CA2185617A1 - Method for recovering immunoglobulin from fractions produced during fractionation of human blood plasma - Google Patents
Method for recovering immunoglobulin from fractions produced during fractionation of human blood plasmaInfo
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- CA2185617A1 CA2185617A1 CA002185617A CA2185617A CA2185617A1 CA 2185617 A1 CA2185617 A1 CA 2185617A1 CA 002185617 A CA002185617 A CA 002185617A CA 2185617 A CA2185617 A CA 2185617A CA 2185617 A1 CA2185617 A1 CA 2185617A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/082—Hepadnaviridae, e.g. hepatitis B virus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- 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
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- 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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
High-titer immunoglobulin preparations are produced by carrying out the following steps in succession: fractionating blood plasma from a plasma pool, whereby at least one industrially usable fraction substantially containingpolyclonal immunoglobulin G is separated, at least one residual fraction being obtained; preparing a protein solution from the protein components contained in the residual fraction obtained in the previous step or in subfractions thereof;
and subjecting the resulting protein solution at least once to affinity chromatography with immobilized ligands of at least one ligand type, the specific plasma proteins being bound to the ligands, and removing the bound plasma proteins, which are converted as active components into a high-titer immunoglobulin preparation. The immunoglobulins obtained can be processed into an IV well-tolerated, storage-stable product. The method leading to a valuable immunoglobulin preparation makes use of human plasma fractions which have hitherto been rejected in conventional industrial plasma fractionation methods.
and subjecting the resulting protein solution at least once to affinity chromatography with immobilized ligands of at least one ligand type, the specific plasma proteins being bound to the ligands, and removing the bound plasma proteins, which are converted as active components into a high-titer immunoglobulin preparation. The immunoglobulins obtained can be processed into an IV well-tolerated, storage-stable product. The method leading to a valuable immunoglobulin preparation makes use of human plasma fractions which have hitherto been rejected in conventional industrial plasma fractionation methods.
Description
. ~ 2i856~7 METHOD OF RECOVERING IMMUNOGLOBULIN FROM FRACTIONS
PRODUCED DURING FRACTIONATION OF HUMAN BLOOD PLASMA
This invention relates to the p~eudldliùn of immunoglobulin, and more particularly to a method of producing a high-titer immunoslobulin 5 ~lepdld~ivl1. This method may be utilized for recovering immuno~lnh~l" ,s fromfractions which, in the methods of plasma r, " , '' ~ customary until now, are not made use of, or at least are not used in the production of immunoglobulin ,UI ~,~dl d~iUI 1~.
There are over one hundred different proteins in human blood 1û plasma. Some of them can be purified by r,d~iu,laliù" from pools of donor plasma and used as therapeutic products as in the following exdmples:
albumin is used for c~ Je~)sdLilly an oncotic deficit in hypu~ lelllia or h~")o./ol~",ia, blood co~g~ ti~n factors Vlll and IX are a-ll";"' ' .ev as helllvl I llayé prophylaxis and therapy in l1~i,n~,ul,''' ~ A and B, respectively;
immuno~ b~ are made use of as infection prophylaxis and therapy in antibody-deficiency diseases, as well as in idiopathic thrombocytopenic purpura; immuno~lob~ 1'' la from selected donors having high titers of specific immuno~!ob~ 1" ,s are used as hyperimmunoglobulin pl~dldliv~ls for the prophylaxis and treatment of specific infections such as hepatitis A or B.
TheMr~l ~" 'Iy usable plasma proteins can be isolated, for example, according to known methods of ethanol r, d~,liUI IdliOI1 (Cohn, E. G., et al., J. Am.
Chem. Soc., 68, 459, 1946; Kistler, P., and t~ dl 111, H., Vox Sang., 7, 414, 1962). With both methods, it is possible to isolate large amounts of functional plasma proteins such as albumin or immunogl~bu" ,c. which, in suitable 25 formulations, can be profitably utilized clinically. However, when working according to these methods, ~, l ,;, :' ' ~ and/or supe" Id~dl Its occur which cannot be used in conventional ,u, UCt:55il 19. The l,u",,uû~i~iui1 of these fractions varies greatly.
Whereas, for example, in the p,. ', :' ''~ 1 of blood plasma with 19%
30 ethanol at a pH of 5.8, human ., ~ 'i, , .: ' 1 A-l (apoA-I) is to be found in a~J,ulUAilll- '~Iy equal parts in the supernatant a (about 5û% of the plasma ~ 2185617 apoA-I) and in ~ , .' ' A (about 40%), the same protein is then found after the next r, ~dtiUI~ steps according to Kistler and ~: ' 'Illldllll in those fractions which have not been used cu"""~" ~ until now: p,. :, ' ' IV and B (about 40% each) (Lerch et al., Protides of the Biological Fluids, 5 36, 409,1 989).
A similar distribution pattern results for the cûpper-binding protein ceruloplasmin. After r, d~liUI, " 20% of the starting material is found in ~, . ., ~ ' IV and 40% in ~ :, ' ' B.
Transferrin is an example of a plasr~a protein which, in the same 10 rld~liull ~ method, is s~uald" "y 100% COI~Ce~l dled in p,- :,: IV, whereas 80% albumin is to be found in the p,~ , .' ' C currently used.
From 5040% of immuno~ h~ ~' ,s may be recovered in p, :,: ' GG by means of Kistler r: ' 2~ dl ll l or Cohn rl d.,Li~dli~l~. The remaining 30-40% is distributed among p,. :,~;' ' IV (5%), p,~ , :' ' B (30%), and filtrate 15 GG (5%) in these methods The dru,~."~:"" ~ed p~ tdye:S are intended to illustrate the order of ",au" ' Icle of the distribution and are not to be taken as restrictive; they are variable dep~l, " ,~ upon the conditions and methods used.
These examples show that in some instances cu,, ,icl~, dL,le amounts 20 of ther~rel ~' - "y usable proteins are to be found in the fractions ,u~ ~,i,uiL~ IV, pl ~ui~uildle: B, supernatant c, and supernatant GG, or in the t,ur, ~apùl l~lil 19 fractiorls of the plasma r,d-~liUIIdLiUI, according to Cohn (fraction IV-1, fraction 11+111, supernatant V, and supernatant 11-1,2). For ethical reasons, but also because of the worldwide shortage of human blood plasma and certain plasma 25 cu",,,.u"~"~, an improvement should be sought in the yield of immur~n~l~b~ ~", " which has not been very high until now.
Immunogl~b~ play a pivotal rôle in warding off infections. Either virus-specific, neutralizing dl ItiL~d;~,3 block the ddSOI IJLiOI I of viruses on the cellular receptors and thus prevent infection, or bacteria-specific dl 1"' _ " 35 ` ~ 21856~7 opsonize the pathogen and thus allow it to be ~ ' ";, I..'~d and killed by neutrophils and Illal~luplldges~ Plasma pools from several thousand donors contain immunno~ohll " of very many different sp~,iri~,ili~s, and immunoglobulin ~ ,Odl dLiul 1~ from such pools consequently also contain 5 measurable titers of immunnoloh~ ~" " directed against epitopes on viruses, bacteria, and toxins, but also against au~uallLiyel~s Hence they are effective against many infections and in the most varied other ,~ " ,oluyi~al conditions.
Now, under certain circumstances, however, it is desirable to make use of an immunoglobulin plt:~Jalaliul~ having high titers of specific a~ ~ " , a so-called hyperimmunoglobulin plt:paldtiUn. Until now, such ~ ualdLiul~s have been prepared at great expenditure of time and money from special plasma pools of donors having increased titers of specific cl, I';L - 'i~ ~ For variousreasons, this involves dffliculties. If, as in the case of an anti-hepatitis B
p, t:~.a, ' ~, there are ~c~y"i~d vacci, laLiul, procedures, then the donors 15 must be inoculated and selected, and the donated blood must be separately p,uce~d. In the case of many other i"~ ' 1S, however, immunization of the donor cannot take place for ethical reasons. Here it is only rarely possibleto locate high-titer blood through an involved selection of the donors (e.g., afbr their having recovered from a specific disease) and to obtain a IJIt:~,al "
20 through ~., u~ i"y of the donated blood.
It is therefore an object of this invention to provide a method of recovering immunoolob~ ~" " by means of which valuable immunoglobulin JaldLiùl~, can be made a~c~ and isolated from the dru,~",~"liu"ed fractions and p,~ci~.;' ' , hardly made use of until now, which become 25 available during industrial plasma-r,d-,Liur,dLio,~ methods. S~hseqllently, it should be possible to process these IJlt:~JdldLiulls, c~ pul~di~ly to hyperimmunoglobulin l.,~pd,.lliuns, into a well-tolerated, especially intravenously (IV), virus-proof, liquid or freeze-dried 1 l t:,Udl " 1.
It has now been found that it is possible to produce 30 hyperimmunoglobulin ~,r~pd, dLiul~s or high-titer immunoglobulin pfeud, dLiol ,s by a method dfflering from the prior art methods. This novel method uses immunooloh~ from the general plasma pools, improves the ~ of the valuable raw material blood plasma through the use of ~wasteU fractions, 21~617 and even permits the production of hyperimmunoglobulin ~l~palaliull;~ having far greater specific activities than previous ~, c ual dtiUI 1~. This means that with small quantities a.l",i";~ d IV, and with low amounts of IV ad~"i"' ' ed proteins, i.e., a c~ a,uOI lui. I~ly low burden on the recipient, high doses of 5 specific immuno~ hl 1" ,~ can be given within a short time. This is made possible in the inventive method through the co,~c~"' aliOI~ of the specific immuno~ bll" ,s through ads~"uliùn on i""" ' ~ antigens, thus through the use of ,ul vl-essed "waste" fractions in affinity ~I Il ul l l ' _ a,ul iic techniques.
To this end, the method of producing immunoglobulin pl~,udldliuils 10 according to the present invention comprises the steps of rldl,liulldlillg blood plasma from a plasma pool, whereby at least one industrially usable fraction sulJ~ldl 1" 'Iy C~l 1' ' 1' Iy polyclonal immunoglobulin G is separated, at least one residual fraction being obtained; preparing a protein solution from the protein .,o,,,,uu, ,~"t~ contained in the residual fraction obtained in the previous step or in sUblla~.liOII~ thereof; and subjecting the resulting protein solution at least once to affinity ,1 1l u", ' _ d,UI Iy with i"""~ ' "' -' ligands of at least one ligand tvpe, the spec'lfic plasma proteins being bound to the ligands, and removing thebound plasma proteins, which are converted as active c~,,,pu,,~ into a high-titer immunoglobulin ,u, epd, dliùl~.
