Sepharose CL6B in the second anion exchange column may be from about 0.025M to about 0.035M sodium acetate, preferably 0.03M sodium acetate, at a pH of from about 4.8 to about 5.6, preferably at about 5.2.
5. The concentrated pure IgG solution obtained as the elua-te from the second anion exchange column may be further stabilized by the addition of mannitol within the range of from about 5% to about 10%, preferably about 7%, and adjustment of the solution to a pH within the range of from about 6.0 to about 6.5, preferably about 6.2. The con-centration of IgG in such a solution is from about 4g~ to lOg% and the stabilized solution contains 0.15M sodium chloride, O.lM glycine and from about 5 to about 10%, pre-ferably about 7%, of mannitol as stabilizers. This stabi-lized IgG solution is then sterile filtered and is thereafter ready for vialing. In certain cases, it may be stored as such ready for use and then used in this form. If desired, the vialled solution may be freeze-dried in order to provide additional stabilization for the IgG product in the form of a solid stabilized composition.
The process of this invention will be described fur-ther with particular reference to the manufacture of human serum immune globulin (HSIG) according to the following pro-cedure:
Ma~eriaZs. Membrane filters and ultrafilter membranes were purchased from Millipore Ltd. (Toronto, Canada). DEAE-Sepharose CL6B and DEAE-Sephadex A-50 were purchased from Pharmacia (Canada) Ltd. (Montreal). DEAE-Biogel was pur-chased from Biorad Laboratories, Richmond, California. All other chemicals were of ACS reagent grade.
EXAMPLE
About 20 litres of ~n;m~l plasma, such as human plasma, is thawed and asceptically pooled. The cryopreci-pitate which is thus formed is then removed by centrifuga-tion at 5200 rpm for 15 minutes to provide a plasma super-natant liquid. This supernatant liquid is treated with DEAE-Sephadex A-50 at an amount of 1 gm/litre by adding the DEAE-Sephadex A-50 to the liquid, mixing and then allow-ing the mixture to settle for about 30 minutes. The mixture is filtered to remove the DEAE-Sephadex A-50 which contains unwanted complex products adsorbed thereto. The filtrate is a plasma solution containing IgG to be used as star-ting material.
The ionic strength of this plasma filtrate is reduced by diafiltration against 4 volumes of pyrogen-free water for injection (WFI). The diafiltered plasma is then sterilized by serial filtration down to 0.20~ Pall filter into a sterile 50 litre polypropylene carboy and there is thus obtained the sterile plasma filtrate containing IgG which is ready for the first anionic exchange fractionation step on an anion exchange column.
The first anion exchange column contains a DEAE-Sepharose CL6B anion exchanger (or a similar kind of an agarose anion exchanger for this first stage of purification).
15 Litres of DEAE-Sepharose CL6B is prepared by an initial wash with a O.lM sodium acetate buffer at pH 6.0, followed by sterilization by autoclaving at 121C., 15 psi for 30 min. The steriIized DEAE-Sepharose CL6B is packed lnto a 15 cm x 370 cm plastic column by the procedure recommended by the supplier of the anion exchanger. The packed column is then equilibrated with a buffer which is a 0.07M sodium acetate buffer at pH 5.2, the buffer passing at a flow rate of 25 litres/hour and at a temperature of 4 to 8C. This equilibration procedure requires approximately 100 litres of buffer.
An aliquot of 10 litres of the sterile plasma fil-trate containing IgG, prepared as above, is applied to the equilibrated column of DEAE-Sepharose CL6B via sterilized polypropylene tubing and a metering pump to provide a flow rate of 25 litres/hour of filtrate to the column. The elu-tion of protein is monitored with a continuous flow U.V.
fi~
monitor using a 280 nm wavelength. The eluting protein, which is an IgG-rich fraction and primarily IgG, is collect-ed in a 50 litre sterilized polypropylene container and this eluate is hereafter referred to as DCl. Following complete application of the 10 litres of plasma filtrate to the column, the elution of IgG from the column is continued using 0.07M sodium acetate buffer at pH 5.2, thus providing more DCl eluate. The plasma proteins not eluted from the column with this 0.07M sodium acetate buffer at pH 5.2 (which include albumin and the other ~-globulins) are sub-sequently eluted from the DEAE-Sepharose CL6B column with sodium acetate buffers of higher ionic strength and lower pH. Following elution with the third buffer the column of DEAE-Sepharose CL6B is re-equilibrated with 0.07M sodium acetate buffer at pH 5.2, in preparation for application of another 10 litres batch of sterile plasma filtrate. The DEAE-Sepharose CL6B column may be re-used in this way for numerous fractionation cycles before fresh DEAE-Sepharose CL6B must be charged to the column.
