TW388763B - Method of producing a hyper immunoglobulin preparation from fractions produced druing fractionation of human blood plasma - Google Patents

Description

The year S # is called “A7” (B7). The present invention relates to the preparation of immunoglobulins, and more specifically to a method for producing high-potency immunoglobulin preparations. The present invention can be used for recovering immunoglobulins from aliquots. So far, such fractions of osmium have not been utilized or at least cannot be used in the preparation of immunoglobulins in the conventional method of plasma tillering. There are more than a hundred different globulins in human plasma. "Some of them can be purified by fractional distillation from the damaged blood plasma pool and used as a therapeutic product in the following example. White eggs are used to compensate for hypoproteinemia. Or swelling deficiency caused by hypovolemia; clotting factors VIII and IX can be administered to hemophilia A and hemophilia B respectively as bleeding prevention and treatment. Immunoglobulins are used for immunocompromised diseases and Idiopathic thrombocytopenic purpura as prevention and treatment of infections; Immunoglobulins from prospective donors with special immunoglobulins with high potency are used as immunoglobulin preparations to prevent or treat hepatitis A Or a special infection of hepatitis B. The plasma proteins available for treatment can be according to known ethanol fractionation methods (Cohn, EG et al., J. An. C he n. Soc., 68, 459, 1946; Kistler, P., and Nitschmann, H., Vox Sang., 7 , 4 1 4, 1 9 6 2). In this way, a large number of functional plasma proteins such as albumin or immunoglobulin can be separated, which can be advantageously used clinically in an appropriate formulation. However, when operating according to these methods, precipitates and / or supernatants occur, which cannot be used in traditional processing methods. The composition of these preserves varies greatly. However, for example, when plasma albumin is precipitated with 19% ethanol at pH 5.8, the paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm). Precautions for re-page setting-Printed by the Central Standards Bureau of the Ministry of Economic Affairs, Shellfish Consumer Cooperatives, printed by the Ministry of Economic Affairs, the Central Bureau of Chess, quasi bureau of Shellfish Consumer Cooperatives, printed date, _ Supplement f7___ V. Description of Invention (2), humans can be found Natural waist protein AI (apoA-I) is approximately equally distributed in the supernatant a (about apoA_I) and the sink ash (about). After treatment with Kistler and Nitsch «ann after the mineral β step R, the same proteins (about 40% each) were found in these fractions (precipitate IV and Shen ttB) that have not been commercially available until now (lerch, etc.) , Protides of the Biological F 1 uids, 36, 40 9, 1 986) »A similar distribution pattern results in the net-bonded protein cyanosporin. After tillering, 20% of the initial mass can be found in Shendian IV and 40% in Shen B. Lotus ferritin is an example of plasma protein. In the same method, it is substantially 10096 concentrated in precipitate IV, and 80% of albumin can be found in Shen C, which is used recently. Using Kistler-Uitschaann or Cohn's seeding method, 50-6096 immunoglobulin β can be recovered from the sinker GG. In these methods, the remaining 30-40% of the immunoglobulin 傜 is distributed in the precipitate IV (596) , Precipitate B (30% > and Filter GG (5% >). The percentages mentioned above show the value of the distribution radiance and are not restrictive; it may vary depending on the conditions or methods used. These Examples show that in some cases a considerable amount of therapeutically available peptone can be found in «parts of sediment IV, lynx B, epithelium C and epithelium, corresponding to the plasma tillering methods of Cohn, GG or Radix Cohn皤 parts (皤 parts IV-1, IIparts II + III, supernatant V and 淸 掖 parts II-1, 2) β are based on Taoist reason, but also due to the global shortage of human plasma and specific blood health ingredients, Ying Chun asked for a method to improve the yield of immunoglobulin (which is still not very high). This paper size is applicable to China National Standards (CNS > Λ4 specification (210X297 mm) --------- 丨 & -------- T · ----- ^ (please read the note on the back of the page first and then this page) Printed by A7 B7, Shellfish Consumer Cooperatives, Central Bureau of Standards, Ministry of Economic Affairs ) Free Epidemic globulin plays a decisive role in preventing infection. Either neutralizing antibodies specific to the virus prevent the cell receiver from adsorbing the virus and thus prevent infection, or antibodies specific to the bacteria condition the pathogen and therefore allow it to be infected. The neutrophil and macrophages are destroyed and killed. Plasma pools from thousands of blood donors contain many different specific immunoglobulins, so the immunoglobulin preparations from this plasma pool contain measurable potency. Immunoglobulins, which directly react to epitopes of viruses, bacteria, and toxins, but also have effects on autoantigens. Therefore, they can effectively fight multiple infections and act under the most diverse pathogenic conditions . Now, under certain circumstances, it is suitable to use immunoglobulin preparations with high valence special antibodies, called