In the inventive method, a fraction obtained in a plasma r,d~liu, Idlio~
process, particularly a waste fraction, is first ,u, u.,~:.ed in such a way that it cdn be used in affinity ~,lllullldluyla,ully. Depending upon the provenance of the fraction, this ,u, U~,C~si, ~y may vary greatly. Thus, for example, the supematant GG may be col~cel ,' ' -' down, dialyzed against a suitable buffer, 25 and filtered. On the other hand, u, . ', " ' ~ or filter cakes (lists of examples of such starting materials are given in Tables 1 and 2 below) must first be suitably suspended, i.e., through variation of the ionic strength, the pH, andlor the le"",e, ' Ire, or through the addition of d~it~i. ye"L;, and salts, in such a way that immunoolob~ are spe~,iri 'Iy 5~ b"' i~ They may, for example, be stirred 30 overnight within a pH range of from 3-9, at a conductance of from 5-2û mS/cm, at 4C and then be clarified by centrifugation andlor filtration. Both s~pe" IdLal ILa and suspel~sio~s may at this stage be subjected a virus-inactivation process according to the solvent-detergent method of Horowitz 2~856~7 (Tl)~ulllbua;~ and l ~ , 65, 1163, 1991), the methylene blue method (Mohr et al., Infusionsther. Infusionsmed. 20, 1911993]), or some other method.
The immuno~l~b~ 1' ,, may also be subjected after suspension to prepurification by p,~d.l~u,,ulion on a matrix in a column or through treatment with a suitable 5 filter aid or adsorbent such as aluminum hydroxide, ,lllu"l ' _ d~JIly on protein A or G for co~c~"' " ~, or one or more ~ s by the common methods of ammonium sulfate, polyethylene glycol, or ethanol pf~-,i,uildLiùll or ~.UI I Ibil IdliOl~s thereof for ~", i~,l " "~:"I, co,~c~"' dliUI), or depletion of disturbing cu",,u~ "t ..
Su,u~ ldldllb Pl~ , :' ' - and Residues Supernatant c Precipitate B
Supernatant GG Precipitate IV
Residue after DEAE treatment Residue after aluminum hydroxide treatment Residues after clarifying filtrations 1, ll and lll Table 1: Examples of possible starting materials coming from rld~liull~
according to Kistler and ~ Idl 111 or from other ~, u~essil ,9 steps resulting from the ,UI~pdldliUII of IV adlllill;~lldble, stable plasma products.
Su,ut:l I Idldl 11~ Pl ~ui,uildlc3 and Residues SupernatantV Precipitate ll+lll Supernatant 11-1,2 Precipitate IV-1 Residue after DEAE treatment Residue after aluminum hydroxide treatment Residues after clarifying filtrations 1, Il, and lll Table 2: Examples of possible starting materials coming from the r~ d~liUIldliU
according to Cohn or from other ,u, u~.~s ,i"~ steps resulting from the Jdldliùll of IVa.llllilli~lldble:, stable plasma products.
21856~7 The following list gives examples of possible ligands for the inventive affinity 1,111 Ul I ~, d,JI Iy Antigenic delel I l lil Idl ,t~ of I lae, I ,opl l ' le influenza b Staphylococcus aureus Staphylococcus ~:,uidel 1 l lidi., Staphylococcus ~ e Sl, IJt. ~COGGI IS pneumoniae Stl-lJt ~,o~,cl le pyogenes and other ~ " ,oye,~ic strains of bacteria tetanus toxin Staphylococcus aureus toxic shock toxin and further r IOyel ,;~ bacterial or other toxins hepatitis A virus hepatitis B virus hepatitis C virus varizella zoster virus cy~"lèydlo virus respiratory syncytial virus 2~ parvovirus B19 herpes simplex virus 1 herpes simplex virus 2 rabies virus and other r ,oge"i~. viruses CD2, CD3, CD4, CD5, CD28, CD4û, CD72 ICAM, LFA-1, LFA-3, DNA
and other potential human ~I ~' ILi~ells Since affinity gels have been prepared by i""" ' " ,y modified or unmodified ligands (examples of such ligands are given in the above list) by means of methods known per se, they are loaded with the ~, u~,essed SU,U~I I Idldl 11~ and Sus~Jell~ions in col~cél ,' dled or diluted form, if necessary also by repeated ~ , ' ' , of the flow. If desired, various affinity gels may be35 activated in s~lccessi~n with the same suspension. The gels are thereafter washed in such a way that u" ,pe,,iri 'Iy binding proteins are removed ,ulepol~deldlllly or to a sufficient extent. This can be done, for example, by 2i85617 i"", t:asi"y the saline c~c~"' " " by addition of a detergent, and/or by shifting the pH in the washing solution. The bound proteins are now separated from the ligands, e.g., by elution at a low or high pH, by addition of .,I I - '. upic saline solutions such as sodium thiocyanate or magnesium chloride, denaturing s agents such as SDS or urea, solvents such as ethylene glycol, by modifying the Itll l,u~ e, or by cul l lbil ldliùl l~ of the foregoing.
In some cases, it may be desirable to modify the ligands to be i"", ' ~ by mutagenesis or by chemical or physical methods in such a way that the specific immunogl~hl ~' Ia can still bind to their epitopes, but with reduced affinity, so that elution can take place under milder conditions than with the u"l"odiried ligands. ~' ' ' ' , of the ligands may also take place in order to facilitate and improve their illll l l L :' ' .1 and/or their epitope pl t:s6'1 Italiù~. Technical details and basic principles of the affinity l,lllulll ' _ d,UIly process in general are described in Cuatrecasas, P., and 15 Anfinsen, C. B. (1971), Ann. Rev. Biochem. 40, 259; Kull, F. C., and C~ ,asas, P. (1981), J. Immunol. 126, 1279; Liebing et al. (1994), Vox Sang. 67, 117.
The specific, separated immuno~l~h~' ,s, also with additional filtration for eli."i, Idlil ,9 viruses, if need be, are p~uc~sed into an end product 20 which can preferably be a.l",i" ' .c:d IV and which is free of pyrogens, virus-proof, and stable with or without the addition of stabilizers such as albumin, amino acids, or carbohydrates in liquid or freeze-dried form. However, formulations may also be used which make possible intramuscular or topical ad",i";~l,dlio,~.
Preferred ~",L,odi."e"l~ of the present invention are described and illustrated below by means of the following examples, which are not intended to be limitative Example 1 HBsAg-Sepharose was prepared by i""" ' :' ' ,9 5 mg of 30 l~collllJilldlll hepatitis B virus surface antigen (HBsAg, Abbott DidyllOalil,~) through coupling of the primary amino groups to 1 ml of activated CH-~ 2185617 Sepharose as directed by the manufacturer (Pl,a""acia Biotech, Uppsala,Sweden). "Placebo~-Sepharose was prepared by carrying out the same coupling process with another gel aliquot, but without adding HBsAg. The finished gels were stored in PBS with 0.02% NlaN3 at 4C.
Seventy grams of ,u,~ :, ' ' B from the Kistler ': ' 'Illldllll plasma r~ d~liUI IdliOI I (NB Lot 4.030.216) were suspended ovemight in 210 ml of 100 mM citric acid, pH 4.0, 0.25% Triton X-100, 10 mM N-ethyl maleimide (NEM), 1 mM phenylmethylsulfonyl fluoride (PMSF) at 4C on a RotaryMix appliance.
After 5~dld~iOI~ of the insoluble cull,,uu,,e:,,t~ by centrifugation (50009, 4C, 10 min.) and filtration (pore size: 1.2 ~lm), 1% tri-n-bu~yl~l lua~ I ' (Merck, Darmstadt, Germany) and 1% Triton X-100 were added at a neutral pH
according to Horowitz et al. (Tl ll ul l lbo ~H and l ldt~ u~ld ~i ~, 65, 1163, 1991 ) and incubated at 30C for 4 hrs. with mixing. A phase 5~dl dliUIl was then carried out overnight at 37C, the clear lower phase was removed, filtered 15 through a 0.45 llm filter, and stored at 4C.
One hundred thirty ml of such an NB suspension were diluted with 280 ml of PBS (plloa,ul~dl~-burfered saline solution: 150 mM NaCI, 10 mM
sodium pllo~,l, ', pH 7.1) and pumped overthe placebo-Sepharose column, then over the HBsAg-Sepharose column at 4C in such a way that the column 20 flow passed back into the storage vessel. The rate of flow was 8 ml/hr for 144 hrs. Thus the NB suspension was pumped over the columns a total of three times. After 90 hrs., the loading was interrupted, and the columns were washed individually, first with PBS and then with 200 mM NaCI, 50 mM tris-HCI, pH 7.4, until the wash solution had an optical density of less than 0.01 at 280 25 nm (OD2go). After conclusion of the activation, washing was again carried out with PBS and 200 mM NaCI, 50 mM tris-HCI, pH 7.4. Bound proteins were separated with 5 ml of 200 mM glycine-HCI, pH 2.5, i""" ' ' !y neutralized, and ~u~ sed.
` ~ 2185617 Gel HBsAg Placebo Tota loading:
rotein ~~5 mg ~ '~5 mg 3G ~ mg ~ mg Anti-HBs IgG ~ 0 mlU A o mlU
Flow:
~nti-HBs IgG 1140 mlU 1140 mlU
Elua e:
~G 0.13 mg 0.08 mg Anti-HBs IgG 5603 mlU 114 mlU
Table 3: Anti-HBsAg affinity ~ lulll ~ ly with NB suspension Example 2 Starting from the Cohn fraction ll+lll (instead of blood plasma as the starting material), a Kistler-Nit,~,l""d"" r~ l;UlldLiUI~ was carried out. Seventy gramsofp~ ,i,uildLt:B(NBLot4.044.488)fromthisKistler~ 'lllldllll r~ d~,liO~ IdliUIl were suspended overnight in 210 ml of 100 mM citric acid, pH 4.0, 0.25% Triton X-100, 10 mM NEM, 1 mM PMSF, at4C on a RotaryMix 10 appliance. Afterclc" iri~dliUIl andpartial~.I;, .;rl.~ JII by~".,c~"'irugatiorl (100,0009, 3 hrs., 4C: the clear phase was withdrawn by piercing the side of the tube with a syringe), the suspension was filtered (0.45 llm) and stored at 4C.