The IgG-rich eluate, DCl, is concentrated to about 20 litres by ultrafiltration with a Millipore Pellicon U.F.
system, followed by diafiltration on the same system against 45 litres of 0.025M potassium phosphate buffer at pH 7.5.
This IgG-rich solu-tion is then ready for the second anion exchange column to achieve fractionation on DEAE-Sephadex A-50 as the second anionic exchanger.
The DEAE-Sephadex A-50 is prepared for column frac-tionation by the following technique. An aliquot of 750 g of DEAE-Sephadex A-50 is transferred to 20 litres of O.lM
potassium phosphate buffer at pH 7.5, incubated for 1 hour and then sterilized by autoclaving at 121C., 15 psi for 30 min. The sterilized DEAE-Sephadex A-50 slurry is trans-3;~
ferred to a 15 cm x 370 cm sectional column and packed ac-cording to the instructions of the supplier, Pharmacia Fine Chemicals. During packing the DEAE-Sephadex A-50 is depyro-genated in the column by washing with g litres of the follow-ing series of solutions: 0.5M hydrochloric acid, WFI, 0.05M
sodium hydroxide, WFI and 0.25M potassium phosphate buffer at pH 7.5. The packed column of DEAE-Sephadex A-50 is finally equilibrated with 0.025M potassium phosphate buffer at pH 7.5 at a fIow rate of about 15 litres/hour. This equilibration requires about 150 litres of buffer. There is thus prepared the second anion exchange column containing DEAE-Sephadex A-50 for purification of the IgG-rich solution.
The IgG-rich solutions, prepared as above, from two 10 litre plasma fractionations on the first anion exchange column are pooled and are applied as one sample to the equilibrated second anion exchange column containing DEAE-Sephadex A-50. Following the complete application of the IgG-rich solution to the column, the eluate obtained from the column contains pure IgG and is collected in a sterili-zed 50 litre polypropylene container. Elution of the column is continued by use of 0.025M potassium phosphate buffer at pH 7.5. The dilute solution of pure IgG thus obtained (about 40 litres) is stabilized by the addition of suffi-cient glycine and sodium chloride to provide a concentration of 0.lM glycine and 0.15M sodium chloride in the dilute solution of IgG. The dilute solution of IgG thus stabilized (about 44 litres) is subsequently concentrated to about one litre by ultrafiltration with a Millipore Pellicon U~Fo System using a PTGC 000 05 cassette membrane (NMWL of 10,000/5f-t. )(Millipore Ontario Canada). Finally, the con-centrated solution of IgG is further stabilized by the addi-tion of 7% mannitol.
_ 9 _ ~?;~ fi~3 The pure concentrated IgG, hereafter referred to as HSIG i.v., was clarified by centrifugation at 5200 rpm for 30 min. at 4C. and was then sterile filtered with a 0.2 membrane filter in preparation for vialling.
The HSIG i.v. prepared as described above was shown to be pure IgG following examination by immunoelectrophore-sis and by double diffusion against anti-human whole serum.
HSIG i.v. contained less than 2% aggregated IgG and less than 1% fragmented IgG. The anti-complementary activity assays of 5g% to 8g% solutions of HSIG i.v. yielded titres of 1/8 or less indicating suitability for intravenous ad-ministration. Several lots of HSIG i.v. have been produced and the quality of control results indicate they meet the pyrogen free, safety, steril:ity, hepatitis B surface anti-gen-free, and purity requirements of the Bureau of Biologics, Health Protection Branch, Health and Weifare, Canada.
It will be appreciated, by one skilled in the art, that the DEAE-Sepharose CL6B or DEAE-Biogel used as the anionic exchanger in the first anion exchange column may be replaced by any other agarose anionic exchanger. Likewise, the DEAE-Sepharose cL6s or DEAE-Sephadex A-50 or DEAE-sioge used as the anionic exchanger in the second anion exchange column may be replaced by any other suitable anionic ex-changer having similar anion exchange properties in being effective in the adsorption of unwanted proteins. It will be understood that the first anionic exchanger removes the bulk of the unwanted proteins, possibly of the order of about 85% or about 90%, from the plasma fraction used as starting material to provide an IgG-rich solution. The second anionic exchanger is effective in removing the residual unwanted proteins from that solution thereby pro-viding a solution containing pure IgG. The second anionic exchanger may thus, for example, be DEAE-Sepharose CL6B, DEAE-Sephadex A-50, DEAE-Biogel, DEAE-Trisacryl, a DEAE-cellulose (granular cellulose support) or DEAE-Sephocel (a beaded form cellulose).