high immunoglobulin preparations. Until now, these preparations have been developed from special antibodies at an expensive time and money cost. It can be prepared in the special blood pool of the increased blood loss. For different reasons, this is quite difficult. For example, anti-hepatitis B system It is known to have a vaccination step, and then the blood-damaged person must be bred and screened, and the blood that has been lost must be treated separately. However, in many other cases, blood-damaged people cannot be treated for moral reasons Immune effect. Here, it is really difficult to obtain this preparation by screening the blood-damaged person (for example, he has just recovered from a particular disease) to find high-priced blood and processing the blood lost to the blood transfusion. Method 傜 provides a method for recovering immunoglobulins, by which the immunoglobulin preparation can be easily prepared and the immunoglobulins are separated from the aforementioned fractions and the precipitates (which have been difficult to apply so far) and in industrial plasma fractionation methods Being able to use this method β, it should be possible to apply the Chinese National Standard (CNS) Α4 (210X297 mm) to this paper size ---------- ^ ------ 1T ----- -i (Please read the precautions on the back first, then this page) Printed by the Central Ministry of Economic Affairs of the Ministry of Economic Affairs and Consumer Cooperatives, printed A7 B7 V. Description of the invention (4) Those who process these equivalent high immunoglobulin preparations become good Tolerance, Do intravenous (IV) or a liquid free of the virus lyophilized formulation "has now found a method different from the previous techniques may be utilized to produce high or immunoglobulin preparation of high titer immunoglobulin preparation. These novel methods use immunoglobulins from common plasma pools to improve the development of useful raw material plasma by using "waste" fractions and even allow the production of high potency immunoglobulin preparations with higher specific activity than traditional systems. This means that the protein administered with a small amount of IV and the protein administered with a low amount of IV, that is, with a very low burden on the recipient, can provide a high dose of immunoglobulin in a very short time. In this inventive method, the special immunoglobulin is concentrated through the adsorption of the immobilized antigen, that is, the "waste" preservatives are processed using affinity chromatography technology to achieve the above purpose. According to this, according to The method for producing an immunoglobulin preparation of the present invention comprises the following steps: fractionating plasma from a plasma pool, wherein at least one fraction that can be used by the industry and contains multiple strains of immunoglobulin G can be separated, and a residual fraction can be obtained ; Preparing the protein component contained in the remaining portion or the second portion obtained from the previous step into a protein solution: dissolving the obtained protein at least once Liquid chromatography on affinity chromatography with at least one ligand type of fixed ligand, special plasma proteins will bind to the ligand and remove the bound plasma protein, which will be converted to high The active ingredient of a potent immunoglobulin preparation. In the method of the present invention, the fractions obtained from the plasma fractionation process, especially the waste fractions, are first processed so that they can be used in affinity chromatography on paper. China National Standard (CNS) A4 specification (210X297mm) ---------- I line (please read the precautions on the back before this page)%, 1 V. Description of the invention (r) Supplementing β depends on the amount of this ingredient, and this processing step may be large. Therefore, for example, the supernatant G6 can be entangled, dialyzed and filtered through a suitable buffer solution. On the other hand, the precipitate or filter The cakes (the listing of these starting materials are shown in Tables 1 and 2) must first be properly suspended, and also by the ionic strength, PI value and / or temperature, or by the addition of lambda cleaners and / or salts, In this way, the immunoglobulin is specifically lysed. Within 3-9, the conductivity is 5-20 mSiemens / cm, stirred overnight at 4 ° C and then clarified by centrifugation and / or filtration. At this stage, the liquid and suspension can be based on Ilorowitz's solvent-cleaning agent Method (Throibosis and Ilaeno stasis, 65, 1163, 1991), methylene. Kilan method (Mohr et al. 'I.nfusionsther. Infusions ^ ed. 20, 19 [1993]), or other methods to receive the virus Deactivation treatment. Immunoglobulins can be pre-purified after suspension. Protein A or G can be chromatographed by pre-adsorption on the matrix of the column or through a suitable filter aid or adsorbent such as hydrogen or aluminum. It can be concentrated, or it can be pre-precipitated one or more times by ammonium sulfate, polyethylene glycol or ethanol precipitation in a general method, or a combination of these methods can be used for animalization, concentration, or to strip off interfering components. (Please read the caution page on the reverse side first.) The policy of the Shellfish Consumer Cooperative of the Central Bureau of the Ministry of Economic Affairs