One hundred twenty-five ml of this NB suspension were diluted with 15 375 ml PBS, the pH adjusted to 7.1 with 0.1 \/1 NaOH, filtered, and pumped, as in Example 1, first over placebo-Sepharose gel and then over HBsAg-Sepharose gel prepared dl I ' J l':ly to Example 3. The gels were then washed sepd, ' !y with PBS and 200 mM NaCI, 50 mM tris-HCI, pH 7.4. The bound proteins were removed with 5 ml of 200 mM glycin-HCI, pH 2.5. Table 4 20 gives a summary of the data.
2~85617 Gel HBsAg Placebo Tota loading:
rotein ~ 400 mg ~ ~ O mg 3G 19 mg ' mg Anti-HBs IgG 0 IU C U
Flow:
Anti-HBs IgG 5 IU 5 IU
Elua :e:
3G 0.18 mg 0.35 mg Anti-HBs IgG 3.3 IU 0.08 IU
Table 4: Anti-HBsAg affinity ~,I"u", ' _ d~JIly with NB suspension (Cohn ll+lll) Example 3 Thirty liters of supernatant GG (Lot No. X95.31.286.1) were diafiltered in PBS and col1ce"' dL~d down to 500 ml. As in Example 1, the col~ce" ' was pumped over placebo and HBsAg columns at 21 ml/hr for 118 hrs. Washing of the columns and S~dld~;UII of the bound proteins likewise took place a,~ ' ~, Isly to Example 1, but an additional washing step with 500 mM NaCI, 50 mM tris-HCI, pH 7.4, was carried out. The results are shown in Table 5.
Gel HBsAg Placebo Total loading:
Protein 8800 mg 8800 mg IgG 320 mg 320 mg Anti-HBs IgG 5000 mlU 5000 mlU
Flow ~nti-HBs IgG ~DL ~DL
Elua .e:
~G 0.05 mg 0.14 mg Anti-HBs IgG 3035 mlU 7 mlU
Table 5: Anti-HBsAg affinity ~ Jllldluyla,ullywith supernatant GG co~lce" dl~
DL: detection limit ~ ~ 2185617 Example 4 DEAE filter cake in an amount of 17.5 9 (Lot 4.422.006.0) was suspended in 52.5 ml of suspension buffer according to Example 1 and p,uc~sed. Forty ml of suspension were diluted with 160 ml of PBS, the pH
5 adjusted to 7.1, and then filtered. As in Example 3, the suspension was pumped over placebo and HBsAg columns at 21 ml/hr for 97 hrs. Washing of the columns and ;._~Jdl dliUI I of the bound proteins also took place dl)~ cly to Example 1. The results are shown in Table 6.
Gel HBsAg Placebo Tota loading:
rotein ~ mg ~ mg ~G mg . mg Anti-HBs IgG ~ mlU ~ mlU
Flow:
~nti-HBs IgG ~DL ~DL
Elua e:
~G 0.07 mg 0.11 mg Anti-HBs IgG 331 mlU 14 mlU
Table 6: Anti-HBsAg affinity Ul~l u", ' _ d,UI Iy with DEAE
filter-cake suspension DL: detection limit Example 5 Tetanus toxoid C-Sepharose was prepared by i""" ' :' ,g 11.5 mg of purified tetanus toxoid (TT) through coupling of the carboxy groups to 1 ml of EAH-Sepharose (Pl,d""acia Biotech, Uppsala, Sweden) with the aid of 0.1 M
15 N-ethyl-N'-(3-dimethy'..."i"u,u,u,uyl)-cd,L ' "ide-HCI, as directed by the manufacturer. The finished gels were stored in PBS with 0.02% NaN3 at 4C.
A cu, Ice, ,I, dlt: of supernatant GG was prepared as in Example 3 and used for affinity .,l " u~ d~,l ,y with TT-Sepharose d" ' ~, Icly to Example 2.
Activation took place at 23.5 ml/hr for 159 hrs. at 4C. The results are shown in 20 Table 7.
2~85617 Gel Tetanus Toxoid C
Tota loading:
rotein ' 000 mg 3G 0 mg Anti-TT IgG ~Lg Flow Anti-TT IgG 16 1l9 Eluate:
IgG 0.16 mg Anti-TT IgG 76 ~9 Table 7: Anti-tetanus toxoid affinity ~l ll u" IdlUUl d,UI Iy with supernatant GG
co,~c~, It Example 6 Tetanus toxoid N-Sepharose was prepared by i"" ' :' ,9 11.5 mg of purlfied tetanus toxoid (TT) through coupling of the primary amino groups to 1 ml of activated CH-Sepharose as directed by the manufacturer (Pharmacia Biotech, Uppsala, Sweden). "Placebo=-Sepharose was prepared by carrying out the same coupling process with another gel aliquot, but without adding TT.
The finished gels were stored in PBS with 0.02% NaN3 at 4C.
Precipitate B was suspended according to Example 2.
After filtration (1.2 ~Lm), 115 ml of this suspension were diluted with 200 ml of PBS, the pH adjusted to 7.1, and pumped first over the placebo-Sepharose at 3.5 ml/hr for 165 hrs. in such a way that its flow passed i""" ' ' 'y thereafter over the TT N-Sepharose column, then back into the storage vessel. The columns were washed sepd, ' Iy with PBS and then with 0.5 M NaCI, 50 mM tris-HCI, pH 7.1, until the OD283 of the flowwas less than 0.01. The results are shown in Table 8.
` ~ 2185617 Gel Tetanus Toxoid N Placebo Tota loading:
rotein ' 334 mg ' 334 mg ~G 33 mg 33 mg Anti-TT IgG .509 mg .509 mg Flow:
Anti-TT IgG 0.213 mg 0.213 mg Elua e:
3G 0.35 mg 0.004 mg Anti-TT IgG 0.091 mg 0.003 mg Table 8: Anti-tetanus toxoid affinity ~ lullldiuuld~,llywith NB suspension Example 7 Precipitate B was suspended according to Example 2.
After filtration (1.2 ~lm), 115 ml of this suspension were diluted with 20û ml of PBS, the pH adjusted to 7.1, and pu~ped first over the HBsAG-Sepharose column at 5 ml/hr for 165 hrs. in such a way that its flow passed i,,,,l, " ' ~y thereafter over the TT-Sepharose column and then back into the storage vessel. The columns were washed 5~ y with PBS and then with 10 0.5 M NaCI, 5û mM tris-HCI, pH 7.1, until the OD28o of the flow was less than 0.01. The results are shown in Table 9.
Gel HBsAg Tetanus Toxoid Tota loading:
rl~tein ' 334 mg ' 334 mg 3~ 33 mg 33 mg An -HBsAg 45 mlU 45 mlU
An -TT IgG .509 mg .509 mg Flow Anti-HBsAg 536 mlU 536 mlU
Anti-TT IgG 0.265 mg 0.265 mg Elua e:
~G 0.13 mg 0.25 mg Anti-HBsAg 1152mlU
Anti-TT IgG 0.18 mg 2~856~7 Table 9: Anti-HBsAb and anti-tetanus toxoid affinity 1,l ll u, l l ' v dUI Iy with NB
suspension Example 8 Fifteen kg of ~,~ , ' ' B from the Kistler ~ 'lllldl 111 r~ d'vliU~ld~iOI~
5 (Lot No. 5.043.303) were suspended in 45 liters of 0.1 M citric acid, pH 4.0, 0.25% Triton X-100, 10 mM NEM, 1 mM PMSF overnight at 4C with a vil,,u,,,i~el. After sepdld~ of the insoluble c~",~,u"~"~v by adding filter aidsand filtering (pore size: 1.2 llm), 1% tri-n-butyl pllo~,ul Idle and 1% Triton X-100 were added at neutral pH for dF l;l 'i~ and virus inactivation according to Horowitz and incubated for 4 hours at 30C with mixing. Thereafter, a phase 5~1Jdl ClliOIl was carried out overnight at 37C, ~he clear lower phase pumped off, filtered through a 0.45 llm filter, and stored at 4C.
The pH of 40 liters of this NB suspension was adjusted with NaOH to a value of 7.1, and it was pumped at 4C over a HBsAg column and a tetanus 15 toxoid Affiprep column in such a way that the column flow passed back into the storage vessel. The Affiprep columns (50x13 r~m) were prepared by coupling 250 mg of It:c~ b;lldlll HBsAg and tetanus to~(oid, ~e:v,ue~.t;lcly, with 25 ml of Arfiprep gel (BioRad Lab. Inc., Hercules, CA 94547. U.S.A.). The rate of flow was 6 IVhr for 62 hrs. Thus the NB suspension was pumped over the columns 20 a total of three times. After conclusion of the activation, the columns were washed sepd, ' 'y, first with PBS and then with 500 mM NaCI, 50 mM tris-HCI, pH 7.4, until the washing solutions had an optical density of less than 0.01 at 280 nm (OD2go). Bound proteins were removed with 200 mM glycin-HCI, pH
2.5, and the pH of the fractions i""" ' ' Iy adjusted to 5.2. The results are 25 compiled in Table 10. The immunoglob~ v were p~uc~vve~ into stable ult:pdldliol~s by pooling the fractions containing IgG, didri" dliol~, and Cul ICt~ ldliUI I with 20 mM NaCI into solutions of 100 lU/ml and 2.5 mg anti-TT-lgG/ml, respectively. Ten percent sac.,l,d,uve was added, and the solutions were Iyophilized in units of 200 IU, and 5 mg of anti-TT-lgG"-, - ' .~ely.
2~85617 Example 9 Fifty grams of p~e~ LdL~ lv from the Kistler ~ dl 111 plasma r, d~,Liul IdLiUI I were suspended in 500 ml of water. The pH was adjusted to 5.0 with citric acid, and the conductance to 13 mS with NaCI. After stirring 5 overnight at 4C, Cldl iri-,dLi,,,. and partial .' ~ 'i, ' ' ~ were achieved by centrifugation for 30 min. at 30,0009 and 4C. The separated protein, IgM, IgA, IgG, L, dl "~rt:" i", and ceruloplasmin content of the suspension was dt:Lt~ ,edand is indicated in Table 11.