It is preferred that the anionic exchanger to be used in the first column is DEAE-Sepharose CL6B and the anionic exchanger to be used in the second column is DEAE-Sephadex A-50.
When the anionic exchanger to be used in the first column is DEAE-Sepharose CL6B, the buffer used for equili-brating the column and for elution may be from about 0.06M
to about 0.08M sodium acetate, preferably 0.07M sodium ace-tate, at a pH within the range of from about 4.8 to about 5.6, preferably at a pH of about 5.2. Under these condi-tiOhS, the anionic exchanger to be used in the second column may be either DEAE-Biogel with a buffer from about 0.015M
to about 0.025M sodium acetate, preferably 0.02M sodium ace-tate, at a pH from about 5.6 to about 6.4, preferably at pH
The process of this invention will be described fur-ther with particular reference to the manufacture of human serum immune globulin (HSIG) according to the following pro-cedure:
Ma~eriaZs. Membrane filters and ultrafilter membranes were purchased from Millipore Ltd. (Toronto, Canada). DEAE-Sepharose CL6B and DEAE-Sephadex A-50 were purchased from Pharmacia (Canada) Ltd. (Montreal). DEAE-Biogel was pur-chased from Biorad Laboratories, Richmond, California. All other chemicals were of ACS reagent grade.
EXAMPLE
About 20 litres of ~n;m~l plasma, such as human plasma, is thawed and asceptically pooled. The cryopreci-pitate which is thus formed is then removed by centrifuga-tion at 5200 rpm for 15 minutes to provide a plasma super-natant liquid. This supernatant liquid is treated with DEAE-Sephadex A-50 at an amount of 1 gm/litre by adding the DEAE-Sephadex A-50 to the liquid, mixing and then allow-ing the mixture to settle for about 30 minutes. The mixture is filtered to remove the DEAE-Sephadex A-50 which contains unwanted complex products adsorbed thereto. The filtrate is a plasma solution containing IgG to be used as star-ting material.
The ionic strength of this plasma filtrate is reduced by diafiltration against 4 volumes of pyrogen-free water for injection (WFI). The diafiltered plasma is then sterilized by serial filtration down to 0.20~ Pall filter into a sterile 50 litre polypropylene carboy and there is thus obtained the sterile plasma filtrate containing IgG which is ready for the first anionic exchange fractionation step on an anion exchange column.
The first anion exchange column contains a DEAE-Sepharose CL6B anion exchanger (or a similar kind of an agarose anion exchanger for this first stage of purification).
15 Litres of DEAE-Sepharose CL6B is prepared by an initial wash with a O.lM sodium acetate buffer at pH 6.0, followed by sterilization by autoclaving at 121C., 15 psi for 30 min. The steriIized DEAE-Sepharose CL6B is packed lnto a 15 cm x 370 cm plastic column by the procedure recommended by the supplier of the anion exchanger. The packed column is then equilibrated with a buffer which is a 0.07M sodium acetate buffer at pH 5.2, the buffer passing at a flow rate of 25 litres/hour and at a temperature of 4 to 8C. This equilibration procedure requires approximately 100 litres of buffer.
An aliquot of 10 litres of the sterile plasma fil-trate containing IgG, prepared as above, is applied to the equilibrated column of DEAE-Sepharose CL6B via sterilized polypropylene tubing and a metering pump to provide a flow rate of 25 litres/hour of filtrate to the column. The elu-tion of protein is monitored with a continuous flow U.V.
fi~
monitor using a 280 nm wavelength. The eluting protein, which is an IgG-rich fraction and primarily IgG, is collect-ed in a 50 litre sterilized polypropylene container and this eluate is hereafter referred to as DCl. Following complete application of the 10 litres of plasma filtrate to the column, the elution of IgG from the column is continued using 0.07M sodium acetate buffer at pH 5.2, thus providing more DCl eluate. The plasma proteins not eluted from the column with this 0.07M sodium acetate buffer at pH 5.2 (which include albumin and the other ~-globulins) are sub-sequently eluted from the DEAE-Sepharose CL6B column with sodium acetate buffers of higher ionic strength and lower pH. Following elution with the third buffer the column of DEAE-Sepharose CL6B is re-equilibrated with 0.07M sodium acetate buffer at pH 5.2, in preparation for application of another 10 litres batch of sterile plasma filtrate. The DEAE-Sepharose CL6B column may be re-used in this way for numerous fractionation cycles before fresh DEAE-Sepharose CL6B must be charged to the column.