The dimensions of this paper are far from the Chinese National Standard (CNS) A4 (210X297 mm).

A7 B7 Supernatant sediments and residues Supernatant C Subsoil B Supernatant GG Substance IV DEAE-treated residue Aluminium hydride-treated residue clarified fractions I, II and III Residues Printed by the Central Government Bureau of the Ministry of Economic Affairs, Shellfish Consumer Cooperatives Table 1: Examples of possible starting materials produced by the Kistler and Nitschnann tillering method or other processing steps in IV plasma-stabilized stable plasma products. Supernatant precipitate and residue Supernatant V Precipitate II + III Supernatant I I-1, 2 Precipitate IV-1 DEAE-treated residue Aluminium hydride-treated residue was filtered Residues of I, II and III Table 2: Examples of possible starting materials 霣 produced by IV-administrable Ditan plasma products based on Cohn fractionation or other processing steps. This paper size applies to China National Standard (CNS) A4 (2! 〇g: 29_7 mm)! 1 Gutter (Please read the precautions on the back before printing this page) Printed by the Employees' Cooperatives of the Central Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (7) The following are examples of possible ligands used in the affinity chromatography of the invention: The following epitope. Escherichia coli type B. Staphylococcus aureus Staphylococcus epidermidis non-porous Staphylococcus pneumococcus streptococcus and other pathogenic strains of bacteria 1 toxoid Staphylococcus aureus toxic shock toxin and further Pathogenous toxins or other toxins Hepatitis A virus Hepatitis B virus Hepatitis C virus varizella vesicular herpes cytomegalovirus respiratory syncytial virus parvovirus B 1 9 Canola simple virus 1 Simple also rash virus 2 Rabies virus This paper ruler applies the Chinese National Standard (CNS) Α4 size (210 X 297 mm) --------—— — ^ .-- (Please read the precautions on the back before: W page)

, 1T line

And other pathogenic viruses CD2, CD3, CD4, CD5, CD28, CD40, CD72 ICAM, LFA-1, LFA-3,

DN A and other potential human autoantigens. Affinity gels are prepared by immobilizing modified or unmodified ligands (examples of such ligands are listed above) by known methods, and are used to concentrate or dilute the treated supernatant or Suspension is used to load the gel, and if necessary, the stream can be repeatedly applied. If necessary, different affinity gels can be activated with the same suspension. These gels are then rinsed to remove non-specifically bound proteins excellently or to a satisfactory extent. This can be achieved, for example, by increasing the concentration of physiological saline, adding detergent and / or shifting the pH value in the cleaning solution. Bonded proteins are now dissolved by low or high pH values, and physiological saline solutions with high chaotropic sequences, such as sodium thioanhydride or magnesium ambinate, denaturants such as SDS or urea, and solvents such as ethylene glycol, are added. Temperature or a combination of the foregoing to separate from the ligand. Printed by the Central Standards Bureau of the Ministry of Economic Affairs, Shellfish Consumer Cooperatives ----------- Installation-'1.1, (Please read the notes on the back page first) In some cases, The fixed ligands are modified by mutation or chemical or physical methods, so that special immunoglobulins are bound to their epitopes with lower affinity, so that the dissociation can be made to the less modified Ligands occur under milder conditions. Modification of a ligand can also occur by promoting and / or improving its immobility or the expression of its epitope. The technical details and basic principles of the affinity chromatography method are described in Cuatrecasas -10- This paper size applies the Chinese National Standard (CNS) A4 ^ (210X297 mm) __B7_ V. Description of the invention (9), P., and Anfinse η, C. B. (1 9 7 1), A η n. Rev. B i 〇che. 40, 2 5 9; Kull, FC and Cuatrecasas, P. (19 81), J. Ι · ιιηο1 .126, 1 2 7 9; Liebing et al. (194 4), Vox Sang. 67, 117〇 After the special and isolated immunoglobulin is filtered to eliminate the virus, it can be processed to make the final product. It is preferably a medicinal IV and is pyrogen-free and virus-free. It can be stabilized with or without the addition of stabilizers such as albumin, amino acids, or water-soluble compounds in a liquid or freeze-dried form. Β, however, can also be used. Formulated for intramuscular or topical application. Preferred embodiments of the present invention will be illustrated and described by the following non-limiting examples. Example 1 A 5Bg recombinant hepatitis B virus surface antigen (HBsAg, Abbott Diagnostics) was prepared by coupling a primary amine group to 1.1 activated CH-Sepharose as shown by the manufacturer (Pharmacia Biotech, Uppsala, Sweden). . H placebo " -Sepharose was prepared by the same coupling method in another gel aliquot without the addition of HBsAg. The finished gel is stored at 4 ° (: 0.02% ^ »13 in? 88). Printed with oxygen by the Central Laboratories of the Ministry of Economic Affairs, Shellfish Consumer Cooperative (please read the precautions on the back before filling out this page). 78 precipitates from Kistler-Nitschmann blood sugar (UB Lot 4.030.216) 8 (1 0 0 B suspended in 2 1 0 η 1 at 4 ° (3 at 1 ° 1 & ^^ 1) Μ citrate, P Η 4.0, 0. 25% T rit ο η X-1 0 0, 'ΙΟβΜ N-ethylmaleimide (ΝΕΜ), 1μΜ benzylsulfonium (PMSF) ). After centrifugation (5000g, 4 ° C, 10 minutes) and filtration (pore size: 1.2 // ·) to separate insoluble components, according to Horowitz et al. (Throibosis, and -11- this paper standard uses Chinese national standards (CNS) Λ4 specification (210X297 mm) A7 B7 V. Description of the invention (β) Add 1 to the method of neutral 以 11 with? 〇81 & 818, 65, 1 1 6 3, 1 9 9 1) % Tri-n-butylphosphonium phosphate (Herc It, Darstadt, Gerany) and 1% Triton X-100 and stirred at 3 ° C for 4 hours. Then phase separation was performed at 37 ° C overnight, and The clear lower phase is shifted out, and Filtered under a 0.45 yu filter and stored at 4 · 0. These NB 懋 suspensions of 130 · 1 were treated with 28 (U1 PBS (phosphate-buffered physiological saline solution: 15 ° M HaCl, 10 mM sodium phosphate pH). 7.1) to dilute and sooth the f-Sepharose column, and then go to the HBsAg-Sepharose column at 4 ° C to allow the column to flow back to the storage tube. The flow rate is 8Bl / hr for 144 hours β then the NB suspension The columns were drawn back a total of three times. After 90 hours, loading was discontinued. When the columns were cleaned separately, first with PBS and then with 200 m Mn N a C 1, 50 β M tris-HC 1, ρ Η 7.4 until the «rinsing solution has an optical density of less than 0.01 (0 D 2so) at 280 nm. After summarizing activation, PBS and 200 · M KaCl, 50rM tris were used again. -HCl, pH 7.4 for washing. The bound protein was separated with 5 · 1 2 0 ·· M glycine HCl, pH 2.5, and immediately neutralized and treated. N (Binding II — I / 4.. {Please read the note on the back before filling this page) Printed paper size of the paper by the Central Rubbing Bureau of the Ministry of Economic Affairs, Shellfish Consumer Cooperative, It uses China National Ladder Standard (CNS) Α4 size (210X297 mm) A7 B7 Central of the Ministry of Economic Affairs Rubbing the Bureau of the C & C Consumer Cooperatives V. Description of Invention (U)