Gel HBsAg Tetanus Toxoid Total loading:
Pr tein ~ 9 ~ g 19~, ~ 9 (,~ 9 An -HBs IgG 0 IU 0 IU
An -TT IgG v mg ~' mg Flow.
Anti-HBs IgG 221 IU 221 IU
Anti-TT IgG 172 mg 172 mg Elua e:
~ 39.8 mg 171 mg An -HBs IgG 1368 IU
An -TT IgG 89 mg Table 10: Anti-HBsAg and anti-tetanus toxoid affinity ,l ll u", ' _ alJl l~l with NB
10 suspension ¦ Total Protein l9~ IgA IgG Transferrin Caeruloplasmin ¦
5.5 0.063 1 0.628 1 0.488 1 2.582 1 0.09 Table 11: Suspension from ,I~ ,itdL~: IV (all values in mg/ml) Example 10 Fifty grams of p~. , ' ' B from the Kistler ~ ' ll l ldl ll l plasma 15 r, d~,liul1dli~ (Lot No. 4030.204.0) in 100 mM citric acid at various pH values and at 4~C were stinred overnight, clarified by ~ ~" d-,~l ,' irugation at 100,0009 and 4C for 3 hrs. and partially d~ l The clear middle phase was removed, and its separated protein, IgM, IgA, IgG, LldllaF~llill, and ceruloplasmin content was d~ :Lt:" "i"ed (Table 12).
i 21~5617 ~
~c .al Protein Ig`1 IgA IgG Transferrin Caenuloplasmin pH4.0 ~. 1. 1.3 1.9 <DL 0.06 pH 5.0 . 1. . 1.0 2.1 <DL <DL
Table 12: Suspension from pl~:l iUildL~ B (all values in mg/ml);
<DL: belowdetection limit The titer of IgG against certain viral (Table 13) and bacterial (Table 14) antigens was .I~:Lt:l " ,i"ed and compared with that in starting plasma and 5 existing immunoglobulin plt~Jdldliol)s.
Plasma Plasma SAGL NB pH 4.0 NB pH 5.0 ' ~2 03 An -H sAg . 2 .05 0. 1 . . 5 'mg IgC
An -C lV . 8 .16 0. . . 4 IE/mg gG
An--V_V . 8 .09 n.. . . mg IgG
An -measles . .2 0. 2 . . 1 /mg IgC
An--HSV1 C37 n.d. n.~. 317 49 AJ/mg IgG
Table 13:
Antiviral IgG in plasma pools SAGL and NB suspel~siol~s Plasma 102/103: plasma pools; SAGL: Sar.do~lob~ NB pH 4.0/5.0: suspension from p~ dL~ B at pH 4.0 and 5.0 resp~ctively; IU: I"~."dLio~1al Units; PEIE:
Paul Ehrlich Institute units; AU: arbitrary units; n.d.: not de l~ d.
Plasma 102 Plasma 103 SAGL NB 4A NB 14 pH 4.0 anti- 162 286 436 100 283 1l9/9 IgG
HiBOAg anti-TT 1855 4159 2740 2928 2923 1l9/9 IgG
anti-SEB 412 689 783 918 670 AU/g IgG
Table 14: A"Lil.a~Lc:rial IgG in plasma pools SAGL and NB SU5pc:1~5iOI15 Plasma 102/103: plasma pools; SAGL: Sandn~ hll" ,~3 NB 4A: suspension from ,~ iuiL.d~ B at pH 4.0; NB 14 pH 4.0:
15 suspension from p, ~ciyiL~._ B at pH 4.0 with virus inactivation; HiBOAg:
l1a~l"uul, " Is influenza B-o~;gosac~l,d,ide antigen; TT: tetanus toxoid; SEB:
Staphylococc~s ~"Lt~ ., B; AU: arbitrary units.
'
PRODUCED DURING FRACTIONATION OF HUMAN BLOOD PLASMA
This invention relates to the p~eudldliùn of immunoglobulin, and more particularly to a method of producing a high-titer immunoslobulin 5 ~lepdld~ivl1. This method may be utilized for recovering immuno~lnh~l" ,s fromfractions which, in the methods of plasma r, " , '' ~ customary until now, are not made use of, or at least are not used in the production of immunoglobulin ,UI ~,~dl d~iUI 1~.
There are over one hundred different proteins in human blood 1û plasma. Some of them can be purified by r,d~iu,laliù" from pools of donor plasma and used as therapeutic products as in the following exdmples:
albumin is used for c~ Je~)sdLilly an oncotic deficit in hypu~ lelllia or h~")o./ol~",ia, blood co~g~ ti~n factors Vlll and IX are a-ll";"' ' .ev as helllvl I llayé prophylaxis and therapy in l1~i,n~,ul,''' ~ A and B, respectively;
immuno~ b~ are made use of as infection prophylaxis and therapy in antibody-deficiency diseases, as well as in idiopathic thrombocytopenic purpura; immuno~lob~ 1'' la from selected donors having high titers of specific immuno~!ob~ 1" ,s are used as hyperimmunoglobulin pl~dldliv~ls for the prophylaxis and treatment of specific infections such as hepatitis A or B.
TheMr~l ~" 'Iy usable plasma proteins can be isolated, for example, according to known methods of ethanol r, d~,liUI IdliOI1 (Cohn, E. G., et al., J. Am.
Chem. Soc., 68, 459, 1946; Kistler, P., and t~ dl 111, H., Vox Sang., 7, 414, 1962). With both methods, it is possible to isolate large amounts of functional plasma proteins such as albumin or immunogl~bu" ,c. which, in suitable 25 formulations, can be profitably utilized clinically. However, when working according to these methods, ~, l ,;, :' ' ~ and/or supe" Id~dl Its occur which cannot be used in conventional ,u, UCt:55il 19. The l,u",,uû~i~iui1 of these fractions varies greatly.
Whereas, for example, in the p,. ', :' ''~ 1 of blood plasma with 19%
30 ethanol at a pH of 5.8, human ., ~ 'i, , .: ' 1 A-l (apoA-I) is to be found in a~J,ulUAilll- '~Iy equal parts in the supernatant a (about 5û% of the plasma ~ 2185617 apoA-I) and in ~ , .' ' A (about 40%), the same protein is then found after the next r, ~dtiUI~ steps according to Kistler and ~: ' 'Illldllll in those fractions which have not been used cu"""~" ~ until now: p,. :, ' ' IV and B (about 40% each) (Lerch et al., Protides of the Biological Fluids, 5 36, 409,1 989).
A similar distribution pattern results for the cûpper-binding protein ceruloplasmin. After r, d~liUI, " 20% of the starting material is found in ~, . ., ~ ' IV and 40% in ~ :, ' ' B.
Transferrin is an example of a plasr~a protein which, in the same 10 rld~liull ~ method, is s~uald" "y 100% COI~Ce~l dled in p,- :,: IV, whereas 80% albumin is to be found in the p,~ , .' ' C currently used.
From 5040% of immuno~ h~ ~' ,s may be recovered in p, :,: ' GG by means of Kistler r: ' 2~ dl ll l or Cohn rl d.,Li~dli~l~. The remaining 30-40% is distributed among p,. :,~;' ' IV (5%), p,~ , :' ' B (30%), and filtrate 15 GG (5%) in these methods The dru,~."~:"" ~ed p~ tdye:S are intended to illustrate the order of ",au" ' Icle of the distribution and are not to be taken as restrictive; they are variable dep~l, " ,~ upon the conditions and methods used.
These examples show that in some instances cu,, ,icl~, dL,le amounts 20 of ther~rel ~' - "y usable proteins are to be found in the fractions ,u~ ~,i,uiL~ IV, pl ~ui~uildle: B, supernatant c, and supernatant GG, or in the t,ur, ~apùl l~lil 19 fractiorls of the plasma r,d-~liUIIdLiUI, according to Cohn (fraction IV-1, fraction 11+111, supernatant V, and supernatant 11-1,2). For ethical reasons, but also because of the worldwide shortage of human blood plasma and certain plasma 25 cu",,,.u"~"~, an improvement should be sought in the yield of immur~n~l~b~ ~", " which has not been very high until now.
Immunogl~b~ play a pivotal rôle in warding off infections. Either virus-specific, neutralizing dl ItiL~d;~,3 block the ddSOI IJLiOI I of viruses on the cellular receptors and thus prevent infection, or bacteria-specific dl 1"' _ " 35 ` ~ 21856~7 opsonize the pathogen and thus allow it to be ~ ' ";, I..'~d and killed by neutrophils and Illal~luplldges~ Plasma pools from several thousand donors contain immunno~ohll " of very many different sp~,iri~,ili~s, and immunoglobulin ~ ,Odl dLiul 1~ from such pools consequently also contain 5 measurable titers of immunnoloh~ ~" " directed against epitopes on viruses, bacteria, and toxins, but also against au~uallLiyel~s Hence they are effective against many infections and in the most varied other ,~ " ,oluyi~al conditions.
Now, under certain circumstances, however, it is desirable to make use of an immunoglobulin plt:~Jalaliul~ having high titers of specific a~ ~ " , a so-called hyperimmunoglobulin plt:paldtiUn. Until now, such ~ ualdLiul~s have been prepared at great expenditure of time and money from special plasma pools of donors having increased titers of specific cl, I';L - 'i~ ~ For variousreasons, this involves dffliculties. If, as in the case of an anti-hepatitis B
p, t:~.a, ' ~, there are ~c~y"i~d vacci, laLiul, procedures, then the donors 15 must be inoculated and selected, and the donated blood must be separately p,uce~d. In the case of many other i"~ ' 1S, however, immunization of the donor cannot take place for ethical reasons. Here it is only rarely possibleto locate high-titer blood through an involved selection of the donors (e.g., afbr their having recovered from a specific disease) and to obtain a IJIt:~,al "
20 through ~., u~ i"y of the donated blood.
It is therefore an object of this invention to provide a method of recovering immunoolob~ ~" " by means of which valuable immunoglobulin JaldLiùl~, can be made a~c~ and isolated from the dru,~",~"liu"ed fractions and p,~ci~.;' ' , hardly made use of until now, which become 25 available during industrial plasma-r,d-,Liur,dLio,~ methods. S~hseqllently, it should be possible to process these IJlt:~JdldLiulls, c~ pul~di~ly to hyperimmunoglobulin l.,~pd,.lliuns, into a well-tolerated, especially intravenously (IV), virus-proof, liquid or freeze-dried 1 l t:,Udl " 1.