The IgG-rich eluate, DCl, is concentrated to about 20 litres by ultrafiltration with a Millipore Pellicon U.F.
system, followed by diafiltration on the same system against 45 litres of 0.025M potassium phosphate buffer at pH 7.5.
This IgG-rich solu-tion is then ready for the second anion exchange column to achieve fractionation on DEAE-Sephadex A-50 as the second anionic exchanger.
The DEAE-Sephadex A-50 is prepared for column frac-tionation by the following technique. An aliquot of 750 g of DEAE-Sephadex A-50 is transferred to 20 litres of O.lM
potassium phosphate buffer at pH 7.5, incubated for 1 hour and then sterilized by autoclaving at 121C., 15 psi for 30 min. The sterilized DEAE-Sephadex A-50 slurry is trans-3;~
ferred to a 15 cm x 370 cm sectional column and packed ac-cording to the instructions of the supplier, Pharmacia Fine Chemicals. During packing the DEAE-Sephadex A-50 is depyro-genated in the column by washing with g litres of the follow-ing series of solutions: 0.5M hydrochloric acid, WFI, 0.05M
sodium hydroxide, WFI and 0.25M potassium phosphate buffer at pH 7.5. The packed column of DEAE-Sephadex A-50 is finally equilibrated with 0.025M potassium phosphate buffer at pH 7.5 at a fIow rate of about 15 litres/hour. This equilibration requires about 150 litres of buffer. There is thus prepared the second anion exchange column containing DEAE-Sephadex A-50 for purification of the IgG-rich solution.
The IgG-rich solutions, prepared as above, from two 10 litre plasma fractionations on the first anion exchange column are pooled and are applied as one sample to the equilibrated second anion exchange column containing DEAE-Sephadex A-50. Following the complete application of the IgG-rich solution to the column, the eluate obtained from the column contains pure IgG and is collected in a sterili-zed 50 litre polypropylene container. Elution of the column is continued by use of 0.025M potassium phosphate buffer at pH 7.5. The dilute solution of pure IgG thus obtained (about 40 litres) is stabilized by the addition of suffi-cient glycine and sodium chloride to provide a concentration of 0.lM glycine and 0.15M sodium chloride in the dilute solution of IgG. The dilute solution of IgG thus stabilized (about 44 litres) is subsequently concentrated to about one litre by ultrafiltration with a Millipore Pellicon U~Fo System using a PTGC 000 05 cassette membrane (NMWL of 10,000/5f-t. )(Millipore Ontario Canada). Finally, the con-centrated solution of IgG is further stabilized by the addi-tion of 7% mannitol.
_ 9 _ ~?;~ fi~3 The pure concentrated IgG, hereafter referred to as HSIG i.v., was clarified by centrifugation at 5200 rpm for 30 min. at 4C. and was then sterile filtered with a 0.2 membrane filter in preparation for vialling.
The HSIG i.v. prepared as described above was shown to be pure IgG following examination by immunoelectrophore-sis and by double diffusion against anti-human whole serum.
HSIG i.v. contained less than 2% aggregated IgG and less than 1% fragmented IgG. The anti-complementary activity assays of 5g% to 8g% solutions of HSIG i.v. yielded titres of 1/8 or less indicating suitability for intravenous ad-ministration. Several lots of HSIG i.v. have been produced and the quality of control results indicate they meet the pyrogen free, safety, steril:ity, hepatitis B surface anti-gen-free, and purity requirements of the Bureau of Biologics, Health Protection Branch, Health and Weifare, Canada.
It will be appreciated, by one skilled in the art, that the DEAE-Sepharose CL6B or DEAE-Biogel used as the anionic exchanger in the first anion exchange column may be replaced by any other agarose anionic exchanger. Likewise, the DEAE-Sepharose cL6s or DEAE-Sephadex A-50 or DEAE-sioge used as the anionic exchanger in the second anion exchange column may be replaced by any other suitable anionic ex-changer having similar anion exchange properties in being effective in the adsorption of unwanted proteins. It will be understood that the first anionic exchanger removes the bulk of the unwanted proteins, possibly of the order of about 85% or about 90%, from the plasma fraction used as starting material to provide an IgG-rich solution. The second anionic exchanger is effective in removing the residual unwanted proteins from that solution thereby pro-viding a solution containing pure IgG. The second anionic exchanger may thus, for example, be DEAE-Sepharose CL6B, DEAE-Sephadex A-50, DEAE-Biogel, DEAE-Trisacryl, a DEAE-cellulose (granular cellulose support) or DEAE-Sephocel (a beaded form cellulose).