Condensing BB s A g placebo knot loaded Dong protein 1 7 7 5ig 1775 * g I gG 4 50 0 ng 4 50 0 g anti-HBs IgG 4560〇1U 4560b1U flow anti-fiBs IgG 1 140〇1U 1140nlU dissociation IgG 0.1 3 ng 0.08 ng anti-HBs IgG 5603nlU 114nlU Table 3: Anti-HBsAg affinity chromatography with NB suspension Example 2 Start with iohn fraction II + III (replace plasma as the starting point Substance), proceed to -13- This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm)! |. Order (please read the note on the back before filling this page) B7 V. Description of the invention ( "≫)

Kistler-Nitschiiann gland. 7 g of precipitate B (NB Lot4.044.488) from Kitsler-N.itbcliiann tiller was suspended at 20.1 ° C in 100 · M betulinic acid at 20.1 in RotaryMix at pH 4.0. , 0.25% Triton X-100, 10 · M-ethyl maleimidine imide (NEM), 1 · M benzylsulfenazine (PMSF). After ultra-concentration (100,000g, 3 hours, 4 ° C: _Pierce the tube wall by a syringe to remove the orange clear phase) for clarification and partial degreasing. After that, the suspension was extinguished (0.45 # B) And this BN suspension stored at 4 ° Ce 1 2 5 1 was diluted with 3 7 5 b 1 PBS, adjusted to pH 7.1 with 0.1M NaOH, filtered and drawn. As in Example 1, first placebo-Sepharose gel The gel was then prepared by HBsAg-SepharoSe gel ', similar to Example 3. These gels were then rinsed with PBS and 20 (^ 1,3) (1,500 (1); 1 (3-1)! (: 1,0117.4). The bound protein was treated with 5 «1 of 2 0 nM glycine. Acid-HC1, PH2.5 are removed. Table 4 summarizes these data, ------------ 政 ------ 1 ------ i (Please read S first (Notes on the back are filled with $. Please fill in this page again.) The Central Government Bureau of the Ministry of Economic Affairs, Shellfish Costs Cooperative Co., Ltd., printed oxygen paper. The paper ruler applies the Chinese National Standard (CNS) A4 specification (210X297mm) A7. Cooperative cooperative printing V. Description of invention (13)