It has now been found that it is possible to produce 30 hyperimmunoglobulin ~,r~pd, dLiul~s or high-titer immunoglobulin pfeud, dLiol ,s by a method dfflering from the prior art methods. This novel method uses immunooloh~ from the general plasma pools, improves the ~ of the valuable raw material blood plasma through the use of ~wasteU fractions, 21~617 and even permits the production of hyperimmunoglobulin ~l~palaliull;~ having far greater specific activities than previous ~, c ual dtiUI 1~. This means that with small quantities a.l",i";~ d IV, and with low amounts of IV ad~"i"' ' ed proteins, i.e., a c~ a,uOI lui. I~ly low burden on the recipient, high doses of 5 specific immuno~ hl 1" ,~ can be given within a short time. This is made possible in the inventive method through the co,~c~"' aliOI~ of the specific immuno~ bll" ,s through ads~"uliùn on i""" ' ~ antigens, thus through the use of ,ul vl-essed "waste" fractions in affinity ~I Il ul l l ' _ a,ul iic techniques.
To this end, the method of producing immunoglobulin pl~,udldliuils 10 according to the present invention comprises the steps of rldl,liulldlillg blood plasma from a plasma pool, whereby at least one industrially usable fraction sulJ~ldl 1" 'Iy C~l 1' ' 1' Iy polyclonal immunoglobulin G is separated, at least one residual fraction being obtained; preparing a protein solution from the protein .,o,,,,uu, ,~"t~ contained in the residual fraction obtained in the previous step or in sUblla~.liOII~ thereof; and subjecting the resulting protein solution at least once to affinity ,1 1l u", ' _ d,UI Iy with i"""~ ' "' -' ligands of at least one ligand tvpe, the spec'lfic plasma proteins being bound to the ligands, and removing thebound plasma proteins, which are converted as active c~,,,pu,,~ into a high-titer immunoglobulin ,u, epd, dliùl~.
In the inventive method, a fraction obtained in a plasma r,d~liu, Idlio~
process, particularly a waste fraction, is first ,u, u.,~:.ed in such a way that it cdn be used in affinity ~,lllullldluyla,ully. Depending upon the provenance of the fraction, this ,u, U~,C~si, ~y may vary greatly. Thus, for example, the supematant GG may be col~cel ,' ' -' down, dialyzed against a suitable buffer, 25 and filtered. On the other hand, u, . ', " ' ~ or filter cakes (lists of examples of such starting materials are given in Tables 1 and 2 below) must first be suitably suspended, i.e., through variation of the ionic strength, the pH, andlor the le"",e, ' Ire, or through the addition of d~it~i. ye"L;, and salts, in such a way that immunoolob~ are spe~,iri 'Iy 5~ b"' i~ They may, for example, be stirred 30 overnight within a pH range of from 3-9, at a conductance of from 5-2û mS/cm, at 4C and then be clarified by centrifugation andlor filtration. Both s~pe" IdLal ILa and suspel~sio~s may at this stage be subjected a virus-inactivation process according to the solvent-detergent method of Horowitz 2~856~7 (Tl)~ulllbua;~ and l ~ , 65, 1163, 1991), the methylene blue method (Mohr et al., Infusionsther. Infusionsmed. 20, 1911993]), or some other method.
The immuno~l~b~ 1' ,, may also be subjected after suspension to prepurification by p,~d.l~u,,ulion on a matrix in a column or through treatment with a suitable 5 filter aid or adsorbent such as aluminum hydroxide, ,lllu"l ' _ d~JIly on protein A or G for co~c~"' " ~, or one or more ~ s by the common methods of ammonium sulfate, polyethylene glycol, or ethanol pf~-,i,uildLiùll or ~.UI I Ibil IdliOl~s thereof for ~", i~,l " "~:"I, co,~c~"' dliUI), or depletion of disturbing cu",,u~ "t ..
Su,u~ ldldllb Pl~ , :' ' - and Residues Supernatant c Precipitate B
Supernatant GG Precipitate IV
Residue after DEAE treatment Residue after aluminum hydroxide treatment Residues after clarifying filtrations 1, ll and lll Table 1: Examples of possible starting materials coming from rld~liull~
according to Kistler and ~ Idl 111 or from other ~, u~essil ,9 steps resulting from the ,UI~pdldliUII of IV adlllill;~lldble, stable plasma products.
Su,ut:l I Idldl 11~ Pl ~ui,uildlc3 and Residues SupernatantV Precipitate ll+lll Supernatant 11-1,2 Precipitate IV-1 Residue after DEAE treatment Residue after aluminum hydroxide treatment Residues after clarifying filtrations 1, Il, and lll Table 2: Examples of possible starting materials coming from the r~ d~liUIldliU
according to Cohn or from other ,u, u~.~s ,i"~ steps resulting from the Jdldliùll of IVa.llllilli~lldble:, stable plasma products.
21856~7 The following list gives examples of possible ligands for the inventive affinity 1,111 Ul I ~, d,JI Iy Antigenic delel I l lil Idl ,t~ of I lae, I ,opl l ' le influenza b Staphylococcus aureus Staphylococcus ~:,uidel 1 l lidi., Staphylococcus ~ e Sl, IJt. ~COGGI IS pneumoniae Stl-lJt ~,o~,cl le pyogenes and other ~ " ,oye,~ic strains of bacteria tetanus toxin Staphylococcus aureus toxic shock toxin and further r IOyel ,;~ bacterial or other toxins hepatitis A virus hepatitis B virus hepatitis C virus varizella zoster virus cy~"lèydlo virus respiratory syncytial virus 2~ parvovirus B19 herpes simplex virus 1 herpes simplex virus 2 rabies virus and other r ,oge"i~. viruses CD2, CD3, CD4, CD5, CD28, CD4û, CD72 ICAM, LFA-1, LFA-3, DNA
and other potential human ~I ~' ILi~ells Since affinity gels have been prepared by i""" ' " ,y modified or unmodified ligands (examples of such ligands are given in the above list) by means of methods known per se, they are loaded with the ~, u~,essed SU,U~I I Idldl 11~ and Sus~Jell~ions in col~cél ,' dled or diluted form, if necessary also by repeated ~ , ' ' , of the flow. If desired, various affinity gels may be35 activated in s~lccessi~n with the same suspension. The gels are thereafter washed in such a way that u" ,pe,,iri 'Iy binding proteins are removed ,ulepol~deldlllly or to a sufficient extent. This can be done, for example, by 2i85617 i"", t:asi"y the saline c~c~"' " " by addition of a detergent, and/or by shifting the pH in the washing solution. The bound proteins are now separated from the ligands, e.g., by elution at a low or high pH, by addition of .,I I - '. upic saline solutions such as sodium thiocyanate or magnesium chloride, denaturing s agents such as SDS or urea, solvents such as ethylene glycol, by modifying the Itll l,u~ e, or by cul l lbil ldliùl l~ of the foregoing.
In some cases, it may be desirable to modify the ligands to be i"", ' ~ by mutagenesis or by chemical or physical methods in such a way that the specific immunogl~hl ~' Ia can still bind to their epitopes, but with reduced affinity, so that elution can take place under milder conditions than with the u"l"odiried ligands. ~' ' ' ' , of the ligands may also take place in order to facilitate and improve their illll l l L :' ' .1 and/or their epitope pl t:s6'1 Italiù~. Technical details and basic principles of the affinity l,lllulll ' _ d,UIly process in general are described in Cuatrecasas, P., and 15 Anfinsen, C. B. (1971), Ann. Rev. Biochem. 40, 259; Kull, F. C., and C~ ,asas, P. (1981), J. Immunol. 126, 1279; Liebing et al. (1994), Vox Sang. 67, 117.
The specific, separated immuno~l~h~' ,s, also with additional filtration for eli."i, Idlil ,9 viruses, if need be, are p~uc~sed into an end product 20 which can preferably be a.l",i" ' .c:d IV and which is free of pyrogens, virus-proof, and stable with or without the addition of stabilizers such as albumin, amino acids, or carbohydrates in liquid or freeze-dried form. However, formulations may also be used which make possible intramuscular or topical ad",i";~l,dlio,~.
Preferred ~",L,odi."e"l~ of the present invention are described and illustrated below by means of the following examples, which are not intended to be limitative Example 1 HBsAg-Sepharose was prepared by i""" ' :' ' ,9 5 mg of 30 l~collllJilldlll hepatitis B virus surface antigen (HBsAg, Abbott DidyllOalil,~) through coupling of the primary amino groups to 1 ml of activated CH-~ 2185617 Sepharose as directed by the manufacturer (Pl,a""acia Biotech, Uppsala,Sweden). "Placebo~-Sepharose was prepared by carrying out the same coupling process with another gel aliquot, but without adding HBsAg. The finished gels were stored in PBS with 0.02% NlaN3 at 4C.
Seventy grams of ,u,~ :, ' ' B from the Kistler ': ' 'Illldllll plasma r~ d~liUI IdliOI I (NB Lot 4.030.216) were suspended ovemight in 210 ml of 100 mM citric acid, pH 4.0, 0.25% Triton X-100, 10 mM N-ethyl maleimide (NEM), 1 mM phenylmethylsulfonyl fluoride (PMSF) at 4C on a RotaryMix appliance.
After 5~dld~iOI~ of the insoluble cull,,uu,,e:,,t~ by centrifugation (50009, 4C, 10 min.) and filtration (pore size: 1.2 ~lm), 1% tri-n-bu~yl~l lua~ I ' (Merck, Darmstadt, Germany) and 1% Triton X-100 were added at a neutral pH
according to Horowitz et al. (Tl ll ul l lbo ~H and l ldt~ u~ld ~i ~, 65, 1163, 1991 ) and incubated at 30C for 4 hrs. with mixing. A phase 5~dl dliUIl was then carried out overnight at 37C, the clear lower phase was removed, filtered 15 through a 0.45 llm filter, and stored at 4C.