It is preferred that the anionic exchanger to be used in the first column is DEAE-Sepharose CL6B and the anionic exchanger to be used in the second column is DEAE-Sephadex A-50.
When the anionic exchanger to be used in the first column is DEAE-Sepharose CL6B, the buffer used for equili-brating the column and for elution may be from about 0.06M
to about 0.08M sodium acetate, preferably 0.07M sodium ace-tate, at a pH within the range of from about 4.8 to about 5.6, preferably at a pH of about 5.2. Under these condi-tiOhS, the anionic exchanger to be used in the second column may be either DEAE-Biogel with a buffer from about 0.015M
to about 0.025M sodium acetate, preferably 0.02M sodium ace-tate, at a pH from about 5.6 to about 6.4, preferably at pH
6.0, or DEAE-Sephadex A-50 with a buffer from about 0.02M
to about 0.03M potassium phosphate~ preferably 0.025M potas-sium phospate, at a pH of from about 7.1 to about 7.9, pre-ferably a pH of about 7.5.
When the anionic exchanger to be used in the first column is DEAE-Biogel, the buffer used for equilibrating the column and for elution may be from about 0.015M to about 0.025M sodium acetate, preferably 0.02M sodium ace-tate at a pH of from about 5.6 to about 6.4, preferably at pH about 6Ø Under these conditions, the anionic exchanger to be used in the second column may be either DEAE-Sepharose CL6B
with a buffer from about 0.025M to about 0.035M sodium ace-tate, preferably 0.03M sodium acetate at a pH from about 4.8 to about 5.6, preferably a pH of about 5.2 or DEAE-Sephadex i3 A-50 with a buffer from about 0.02M to about 0.03M potassium phosphate, preferably about 0.025M potassium phosphate, at a pH from about 7.1 to about 7.9, preferably at a pH of about
to about 0.03M potassium phosphate~ preferably 0.025M potas-sium phospate, at a pH of from about 7.1 to about 7.9, pre-ferably a pH of about 7.5.
When the anionic exchanger to be used in the first column is DEAE-Biogel, the buffer used for equilibrating the column and for elution may be from about 0.015M to about 0.025M sodium acetate, preferably 0.02M sodium ace-tate at a pH of from about 5.6 to about 6.4, preferably at pH about 6Ø Under these conditions, the anionic exchanger to be used in the second column may be either DEAE-Sepharose CL6B
with a buffer from about 0.025M to about 0.035M sodium ace-tate, preferably 0.03M sodium acetate at a pH from about 4.8 to about 5.6, preferably a pH of about 5.2 or DEAE-Sephadex i3 A-50 with a buffer from about 0.02M to about 0.03M potassium phosphate, preferably about 0.025M potassium phosphate, at a pH from about 7.1 to about 7.9, preferably at a pH of about
7.5.
When the anionic exchanger to be used in the second column is DEAE-Trisacryl, a suitable buffer may be 0.025M
potassium phosphate at a pH of about 7.5.
When the anionic exch~ger to be used in the second column is DEAE-cellulose ~granular cellulose support) or DEAE-Sephacel (a beaded form cellulose), a suitable buffer may be 0.025M potassium phosphate at a pH of about 7.5 or 0.03M sodium acetate at a pH of about 502 or 0.02M sodium acetate at a pH of about 6Ø
~, - 12 -* Trade Mark
When the anionic exchanger to be used in the second column is DEAE-Trisacryl, a suitable buffer may be 0.025M
potassium phosphate at a pH of about 7.5.
When the anionic exch~ger to be used in the second column is DEAE-cellulose ~granular cellulose support) or DEAE-Sephacel (a beaded form cellulose), a suitable buffer may be 0.025M potassium phosphate at a pH of about 7.5 or 0.03M sodium acetate at a pH of about 502 or 0.02M sodium acetate at a pH of about 6Ø
~, - 12 -* Trade Mark
Claims (28)
1. An improved process for the preparation of puri-fied immune globulin (IgG) which comprises (a) contacting an aqueous animal plasma fraction containing IgG with a first anionic exchanger to produce an IgG-rich fraction and then (b) contacting said IgG-rich fraction with a second different anionic exchanger to produce a purified IgG-rich fraction.
2. An improved process for the preparation of puri-fied immune globulin (IgG) which comprises (a) passing an aqueous animal plasma fraction containing IgG through a first chromatographic separation column containing a first anionic exchanger to produce an IgG-rich fraction and then (b) pass-ing said IgG-rich fraction through a second chromatographic separation column containing a second different anionic ex-changer to produce a purified IgG-rich fraction.