Coagulation 6 s A g Placebo knot loading protein 1 4 0 0 g 1 4 0 0 g I gG 6 1 9> g 61hg anti-HBs IgG 1 0 I I 1 0 IU fluid anti-HBs IgG 5 IU 5 1 IU eluate IgG 0.1 8 ffl g 0. 3 5 μg anti-HBs IgG 3.3 IU 0. 08 IU Table 4: Anti-HBsAg affinity layer with NB suspension (Cohn II + III) Analysis Example 3 Percolate 30 liters of liquid GG (Lot NO.X95.31.286.1) in PBS-1 5- This paper ruler is applicable to China National Standards (CNS) 8 4 wash grid (210X297 mm) I Gutter (please read the note on the back page first) A7 B7 printed by Shelley Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (l4) (diafiltered) and concentrated to 500 · 1. As in Example 1, the concentrate was drawn into the placebo and IIBsAg column at a rate of 21 * l / hr for 118 hours. The column was rinsed similar to Example 1 and the bound proteins were separated, but an additional rinse was performed with 500 m NaCl and 50 · M tris-HCl, pH 7.4. The results are shown in Table 5. m. ^ ia— HI HI In I 1 ^ 1 ml n ^ i ml ^^^ 1 ^ Ά (Please read the notes on the back before filling this page)

Gel Η B s A g Total placebo loading protein 88 0 0 mg 8 8 0 0 Pass g IgG 3 2 0 ig 3 2 0 Mg Anti-HBs IgG 5000b1U 5000nlU Flow Anti-HBs I gG < DL < DL eluate IgG 0 · 0 5 g. 0. 14 g anti-HBs IgG 3035 · ΐυ 7 alU ~ 1 6-This paper uses Chinese National Standard (CNS) A4 specifications (210 > < 297 mm) ) A7 B7

VN V. Description of the invention (, r) Supplementary Table 5: Anti-HBsAg Affinity Distillation DL Concentrated 液 of the above serum GG ·: 17.5g DEAE filter cake (Lot 4.422.) 0 0 6.0) Suspension in 52.5 · 1 suspension buffer as in Example 1 and treatment. 40 * 1 suspension solution was diluted with 160 · 1 PBS and pH adjusted to 7 · 1, and then diluted. Off. As in Example 3, the suspension was drawn to the placebo and HBsAg column at a rate of 21 b1 / hr for 97 hours, and the column was washed similar to Example 1 and the bound proteins were separated. The results are shown in Table 6 »Gel RB s A g Placebo Total Loaded Protein 548 ^ g 548Hg IgG 28 0 ag 280ag Anti-HBs IgG 4 0 0 1U .......-400ilU Fluid Anti- HBs IgG < DL < DL eluate IgG 0. 0 7 ug 0. 1 1 B g anti-HBs IgG 331 · ΐυ ΐ4 · ιυ The policy of the Bayong Consumer Cooperative of the Central Fengfeng Bureau of the Ministry of Economic Affairs -------- ---- Equipment-(Please read the note on the back page first) Line-Table 6: Anti-HBsAg Affinity Chromatography DL with DEAE Filter Cake Suspension Limit: Detection Limit-17 Free use of China National Standards (CNS) A4 specifications (210X297 mm)

V. Description of the invention (16) Example 5 As shown by the manufacturer, tetanus toxoid C-Sepharose, as shown by the manufacturer, you will pass 11.5mg of pure tetanus toxoid (TT) through EAH-Sepharose (Phariacia) Biotech, Uppsala. Sweden), was prepared by the fixed preparation with the aid of 0.1 H of N-ethyl-N '-(3-dimethylaminopropyl) sulfodiimide-HC1. The concentrated solution of supernatant GG was prepared as in Example 3 and similar to Example 2 for affinity chromatography with 1'1'-36? 1 ^ 1 * 〇86 <) at 4 ° (: Activation was performed at 23.5 nl / hr for 159 hours. The results are shown in Table 70. Gel toxoid C junction loading protein 15000 ^ g Ig6 3 2 0 ng Anti-TT IgG 36 μg Flow-through anti-TT IgG 16 g Eluate IgG 0.1 .6 ng anti-TT IgG 76 / ^ g Table 7: The above supernatant GG «condensed for anti-toxoid affinity chromatography " 18 " This paper size applies to China National Standard (CNS) A4 specifications (210X297 mm) gutter (please read the note on the back side first, then ^-ί this page), the Central Bureau of Procurement of the Ministry of Economic Affairs