One hundred thirty ml of such an NB suspension were diluted with 280 ml of PBS (plloa,ul~dl~-burfered saline solution: 150 mM NaCI, 10 mM
sodium pllo~,l, ', pH 7.1) and pumped overthe placebo-Sepharose column, then over the HBsAg-Sepharose column at 4C in such a way that the column 20 flow passed back into the storage vessel. The rate of flow was 8 ml/hr for 144 hrs. Thus the NB suspension was pumped over the columns a total of three times. After 90 hrs., the loading was interrupted, and the columns were washed individually, first with PBS and then with 200 mM NaCI, 50 mM tris-HCI, pH 7.4, until the wash solution had an optical density of less than 0.01 at 280 25 nm (OD2go). After conclusion of the activation, washing was again carried out with PBS and 200 mM NaCI, 50 mM tris-HCI, pH 7.4. Bound proteins were separated with 5 ml of 200 mM glycine-HCI, pH 2.5, i""" ' ' !y neutralized, and ~u~ sed.
` ~ 2185617 Gel HBsAg Placebo Tota loading:
rotein ~~5 mg ~ '~5 mg 3G ~ mg ~ mg Anti-HBs IgG ~ 0 mlU A o mlU
Flow:
~nti-HBs IgG 1140 mlU 1140 mlU
Elua e:
~G 0.13 mg 0.08 mg Anti-HBs IgG 5603 mlU 114 mlU
Table 3: Anti-HBsAg affinity ~ lulll ~ ly with NB suspension Example 2 Starting from the Cohn fraction ll+lll (instead of blood plasma as the starting material), a Kistler-Nit,~,l""d"" r~ l;UlldLiUI~ was carried out. Seventy gramsofp~ ,i,uildLt:B(NBLot4.044.488)fromthisKistler~ 'lllldllll r~ d~,liO~ IdliUIl were suspended overnight in 210 ml of 100 mM citric acid, pH 4.0, 0.25% Triton X-100, 10 mM NEM, 1 mM PMSF, at4C on a RotaryMix 10 appliance. Afterclc" iri~dliUIl andpartial~.I;, .;rl.~ JII by~".,c~"'irugatiorl (100,0009, 3 hrs., 4C: the clear phase was withdrawn by piercing the side of the tube with a syringe), the suspension was filtered (0.45 llm) and stored at 4C.
One hundred twenty-five ml of this NB suspension were diluted with 15 375 ml PBS, the pH adjusted to 7.1 with 0.1 \/1 NaOH, filtered, and pumped, as in Example 1, first over placebo-Sepharose gel and then over HBsAg-Sepharose gel prepared dl I ' J l':ly to Example 3. The gels were then washed sepd, ' !y with PBS and 200 mM NaCI, 50 mM tris-HCI, pH 7.4. The bound proteins were removed with 5 ml of 200 mM glycin-HCI, pH 2.5. Table 4 20 gives a summary of the data.
2~85617 Gel HBsAg Placebo Tota loading:
rotein ~ 400 mg ~ ~ O mg 3G 19 mg ' mg Anti-HBs IgG 0 IU C U
Flow:
Anti-HBs IgG 5 IU 5 IU
Elua :e:
3G 0.18 mg 0.35 mg Anti-HBs IgG 3.3 IU 0.08 IU
Table 4: Anti-HBsAg affinity ~,I"u", ' _ d~JIly with NB suspension (Cohn ll+lll) Example 3 Thirty liters of supernatant GG (Lot No. X95.31.286.1) were diafiltered in PBS and col1ce"' dL~d down to 500 ml. As in Example 1, the col~ce" ' was pumped over placebo and HBsAg columns at 21 ml/hr for 118 hrs. Washing of the columns and S~dld~;UII of the bound proteins likewise took place a,~ ' ~, Isly to Example 1, but an additional washing step with 500 mM NaCI, 50 mM tris-HCI, pH 7.4, was carried out. The results are shown in Table 5.
Gel HBsAg Placebo Total loading:
Protein 8800 mg 8800 mg IgG 320 mg 320 mg Anti-HBs IgG 5000 mlU 5000 mlU
Flow ~nti-HBs IgG ~DL ~DL
Elua .e:
~G 0.05 mg 0.14 mg Anti-HBs IgG 3035 mlU 7 mlU
Table 5: Anti-HBsAg affinity ~ Jllldluyla,ullywith supernatant GG co~lce" dl~
DL: detection limit ~ ~ 2185617 Example 4 DEAE filter cake in an amount of 17.5 9 (Lot 4.422.006.0) was suspended in 52.5 ml of suspension buffer according to Example 1 and p,uc~sed. Forty ml of suspension were diluted with 160 ml of PBS, the pH
5 adjusted to 7.1, and then filtered. As in Example 3, the suspension was pumped over placebo and HBsAg columns at 21 ml/hr for 97 hrs. Washing of the columns and ;._~Jdl dliUI I of the bound proteins also took place dl)~ cly to Example 1. The results are shown in Table 6.
Gel HBsAg Placebo Tota loading:
rotein ~ mg ~ mg ~G mg . mg Anti-HBs IgG ~ mlU ~ mlU
Flow:
~nti-HBs IgG ~DL ~DL
Elua e:
~G 0.07 mg 0.11 mg Anti-HBs IgG 331 mlU 14 mlU
Table 6: Anti-HBsAg affinity Ul~l u", ' _ d,UI Iy with DEAE
filter-cake suspension DL: detection limit Example 5 Tetanus toxoid C-Sepharose was prepared by i""" ' :' ,g 11.5 mg of purified tetanus toxoid (TT) through coupling of the carboxy groups to 1 ml of EAH-Sepharose (Pl,d""acia Biotech, Uppsala, Sweden) with the aid of 0.1 M
15 N-ethyl-N'-(3-dimethy'..."i"u,u,u,uyl)-cd,L ' "ide-HCI, as directed by the manufacturer. The finished gels were stored in PBS with 0.02% NaN3 at 4C.
A cu, Ice, ,I, dlt: of supernatant GG was prepared as in Example 3 and used for affinity .,l " u~ d~,l ,y with TT-Sepharose d" ' ~, Icly to Example 2.
Activation took place at 23.5 ml/hr for 159 hrs. at 4C. The results are shown in 20 Table 7.
2~85617 Gel Tetanus Toxoid C
Tota loading:
rotein ' 000 mg 3G 0 mg Anti-TT IgG ~Lg Flow Anti-TT IgG 16 1l9 Eluate:
IgG 0.16 mg Anti-TT IgG 76 ~9 Table 7: Anti-tetanus toxoid affinity ~l ll u" IdlUUl d,UI Iy with supernatant GG
co,~c~, It Example 6 Tetanus toxoid N-Sepharose was prepared by i"" ' :' ,9 11.5 mg of purlfied tetanus toxoid (TT) through coupling of the primary amino groups to 1 ml of activated CH-Sepharose as directed by the manufacturer (Pharmacia Biotech, Uppsala, Sweden). "Placebo=-Sepharose was prepared by carrying out the same coupling process with another gel aliquot, but without adding TT.
The finished gels were stored in PBS with 0.02% NaN3 at 4C.
Precipitate B was suspended according to Example 2.
After filtration (1.2 ~Lm), 115 ml of this suspension were diluted with 200 ml of PBS, the pH adjusted to 7.1, and pumped first over the placebo-Sepharose at 3.5 ml/hr for 165 hrs. in such a way that its flow passed i""" ' ' 'y thereafter over the TT N-Sepharose column, then back into the storage vessel. The columns were washed sepd, ' Iy with PBS and then with 0.5 M NaCI, 50 mM tris-HCI, pH 7.1, until the OD283 of the flowwas less than 0.01. The results are shown in Table 8.
` ~ 2185617 Gel Tetanus Toxoid N Placebo Tota loading:
rotein ' 334 mg ' 334 mg ~G 33 mg 33 mg Anti-TT IgG .509 mg .509 mg Flow:
Anti-TT IgG 0.213 mg 0.213 mg Elua e:
3G 0.35 mg 0.004 mg Anti-TT IgG 0.091 mg 0.003 mg Table 8: Anti-tetanus toxoid affinity ~ lullldiuuld~,llywith NB suspension Example 7 Precipitate B was suspended according to Example 2.
After filtration (1.2 ~lm), 115 ml of this suspension were diluted with 20û ml of PBS, the pH adjusted to 7.1, and pu~ped first over the HBsAG-Sepharose column at 5 ml/hr for 165 hrs. in such a way that its flow passed i,,,,l, " ' ~y thereafter over the TT-Sepharose column and then back into the storage vessel. The columns were washed 5~ y with PBS and then with 10 0.5 M NaCI, 5û mM tris-HCI, pH 7.1, until the OD28o of the flow was less than 0.01. The results are shown in Table 9.
Gel HBsAg Tetanus Toxoid Tota loading:
rl~tein ' 334 mg ' 334 mg 3~ 33 mg 33 mg An -HBsAg 45 mlU 45 mlU
An -TT IgG .509 mg .509 mg Flow Anti-HBsAg 536 mlU 536 mlU
Anti-TT IgG 0.265 mg 0.265 mg Elua e:
~G 0.13 mg 0.25 mg Anti-HBsAg 1152mlU
Anti-TT IgG 0.18 mg 2~856~7 Table 9: Anti-HBsAb and anti-tetanus toxoid affinity 1,l ll u, l l ' v dUI Iy with NB
suspension Example 8 Fifteen kg of ~,~ , ' ' B from the Kistler ~ 'lllldl 111 r~ d'vliU~ld~iOI~
5 (Lot No. 5.043.303) were suspended in 45 liters of 0.1 M citric acid, pH 4.0, 0.25% Triton X-100, 10 mM NEM, 1 mM PMSF overnight at 4C with a vil,,u,,,i~el. After sepdld~ of the insoluble c~",~,u"~"~v by adding filter aidsand filtering (pore size: 1.2 llm), 1% tri-n-butyl pllo~,ul Idle and 1% Triton X-100 were added at neutral pH for dF l;l 'i~ and virus inactivation according to Horowitz and incubated for 4 hours at 30C with mixing. Thereafter, a phase 5~1Jdl ClliOIl was carried out overnight at 37C, ~he clear lower phase pumped off, filtered through a 0.45 llm filter, and stored at 4C.