3. The process as in claim 2 wherein the first anionic exchanger is an agarose cross-linked support anionic exchanger.
4. The process as in claim 2 wherein the first anionic exchanger is DEAE-Sepharose CL6B or DEAE-Biogel.
5. The process as in claim 2 wherein the first anionic exchanger is DEAE-Sepharose CL6B and the IgG-rich fraction is obtained therefrom as an eluate by use of a buffer which con-tains from about 0.06M to about 0.08M sodium acetate at a pH
within the range of from 4.8 to 5.6.
within the range of from 4.8 to 5.6.
6. The process as in claim 2 wherein the first anionic exchanger is DEAE-Sepharose CL6B and the IgG-rich fraction is obtained therefrom as an eluate by use of a buffer which is 0.07M sodium acetate at a pH of 5.2.
7, The process as in claim 2 wherein the first anio-nic exchanger is DEAE-Biogel and the IgG-rich fraction is obtained therefrom as an eluate by use of a buffer which con-tains from about 0.015M to about 0.025M sodium acetate at a pH within the range of from 5.6 to 6.4.
8. The process as in claim 2 wherein the first anio-nic exchanger is DEAE-Biogel and the IgG-rich fraction is ob-tained therefrom as an eluate by use of a buffer which is 0.02M sodium acetate at a pH of 6Ø
9. The process as in claim 2 wherein the second an-ionic exchanger is selected from the group consisting of DEAE-Sepharose CL6B, DEAE-Biogel and DEAE-Sephadex A-50.
10. The process as in claim 2 wherein the second an-ionic exchanger is DEAE-Sepharose CL6B and the purified IgG-rich fraction is obtained therefrom as an eluate by use of a buffer which contains from about 0.025M to about 0.035M
sodium acetate at a pH within the range of 4.8 to 5.6.
sodium acetate at a pH within the range of 4.8 to 5.6.
11. The process as in claim 2 wherein the second an-ionic exchanger is DEAE-Sepharose CL6B and the purified IgG-rich fraction is obtained therefrom as an eluate by use of a buffer which is 0.03M sodium acetate at a pH of 5.2.
12. The process as in claim 2 wherein the second an-ionic exchanger is DEAE-Biogel and the purified IgG-rich fraction is obtained therefrom as an eluate by use of a buf-fer which contains from about 0.015M to about 0.025M sodium acetate at a pH within the range of 5.6 to 6.4.
13. The process as in claim 2 wherein the second an-ionic exchanger is DEAE-Biogel and the purified IgG-rich fraction is obtained therefrom as an eluate by use of a buf-fer which is 0.02M sodium acetate at a pH of 6Ø
14. The process as in claim 2 wherein the second an-ionic exchanger is DEAE-Sephadex A-50 and the purified IgG-rich fraction is obtained therefrom as an eluate by use of a buffer which contains from about 0.02M to about 0.03M potassium phosphate at a pH within the range of 7.1 to 7.9.
15. The process as in claim 2 wherein the second an-ionic exchanger is DEAE-Sephadex A-50 and the purified IgG-rich fraction is obtained therefrom as an eluate by use of a buffer which is 0.025M potassium phosphate at a pH of 7.5.
16. The process as in claim 2 wherein the first an-ionic exchanger is DEAE-Sepharose CL6B, the IgG-rich fraction being obtained therefrom as an eluate by use of a buffer which contains from about 0.06M to about 0.08M sodium acetate at a pH within the range of 4.8 to 5.6, and the second anionic exchanger is either DEAE-Biogel, the purified IgG-rich frac-tion being obtained therefrom as an eluate by use of a buffer which contains from about 0.015M to about 0.025M sodium ace-tate at a pH within the range of 5.6 to 6.4, or DEAE-Sephadex A-50, the purified IgG-rich fraction being obtained therefrom as an eluate by use of a buffer which contains from about 0.02M to about 0.03M potassium phosphate at a pH within the range of 7.1 to 7.9.
17. The process as in claim 2 wherein the first an-ionic exchanger is DEAE-Sepharose CL6B, the IgG-rich frac-tion being obtained therefrom as an eluate by use of a buffer which is 0.07M sodium acetate at a pH of 5.2, and the second anionic exchanger is either DEAE-Biogel, the purified IgG-rich fraction being obtained therefrom as an eluate by use of a buffer which is 0.02M sodium acetate at pH 6.0, or DEAE-Sephadex A-50, the purified IgG-rich fraction being obtained therefrom as an eluate by use of a buffer which is 0.025M
potassium phosphate at a pH of 7.5.
potassium phosphate at a pH of 7.5.