V. Description of the invention (17) Example 6 As shown by the manufacturer CPharmacia Biotech, Uppsala, Sweden), the toxoid H-Sepharose 鞴 is treated by immersing 11.5 · 8 姹 toxoid (TT) in a primary basis. "To 1.1 activated CH-Sepharose to fix" "Placebo" -Sepharose was prepared with another aliquot in the same coupling process without adding TT. The completed gel was stored at 4-C in germanium in PBS with 0.02% NaN3. Precipitate B was suspended according to Example 2. After the filtration (1.2 #), the 115 · 1 suspension was diluted with 200 · 1 PBS and PBS to 7.1, and then the placebo-Sepharose was drawn in at 3.5 · 1 / Ιχγ for 165 hours, then The tower opens into the TTU-Seph_arose column and returns to the remaining tube. The columns were washed with PBS and 0.5M NaCl, 50 · M tris-HCl, PH7.1, respectively, until the OD value of the flow was below 0.01. The results are shown in Table 8. --------- ^ ------ # ----- ί ^ (Please read the note on the back of this page first) Printed by the Cooperate Cooperative of the Central Bureau of the Ministry of Economic Affairs Warm toxin N comforter loaded Dong protein 1334ag 133 "g IgG 333 Lig 3 3 3ag anti-TT IgG 0 · 5 0 9 Lig 0.5 .5 0 9 ag Flow Anti-TT IgG 0.2 13ig 0.2 1 3ig eluate IgG 0. 3 5 g 0 · 0 0 4 g anti-TTI g G 0 · 0 9 1 ag 0. 0 0 3ig Applicable to China National Standards (CNS) A4 grid (210X297 mm) A7 B7 V. Description of the invention (ιβ). Example 7 Suspend the deposit B according to implementation 2. After ϋϋ (1 · 2Α ·), The suspension of ι! 5.1 · was diluted with 200 * 1 PBS, PHJS S to 7.1, and was first absorbed into 〇sAg-Sepharose at 5Bl / hr. Β5 畤 was then immediately passed into a TT_Sephar〇se tube Column • Then return to the remaining tube. The columns were washed with PBS and 0.5M MaCl 'tris_HC1, pH 71, respectively, until the 0 D 280 value of the flowing liquid was lower than 0. 〇1. The results are shown in Table 9 . Gel HBsAg toxoid load protein 1334ng 1 3 3 4 eg IgG 3 3 3ig 3 3 3ag anti-HBs igG 9 4 5 · 11Ι 945alU anti-TT IgG 0.5 .9 g 0. 5 0 9 mg flow rate anti-HBs IgG 536ilU 536 · 1U anti-TT IgG 0.2 6 5 B g 0. 2 6 5 ng Refractory IgG 0. 1 3 B g 0. 2 5 B g anti-HBs Ig6 1 l 52alU anti-TT IgG 0. 18ng Table 9: Anti-HBsAg and Anti-Toxoid Affinity Chromatography-20- This paper size is in accordance with Chinese National Standard (CNS) A4 specification (210X297 mm) ---------- ^-xfi (Please read "Notes on the back" Please fill in this page again.) Order Λ. Printed by the Central Government Bureau of the Ministry of Economy Shellfish Consumer Cooperative Co., Ltd. Printed by the Central Ministry of Economic Affairs of the Ministry of Economy Shellfish Consumer Cooperative Co., Ltd. Yin Ben A? B7 5. Description of the invention (· 9) Example 8 will come from Kistler -Nitschnann decoction of 15 kg of antiseptic B (Lot No. 5.043.303) at 4 · 0: suspended in 45 liters of 0.1M citric acid in a vibration mixer. PH4.0, 0.25% Triton χ- 100, 10 · M-ethylmaleimide (NEM), UM benzylsulfonium chloride (PMSF) overnight. After adding the infiltration rib filter and filtering (pore size: 1 · 2 # β) to separate the insoluble components, according to the Horowitz method, add 1% third n-butyl phosphate and 1% Triton X-100 at neutral pH. Incubate at 30 ° C for 4 hours to remove lipids and deactivate the virus. Then, phase separation was performed overnight at 37eC, and the lower phase was extracted, filtered through a 0.45 / L filter, and stored at 4 ° C. The pH value of 40 liters of NB suspension was adjusted to 7.1 with NaOH and was drawn into the HBsAg column and the toxoid Affiprep column at 4 »C to allow the column flow to return to the storage tube. Affiprep column (50X 13bb) was prepared by combining 250eg of recombinant HBsAg and toxoid with 25nl of Affiprep (BioRad Lab. Inc., Hercules, CA 94547, USA) to prepare a β flow rate of 6 liters / hr. Up to 62 hours. Then the NB suspension was drawn into the columns three times. After the entanglement was activated, the columns were washed separately, first with PBS and then with 500 · M NaCl, 50iM tris-BCl, pH 7.4, until washed. The optical density (ODiso) of the solution at 280η · is less than 0.01. The binding protein was removed with 2 g of glycine-HC1, pH 2.5 and the pH of the fraction was immediately trimmed to 5.2. The results are collected in Table 10. _ Processing of immunoglobulins into stable preparations by collecting IgG-containing fractions, diafiltration and concentrating to 20 IU / 11 and 2.5 ng anti-TT IG / nl solutions with 20 · M HaCl, respectively. Add 10% cheap sugar, and freeze-dry the solution separately 2-1 The paper size applies to Chinese National Standard (CNS) A4 specification (210X297), ^ — • ϋ m · n an a ^ i Hal-. In mma -i ^^ 1 n an— (please read "Note $ on the back side before filling out this page) A7 B7 V. Description of the invention (w). It is 2 0 0 IU units and 5 g anti-TT-I g G. Example 9 50 g of S from the Kistler-Nitschiann blood purple branch was suspended in 50 0 · 1 water β. The pH was adjusted to 5.0 with citric acid and the temperature was adjusted to 13 iSe with H 霣 degree at 4 * C overnight. • Clarify and partially delipidize by centrifugation at 4 ° C for 30 minutes. The proteins of suspension P, IgM, IgA, IgG, lotus ferritin, and cyanosporin are identified and shown in Test 11.

