. ~ 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
11
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
12
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
13
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
14
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 ~
16
~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.
'