The pH of 40 liters of this NB suspension was adjusted with NaOH to a value of 7.1, and it was pumped at 4C over a HBsAg column and a tetanus 15 toxoid Affiprep column in such a way that the column flow passed back into the storage vessel. The Affiprep columns (50x13 r~m) were prepared by coupling 250 mg of It:c~ b;lldlll HBsAg and tetanus to~(oid, ~e:v,ue~.t;lcly, with 25 ml of Arfiprep gel (BioRad Lab. Inc., Hercules, CA 94547. U.S.A.). The rate of flow was 6 IVhr for 62 hrs. Thus the NB suspension was pumped over the columns 20 a total of three times. After conclusion of the activation, the columns were washed sepd, ' 'y, first with PBS and then with 500 mM NaCI, 50 mM tris-HCI, pH 7.4, until the washing solutions had an optical density of less than 0.01 at 280 nm (OD2go). Bound proteins were removed with 200 mM glycin-HCI, pH
2.5, and the pH of the fractions i""" ' ' Iy adjusted to 5.2. The results are 25 compiled in Table 10. The immunoglob~ v were p~uc~vve~ into stable ult:pdldliol~s by pooling the fractions containing IgG, didri" dliol~, and Cul ICt~ ldliUI I with 20 mM NaCI into solutions of 100 lU/ml and 2.5 mg anti-TT-lgG/ml, respectively. Ten percent sac.,l,d,uve was added, and the solutions were Iyophilized in units of 200 IU, and 5 mg of anti-TT-lgG"-, - ' .~ely.
2~85617 Example 9 Fifty grams of p~e~ LdL~ lv from the Kistler ~ dl 111 plasma r, d~,Liul IdLiUI I were suspended in 500 ml of water. The pH was adjusted to 5.0 with citric acid, and the conductance to 13 mS with NaCI. After stirring 5 overnight at 4C, Cldl iri-,dLi,,,. and partial .' ~ 'i, ' ' ~ were achieved by centrifugation for 30 min. at 30,0009 and 4C. The separated protein, IgM, IgA, IgG, L, dl "~rt:" i", and ceruloplasmin content of the suspension was dt:Lt~ ,edand is indicated in Table 11.
Gel HBsAg Tetanus Toxoid Total loading:
Pr tein ~ 9 ~ g 19~, ~ 9 (,~ 9 An -HBs IgG 0 IU 0 IU
An -TT IgG v mg ~' mg Flow.
Anti-HBs IgG 221 IU 221 IU
Anti-TT IgG 172 mg 172 mg Elua e:
~ 39.8 mg 171 mg An -HBs IgG 1368 IU
An -TT IgG 89 mg Table 10: Anti-HBsAg and anti-tetanus toxoid affinity ,l ll u", ' _ alJl l~l with NB
10 suspension ¦ Total Protein l9~ IgA IgG Transferrin Caeruloplasmin ¦
5.5 0.063 1 0.628 1 0.488 1 2.582 1 0.09 Table 11: Suspension from ,I~ ,itdL~: IV (all values in mg/ml) Example 10 Fifty grams of p~. , ' ' B from the Kistler ~ ' ll l ldl ll l plasma 15 r, d~,liul1dli~ (Lot No. 4030.204.0) in 100 mM citric acid at various pH values and at 4~C were stinred overnight, clarified by ~ ~" d-,~l ,' irugation at 100,0009 and 4C for 3 hrs. and partially d~ l The clear middle phase was removed, and its separated protein, IgM, IgA, IgG, LldllaF~llill, and ceruloplasmin content was d~ :Lt:" "i"ed (Table 12).
i 21~5617 ~
~c .al Protein Ig`1 IgA IgG Transferrin Caenuloplasmin pH4.0 ~. 1. 1.3 1.9 <DL 0.06 pH 5.0 . 1. . 1.0 2.1 <DL <DL
Table 12: Suspension from pl~:l iUildL~ B (all values in mg/ml);
<DL: belowdetection limit The titer of IgG against certain viral (Table 13) and bacterial (Table 14) antigens was .I~:Lt:l " ,i"ed and compared with that in starting plasma and 5 existing immunoglobulin plt~Jdldliol)s.
Plasma Plasma SAGL NB pH 4.0 NB pH 5.0 ' ~2 03 An -H sAg . 2 .05 0. 1 . . 5 'mg IgC
An -C lV . 8 .16 0. . . 4 IE/mg gG
An--V_V . 8 .09 n.. . . mg IgG
An -measles . .2 0. 2 . . 1 /mg IgC
An--HSV1 C37 n.d. n.~. 317 49 AJ/mg IgG
Table 13:
Antiviral IgG in plasma pools SAGL and NB suspel~siol~s Plasma 102/103: plasma pools; SAGL: Sar.do~lob~ NB pH 4.0/5.0: suspension from p~ dL~ B at pH 4.0 and 5.0 resp~ctively; IU: I"~."dLio~1al Units; PEIE:
Paul Ehrlich Institute units; AU: arbitrary units; n.d.: not de l~ d.
Plasma 102 Plasma 103 SAGL NB 4A NB 14 pH 4.0 anti- 162 286 436 100 283 1l9/9 IgG
HiBOAg anti-TT 1855 4159 2740 2928 2923 1l9/9 IgG
anti-SEB 412 689 783 918 670 AU/g IgG
Table 14: A"Lil.a~Lc:rial IgG in plasma pools SAGL and NB SU5pc:1~5iOI15 Plasma 102/103: plasma pools; SAGL: Sandn~ hll" ,~3 NB 4A: suspension from ,~ iuiL.d~ B at pH 4.0; NB 14 pH 4.0:
15 suspension from p, ~ciyiL~._ B at pH 4.0 with virus inactivation; HiBOAg:
l1a~l"uul, " Is influenza B-o~;gosac~l,d,ide antigen; TT: tetanus toxoid; SEB:
Staphylococc~s ~"Lt~ ., B; AU: arbitrary units.
'
Claims (16)
1. A method of producing a high-titer immunoglobulin preparation, wherein the following steps are carried out in succession:
(a) fractionating blood plasma from a plasma pool, whereby at least one industrially usable fraction substantially containing polyclonal immunoglobulin G is separated, at least one residual fraction being obtained;
(b) preparing a protein solution from the protein components contained in the residual fraction obtained in step (a) or in subfractions thereof; and (c) subjecting the resulting protein solution at least once to affinity chromatography with immobilized ligands of at least one ligand type, the specific plasma proteins being bound to the ligands, and removing the bound plasma proteins, which are converted as active components into a high-titer immunoglobulin preparation.
(a) fractionating blood plasma from a plasma pool, whereby at least one industrially usable fraction substantially containing polyclonal immunoglobulin G is separated, at least one residual fraction being obtained;
(b) preparing a protein solution from the protein components contained in the residual fraction obtained in step (a) or in subfractions thereof; and (c) subjecting the resulting protein solution at least once to affinity chromatography with immobilized ligands of at least one ligand type, the specific plasma proteins being bound to the ligands, and removing the bound plasma proteins, which are converted as active components into a high-titer immunoglobulin preparation.
2. A method according to claim 1, wherein the fractionation of blood plasma is carried out on an industrial scale, the residual fraction being a waste fraction.
3. A method according to claim 1 or 2, wherein the residual fraction according to step (a) is a precipitate or a filter cake, and the protein components are put into solution by treating the precipitate or the filter cake with an aqueous buffer solution having an ionic strength of <5M
and a pH of from 3.0 to 9.0, a solution or suspension being formed.
and a pH of from 3.0 to 9.0, a solution or suspension being formed.
4. A method according to claim 3, wherein the buffer solution is a phosphate buffer, a tris-HCI buffer, or a citrate buffer.
5. A method according to claim 3 or 4, wherein the buffer solution contains a detergent, one or more protease inhibitors, and/or a salt.
6. A method according to one of the claims 3 to 5, wherein the solution or suspension is filtered or is pretreated with an adsorbent such as aluminum hydroxide or an adsorbent containing DEAE groups.
7. A method according to one of the claims 3 to 5, wherein the solution or suspension is reacted with ammonium sulfate, polyethylene glycol, or ethanol, a precipitate being formed, and the supernatant or the precipitate being further processed.
8. A method according to claim 1 or 2, wherein the residual fraction according to step (a) is a supernatant, and the protein solution is prepared by filtration and concentration, e.g., by diafiltration
9. A method according to claim 8, wherein the supernatant is filtered or is pretreated with an adsorbent such as aluminum hydroxide or an adsorbent containing DEAE groups.
10. A method according to one of the claims 1 to 9, wherein the immobilized ligands are natural or recombinant, viral, bacterial, or cellular antigens.
11. A method according to claim 10, wherein the ligands are selected from the group of antigen determinants of Haemophilus influenza b, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, tetanus toxin, Staphylococcus aureus toxic shock toxin, hepatitis A
virus, hepatitis B virus, hepatitis C virus, varizella zoster virus, cytomegalo virus, respiratory syncytial virus, parvovirus B19, herpes simplex virus 1 and 2, rabies virus, and the potential human autoantigens CD2, CD3, CD4, CD5, CD28, CD40, CD72 ICAM, LFA-1, LFA-3, DNA, and phospholipids.
virus, hepatitis B virus, hepatitis C virus, varizella zoster virus, cytomegalo virus, respiratory syncytial virus, parvovirus B19, herpes simplex virus 1 and 2, rabies virus, and the potential human autoantigens CD2, CD3, CD4, CD5, CD28, CD40, CD72 ICAM, LFA-1, LFA-3, DNA, and phospholipids.
12. A method according to claim 10 or 11, wherein that the ligands have been modified by mutagenesis or chemical or physical methods.
13. A method according to one of the claims 1 to 12, wherein the high-titer immunoglobulin preparation obtained consists only of immunoglobulins of the classes G or A or M or any combination thereof.
14. A method according to one of the claims 1 to 13, wherein the high-titer immunoglobulin preparation obtained is subjected to virus inactivation and, if necessary, stabilized, a stabilizer such as albumin, amino acids, or carbohydrates being added and/or the product being freeze-dried.
15. A method according to one of the claims 1 to 14, wherein the high-titer immunoglobulin preparation obtained is converted into a pharmaceutically acceptable product, e.g., into an intravenously, intramuscularly, or topically administrable preparation.
16. A method of producing a high-titer immunoglobulin preparation, wherein the following steps are carried out in succession:
(a) preparing a protein solution from the protein components of a residual fraction, or subfraction thereof, obtained during the fractionation of blood plasma, and (b) subjecting the resultant protein solution at least once to affinity chromatography with immobilized ligands of at least one ligand type, the specific plasma proteins being bound to the ligands, and separation of the bound plasma proteins, which are converted as active components into a high-titer immunoglobulin preparation.