Ivifo.oli will be 30 minutes, cut C, 'all UL β Γ Λ} content printed by the Ministry of Economic Affairs Central Bureau of Labor and Consumer Cooperatives printed HBsAg soothing knot load Dong protein 448g 448g Ig6 2 0 4 g 2 0 4 g anti-HBs IgG 1 1 2 0 IU 1120IU anti-TT IgG 3 1 7ag 3 1 7ng fluid anti-HBs IgG 221IU 22 1IU anti-HB IgG 172lg 172ag eluate IgG 3 9. 8 D g 1 7 1鼸 g anti-HBs IgG 1368IU anti-TT IgG 8 9 B g ---------- ^-〆.t (Please read the notes on the back before filling this page) Order Form 10: NB suspension for anti-HBsAg and anti-steroids-22- delamination and it This paper ruler uses China National Standard (CNS) A4 this grid (210X297 cm) A7 B7 1. Description of the invention (21)- --- Absolute protein IgM IgA IgG face protein shovel cytosin 5.5 0.063 0.628 0.488 2.582 0.09 Table 11: Suspension from precipitate IV (all values are in units. Example 10 will be from Kistler-Nitscheann blood 50 grams of caisson B (Lot No. 4030.204.0) was sown overnight at 100 * M citric acid with different pH and 4Ό, and clarified by ultracentrifugation at 10, 0, 0 g and 4 eC for 3 hours. Partially delipidized β will remove the clarified intermediate phase and isolate the content of proteins, IgH, IgA, IgG, androgen and cyanogenin (Table 12). Nai > B ^-· flm — ^^ 1 ϋ · —ϋ I «_Β · ϋ ι_ · 9 ι ^ 4 (please read the notes on the back first and then write 4 copies of Jr) Printed by the Central Consumer Samples Bureau, Ministry of Economic Affairs

Absolute protein IgH IgA I Purine androgen cytoplasmin PH4.0 4.9 1.2 1.3 1.9 < DL 0.06 ρΗ5 · 0 6.1 1.2 1.0 2.1 < DL < DL -23- This paper standard applies to Chinese National Standard (CNS > A4 specification (210X297 mm) 388763 V. Description of the invention (22) Table 12: Suspension from sinker B (all values are in the unit of 8 / · 1) < DL: Congestion below detection limit Undetermined virus (Table 13) and bacteria (Table 10 antigen IgG titer and compared with the original blood paddle and existing immune #protein system_

Plasma 102 plasma 103 SAGL NB pH 4.0 NB pH 5.0 anti-HBsAg 0.02 0.05 0.01 0.05 0.05 IU / «g lg6 anti-CMV 0.28 0.16 0.3 0.52 0.34 PEIE / mg IgG anti-VZV 0.08 0.09 nd 0.21 0.1 ID / ag Ig6 anti- Measles 0.9 0.2 0.02 2.1 0.81 ID / ag IgG anti-BSV1 1037 ndnd 2317 749 AU / ag IgG (Please read the note f on the back before filling out this page), 11 Printed by the Bayer Consumer Cooperative in the Ministry of Economic Affairs Table 13: Antiviral IgG, SAGL, and NB suspended plasma in the blood paddle pool 1 2 0/103: blood, tired pool; 8 pills 61; 8 & 11 (1 (^ 1〇1) 111111 « ); PH4.0 / 5.0: Suspension of precipitate B at pH 4.0 and 5.0 respectively; IU: Intermediate unit: PEIE: Paul Ehrlich-2 4-This paper is scale A in China National standard rate (CNS) A4 specification (210X297 mm) 388763 s 5. Description of invention (23)