18. The process as in claim 2 wherein the first an-ionic exchanger is DEAE-Biogel, the IgG-rich fraction being obtained therefrom as an eluate by use of a buffer which con-tains from about 0.015M to about 0.025M sodium acetate within a pH range of 5.6 to 6.4, and the second anionic exchanger is either DEAE-Sepharose CL6B, the purified IgG-rich fraction being obtained therefrom as an eluate by use of a buffer which contains from about 0.025M to about 0.035M sodium acetate with-in a pH range of 4.8, to 5.6, or DEAE-Sephadex A-50, the pur-ified IgG-rich fraction being obtained as an eluate by use of a buffer which contains from about 0.02M to about 0.03M
potassium phosphate within a pH range of 7.1 to 7.9.
potassium phosphate within a pH range of 7.1 to 7.9.
19. The process as in claim 2 wherein the first an-ionic exchanger is DEAE-Biogel, the IgG-rich fraction being obtained therefrom as an eluate by use of a buffer which is 0.02M sodium acetate at a pH of 6.0, and the second anionic exchanger is either DEAE-Sepharose CL6B, the purified IgG-rich fraction being obtained therefrom as an eluate by use of a buffer which is 0.03M sodium acetate at a pH of 5.2, or DEAE-Sephadex A-50, the purified IgG-rich fraction being obtained as an eluate by use of a buffer which is 0.025M
potassium phosphate at a pH of 7.5.
potassium phosphate at a pH of 7.5.
20. The process as in claim 2 wherein the second an-ionic exchanger is selected from the group consisting of DEAE-Trisacryl, a DEAE-cellulose and DEAE-Sephocel.
21. The process as in claim 2 wherein the purified IgG-rich fraction is stabilized by the addition of sodium chloride and glycine.
22. The process as in claim 2 wherein the purified IgG-rich fraction is stabilized by the addition of sodium chloride, glycine and mannitol.
23. The process as in claim 2 wherein the purified IgG-rich fraction is stabilized and concentrated to produce a product containing pure IgG within the range of from about 4g% to about 10g% containing 0.15M sodium chloride and 0.1M
glycine.
glycine.
24. The process as in claim 2 wherein the purified IgG-rich fraction is stabilized and concentrated to produce a product containing pure IgG within the range of from about 4g% to about 10g% containing 0.15M sodium chloride, 0.1M
glycine and from about 5% to about 10% mannitol.
glycine and from about 5% to about 10% mannitol.
25. The process as in claim 2 wherein the aqueous animal plasma fraction used as starting material is an aqueous human plasma fraction.
26. The process as in claim 25 wherein the human plasma fraction is either normal human plasma or human plasma containing a high-titer of Rh factor antibodies, rabies anti-bodies, tetanus antibodies or zoster antibodies.
27. An improved process for the preparation of puri-fied immune globulin (IgG) which comprises (a) passing an aqueous animal plasma fraction containing IgG through a first chromatographic separation column containing a first anionic exchanger to produce an IgG-rich fraction, (b) subjecting said IgG-rich fraction to concentration and equilibration for a second chromatographic separation column and then (c) passing said IgG-rich fraction through a second chromatographic sepa-ration column containing a second different anionic exchanger to produce a purified IgG-rich fraction.