(a) preparing a protein solution from the protein components of a residual fraction, or subfraction thereof, obtained during the fractionation of blood plasma, and (b) subjecting the resultant protein solution at least once to affinity chromatography with immobilized ligands of at least one ligand type, the specific plasma proteins being bound to the ligands, and separation of the bound plasma proteins, which are converted as active components into a high-titer immunoglobulin preparation.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP95810596.7 | 1995-09-22 | ||
| EP95810596A EP0764658B1 (en) | 1995-09-22 | 1995-09-22 | Process for obtaining immunoglobulins from fractions originating from the fractionation of human blood plasma |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2185617A1 true CA2185617A1 (en) | 1997-03-23 |
Family
ID=8221797
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002185617A Abandoned CA2185617A1 (en) | 1995-09-22 | 1996-09-16 | Method for recovering immunoglobulin from fractions produced during fractionation of human blood plasma |
Country Status (21)
| Country | Link |
|---|---|
| EP (1) | EP0764658B1 (en) |
| JP (1) | JP2952572B2 (en) |
| KR (1) | KR100236762B1 (en) |
| CN (1) | CN1089609C (en) |
| AT (1) | ATE211486T1 (en) |
| AU (1) | AU715427B2 (en) |
| BR (1) | BR9603826A (en) |
| CA (1) | CA2185617A1 (en) |
| CZ (1) | CZ286885B6 (en) |
| DE (1) | DE59509979D1 (en) |
| DK (1) | DK0764658T3 (en) |
| ES (1) | ES2170788T3 (en) |
| FI (1) | FI963719A (en) |
| HU (1) | HUP9602570A3 (en) |
| MX (1) | MX9604256A (en) |
| NO (1) | NO963952L (en) |
| NZ (1) | NZ299394A (en) |
| PL (1) | PL316126A1 (en) |
| PT (1) | PT764658E (en) |
| RO (1) | RO117921B1 (en) |
| RU (1) | RU2157240C2 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6159471A (en) * | 1997-10-23 | 2000-12-12 | Yoshitomi Pharmaceutical Industries, Ltd. | Room temperature storable immunoglobulin preparation for intravenous injection |
| US8198407B1 (en) | 2004-08-20 | 2012-06-12 | Prometic Biosciences, Ltd. | Sequential protein isolation and purification schemes by affinity chromatography |
| US8632773B2 (en) | 2009-03-26 | 2014-01-21 | Csl Behring Ag | Antibody composition with altered Fab sialylation |
| US8900806B2 (en) | 2010-04-22 | 2014-12-02 | Biotest Ag | Antibody preparations |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IT1293119B1 (en) * | 1997-06-12 | 1999-02-11 | Mini Ricerca Scient Tecnolog | PROCESS FOR THE SEPARATION OF SPECIFIC IMMUNOGLOBULINS FROM STANDARD PLASMA AND THEIR USE FOR THERAPEUTIC TREATMENTS |
| WO2010148117A1 (en) * | 2009-06-17 | 2010-12-23 | Scantibodies Laboratory, Inc. | Therapeutic and diagnostic affinity purified specific polyclonal antibodies |
| ITRM20090558A1 (en) * | 2009-11-03 | 2011-05-04 | Michele Pitaro | PROCEDURE FOR THE PRODUCTION OF IMMUNOGLOBULINES EXTRACTED FROM HUMAN PLASMA FOR THERAPEUTIC USE NEUTRALIZING THE VIRUS OF EPSTEIN BARR AND DRUG CONTAINING DETTE IMMUNOGLOBULINE |
| EA201390156A1 (en) * | 2010-07-23 | 2014-01-30 | Бакстер Интернэшнл Инк. | PRODUCTION OF INTER-ALPHA INHIBITOR PROTEINS (IaIp) FROM PLASMA |
| ES2531459B1 (en) * | 2013-06-10 | 2015-12-28 | Antonio VILA SANJURJO | Method for the physical separation of convoluted "traductomas" |
| FR3018450B1 (en) * | 2014-03-11 | 2016-04-15 | Lab Francais Du Fractionnement | PROCESS FOR THE PREPARATION OF HUMAN PLASMA PROTEINS |
| KR20150115639A (en) * | 2014-04-04 | 2015-10-14 | 전숙영 | An acidic buffer solution for manufacturing therapeutic immunoglobulin preparation, manufacturing method of therapeutic immunoglobulin preparation using the acidic buffer solution and therapeutic immunoglobulin preparation maid by this method |
| GB201413227D0 (en) * | 2014-07-25 | 2014-09-10 | Bioproducts Lab Ltd | Process |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4232004A (en) * | 1977-09-21 | 1980-11-04 | American National Red Cross | Antibody-specific solid phase immunoadsorbent, preparation thereof, and antibody purification therewith |
| DE3516119A1 (en) * | 1985-05-04 | 1986-11-06 | Biotest Pharma GmbH, 6000 Frankfurt | POLYVALENT HYPERIMMIMMLOBULIN PREPARATE |
| IL90281A (en) * | 1988-06-06 | 1994-10-07 | Miles Inc | Composition consisting of antibodies of the igm type |
-
1995
- 1995-09-22 DE DE59509979T patent/DE59509979D1/en not_active Expired - Lifetime
- 1995-09-22 DK DK95810596T patent/DK0764658T3/en active
- 1995-09-22 AT AT95810596T patent/ATE211486T1/en not_active IP Right Cessation
- 1995-09-22 PT PT95810596T patent/PT764658E/en unknown
- 1995-09-22 ES ES95810596T patent/ES2170788T3/en not_active Expired - Lifetime
- 1995-09-22 EP EP95810596A patent/EP0764658B1/en not_active Expired - Lifetime
-
1996
- 1996-09-16 CA CA002185617A patent/CA2185617A1/en not_active Abandoned
- 1996-09-16 NZ NZ299394A patent/NZ299394A/en unknown
- 1996-09-18 KR KR1019960040589A patent/KR100236762B1/en not_active IP Right Cessation
- 1996-09-18 CZ CZ19962748A patent/CZ286885B6/en not_active IP Right Cessation
- 1996-09-18 PL PL96316126A patent/PL316126A1/en unknown
- 1996-09-19 HU HU9602570A patent/HUP9602570A3/en unknown
- 1996-09-19 RO RO96-01839A patent/RO117921B1/en unknown
- 1996-09-19 AU AU65725/96A patent/AU715427B2/en not_active Ceased
- 1996-09-19 FI FI963719A patent/FI963719A/en unknown
- 1996-09-20 NO NO963952A patent/NO963952L/en unknown
- 1996-09-20 BR BR9603826A patent/BR9603826A/en active Search and Examination
- 1996-09-21 CN CN96122771A patent/CN1089609C/en not_active Expired - Fee Related
- 1996-09-23 RU RU96118916/14A patent/RU2157240C2/en not_active IP Right Cessation
- 1996-09-23 MX MX9604256A patent/MX9604256A/en unknown
- 1996-09-24 JP JP8251895A patent/JP2952572B2/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6159471A (en) * | 1997-10-23 | 2000-12-12 | Yoshitomi Pharmaceutical Industries, Ltd. | Room temperature storable immunoglobulin preparation for intravenous injection |
| US6485725B1 (en) | 1997-10-23 | 2002-11-26 | Welfide Corporation | Room temperature storable immunoglobulin preparation for intravenous injection |
| US8198407B1 (en) | 2004-08-20 | 2012-06-12 | Prometic Biosciences, Ltd. | Sequential protein isolation and purification schemes by affinity chromatography |
| US8632773B2 (en) | 2009-03-26 | 2014-01-21 | Csl Behring Ag | Antibody composition with altered Fab sialylation |
| US8900806B2 (en) | 2010-04-22 | 2014-12-02 | Biotest Ag | Antibody preparations |
| US9243056B2 (en) | 2010-04-22 | 2016-01-26 | Biotest Ag | Process for preparing an immunoglobulin composition |
| US9518110B2 (en) | 2010-04-22 | 2016-12-13 | Biotest Ag | Antibody preparations |
| US10059759B2 (en) | 2010-04-22 | 2018-08-28 | Biotest Ag | Antibody preparations |
| US10954290B2 (en) | 2010-04-22 | 2021-03-23 | Biotest Ag | IgG, IgA and IgM antibody preparations, method of making and method of use in treatment |
| US11780909B2 (en) | 2010-04-22 | 2023-10-10 | Biotest Ag | Methods of treating viral infections by administering an antibody preparation comprising IGG, IGA and IGM |
Also Published As
| Publication number | Publication date |
|---|---|
| ES2170788T3 (en) | 2002-08-16 |
| JP2952572B2 (en) | 1999-09-27 |
| BR9603826A (en) | 1998-06-02 |
| RO117921B1 (en) | 2002-09-30 |
| HU9602570D0 (en) | 1996-11-28 |
| NO963952L (en) | 1997-03-24 |
| AU6572596A (en) | 1997-04-10 |
| KR100236762B1 (en) | 2000-01-15 |
| HUP9602570A2 (en) | 1997-08-28 |
| DE59509979D1 (en) | 2002-02-07 |
| KR970015745A (en) | 1997-04-28 |
| AU715427B2 (en) | 2000-02-03 |
| EP0764658B1 (en) | 2002-01-02 |
| JPH09124508A (en) | 1997-05-13 |
| PL316126A1 (en) | 1997-04-01 |
| FI963719A0 (en) | 1996-09-19 |
| CZ274896A3 (en) | 1997-04-16 |
| CN1089609C (en) | 2002-08-28 |
| NO963952D0 (en) | 1996-09-20 |
| CZ286885B6 (en) | 2000-07-12 |
| HUP9602570A3 (en) | 1998-01-28 |
| RU2157240C2 (en) | 2000-10-10 |
| FI963719A (en) | 1997-03-23 |
| NZ299394A (en) | 1997-12-19 |
| CN1153063A (en) | 1997-07-02 |
| MX9604256A (en) | 1998-04-30 |
| ATE211486T1 (en) | 2002-01-15 |
| EP0764658A1 (en) | 1997-03-26 |
| DK0764658T3 (en) | 2002-04-22 |
| PT764658E (en) | 2002-06-28 |
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| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |
Effective date: 20040916 |