28. An improved process for the preparation of puri-fied immune globulin (IgG) which comprises (a) passing an aqueous animal plasma fraction containing IgG through a first chromatographic separation column containing a first anionic exchanger and eluting said column with an aqueous buffer solution to provide an IgG-rich fraction, (b) subjecting said IgG-rich fraction to concentration and diafiltration to pro-vide an equilibrated solution and then (c) passing said equilibrated solution through a second chromatographic sepa-ration column containing a second different anionic exchanger and eluting said second column to provide a purified IgG-rich fraction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000407649A CA1201063A (en) | 1982-07-20 | 1982-07-20 | Process for preparing purified immune globulin (igg) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000407649A CA1201063A (en) | 1982-07-20 | 1982-07-20 | Process for preparing purified immune globulin (igg) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1201063A true CA1201063A (en) | 1986-02-25 |
Family
ID=4123251
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000407649A Expired CA1201063A (en) | 1982-07-20 | 1982-07-20 | Process for preparing purified immune globulin (igg) |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1201063A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0268973A2 (en) * | 1986-11-27 | 1988-06-01 | Biotest Pharma GmbH | Process for manufacturing a virus-free, stable and intravenously tolerant immunoglobulin-G preparation |
| EP0270025A3 (en) * | 1986-12-02 | 1989-05-10 | Schwab & Co. Ges.M.B.H. | Process for the preparation of an intravenously administrable immunoglobulin stable in the liquid phase |
| WO1998005686A1 (en) * | 1996-08-07 | 1998-02-12 | Csl Limited | Purification of immunoglobulins |
| US5886154A (en) * | 1997-06-20 | 1999-03-23 | Lebing; Wytold R. | Chromatographic method for high yield purification and viral inactivation of antibodies |
| US6955917B2 (en) | 1997-06-20 | 2005-10-18 | Bayer Healthcare Llc | Chromatographic method for high yield purification and viral inactivation of antibodies |
| US7847071B2 (en) | 2003-02-28 | 2010-12-07 | Lonza Biologics Plc. | Antibody purification by protein a and ion exchange chromatography |
| EP2277915A1 (en) | 2004-02-27 | 2011-01-26 | Octapharma AG | A purified, virus safe antibody preparation |
| DE102010054767A1 (en) | 2010-12-16 | 2012-06-21 | Previpharma Ag | Disconnecting, concentrating and/or cleaning (blood)plasma protein, virus or virus component, by dissolving/suspending first liquid and third liquid, and equilibrizing (blood)plasma protein, virus or virus component with further liquids |
| WO2012080422A1 (en) | 2010-12-16 | 2012-06-21 | Previpharma Ag | Method for separating, concentrating or purifying a (blood) plasma protein or viral component from a mixture |
-
1982
- 1982-07-20 CA CA000407649A patent/CA1201063A/en not_active Expired
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0268973A3 (en) * | 1986-11-27 | 1989-12-27 | Biotest Pharma GmbH | Process for manufacturing a virus-free, stable and intravenously tolerant immunoglobulin-g preparation |
| EP0268973A2 (en) * | 1986-11-27 | 1988-06-01 | Biotest Pharma GmbH | Process for manufacturing a virus-free, stable and intravenously tolerant immunoglobulin-G preparation |
| EP0270025A3 (en) * | 1986-12-02 | 1989-05-10 | Schwab & Co. Ges.M.B.H. | Process for the preparation of an intravenously administrable immunoglobulin stable in the liquid phase |
| US4880913A (en) * | 1986-12-02 | 1989-11-14 | Schwab & Co. Ges.M.B.H. | Process for the preparation of an immunoglobulin which can be administered intravenously and is stable in liquid form |
| US6093324A (en) * | 1996-08-07 | 2000-07-25 | Csl Limited | Purification of immunoglobulins |
| WO1998005686A1 (en) * | 1996-08-07 | 1998-02-12 | Csl Limited | Purification of immunoglobulins |
| US6955917B2 (en) | 1997-06-20 | 2005-10-18 | Bayer Healthcare Llc | Chromatographic method for high yield purification and viral inactivation of antibodies |
| US6307028B1 (en) | 1997-06-20 | 2001-10-23 | Bayer Corporation Incorporated | Chromatographic method for high yield purification and viral inactivation of antibodies |
| US5886154A (en) * | 1997-06-20 | 1999-03-23 | Lebing; Wytold R. | Chromatographic method for high yield purification and viral inactivation of antibodies |
| USRE43655E1 (en) | 1997-06-20 | 2012-09-11 | Bayer Healthcare Llc | Chromatographic method for high yield purification and viral inactivation of antibodies |
| USRE44558E1 (en) | 1997-06-20 | 2013-10-22 | Bayer Healthcare Llc | Chromatographic method for high yield purification and viral inactivation of antibodies |
| US7847071B2 (en) | 2003-02-28 | 2010-12-07 | Lonza Biologics Plc. | Antibody purification by protein a and ion exchange chromatography |
| EP2277915A1 (en) | 2004-02-27 | 2011-01-26 | Octapharma AG | A purified, virus safe antibody preparation |
| DE102010054767A1 (en) | 2010-12-16 | 2012-06-21 | Previpharma Ag | Disconnecting, concentrating and/or cleaning (blood)plasma protein, virus or virus component, by dissolving/suspending first liquid and third liquid, and equilibrizing (blood)plasma protein, virus or virus component with further liquids |
| WO2012080422A1 (en) | 2010-12-16 | 2012-06-21 | Previpharma Ag | Method for separating, concentrating or purifying a (blood) plasma protein or viral component from a mixture |
| DE102010054766A1 (en) | 2010-12-16 | 2012-06-21 | Previpharma Ag | A method of separating, concentrating or purifying a (blood) plasma protein or viral component from a mixture |
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