Institute) niches; Αϋ: arbitrary units; n.d .: not determined β

Plasma 102 Plasma 103 SAGL NB 4A NB 4B pH 4.0 Anti-HiBOAg 162 286 436 100 283 M g / g IgG Anti-ττ 1855 4159 2740 2928 2923 M g / g IgG Anti-SEB 412 689 783 918 670 AU / g IgG ( Please read the note on the back of the page before filling in this page.) Binding and ordering printed by the Shellfish Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs *. Table 14: Anti-Bacterial IgG, SAGL, and NB Suspension 1 02/103: blood pool; SAGL; S and 〇g 1 〇bu 1 i η ®; Ν B 4 A from the precipitate B in pH 4.0 suspension f; 014 PH4.0: virus loss Suspension of activated precipitate βP4.0; HiBOAg: Epidemic Hexabacillus beta-oligosaccharide antigen; TT: Toxoid; SEB: Neissin Staphylococcus B; Αϋ: Any unit. -25- This paper size is applicable to China National Standard (CNS) A4 specification (210 × 297 mm)

Claims (1)

Printed value ha-shirt printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs * Be_ Supplement 丨 gs___ VI. Application scope of patent 88S763 No. 85 1 10788 `` A kind of high-power immunity generated from the distillate produced when the human 醴 plasma is divided "Method of globulin preparation" patent (revised in October 88) Qiao Li Fan Yuan 1. A method for producing high-potency immunoglobulin preparations, which includes steps T: (a) by Cohn Or the Kistler-Nietchnan method was used to separate the plasma from the blood pool, thereby obtaining a variety of residual fractions, and the residual fractions include the milk fraction II + III, the Keller-Nimmon method Selection of compiling parts _ Box B, liquid GG, DEAE filter or precipitate IV; (b) the protein content contained in the extra aliquots obtained in step ® U) or its sub-JB is obtained by using _ A buffered aqueous solution with a sub-strength PH of 13.0 to 9.0 is used to process the protein components to form a protein solution or suspension; (c) the obtained protein is dissolved or desired to be floated with-at least one type of ligand Sisters of sexual ligands JI analysis into Ji processing at least once, special plasma-protein > Sell binding to the coordination 碁, and / or remove the binding protein, in which the fixed ligand 'ν ..... position is an antigen. Silkworm comes from natural or recombinant antigens, viral antigens, bacterial properties Antigens and cellular antigens; and (d) the plasma peptone obtained is made into a free valence immunoglobulin protein. The small I paper scale uses the Chinese National Standard (CNS) M specification (210x297 mm) A8 B8 C8 D8 The method printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs and applied for a patent scope of 2 · $ 1. Among them, the blood paddle tiller is provided for production and the remaining portion is waste cold. Please refer to the method of the first item of the patent scope, wherein the residues of step "U) @« 8 parts are Shendian or filter cake. The method of item 3 in the scope of benefit, wherein the buffer solution is phosphorus buffer solution, tris-HCl buffer solution, or dilute acid buffer solution. The method according to item 3 of the patent, wherein the buffer solution comprises a detergent, one or more protein inhibitors, and / or salts. 6. The method of item 3 of the patented patent park, wherein the solvent or suspension is treated by filtration or pre-treated with an adsorbent such as aluminum cedodide or an adsorbent containing DEAE group. 7. The method according to item 3 of the scope of the claim, wherein the solution or suspension is reacted with ammonium sulfate, polyethylene glycol or acetic acid to form a precipitate. 8. The method according to item 1 of the Shen Ma patent patent, wherein the residual portion of step (a) is a mash, and the protein solution of step (b) is prepared by filtration and concentrated by dialysis. 9. The method according to item 8 of the scope of patent application, in which the supernatant liquid 傜 is treated with 礴 预处理 or pretreated with an adsorbent such as aluminum hydroxide or an adsorbent containing a DEAE base. 1 0 ·. For the method of the first item of the patent application, where the coordination and base are from the following epitope: Influenza b-type H. influenzae, please read the note $ on the back and then Θ (Page)-Binding. \ / · $ Paper size (using China National Standards (CNS) A4iUM 210X297 mm) 388763 A8 B8 C8 D8 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, patent application scope Staphylococcus aureus, staphylococcus epidermidis, milk-free "staphylococcus aureus, pneumococcus pneumoniae, pneumococcus pneumoniae, toxoids, golden chrysanthemum spheres have _ shock toxin, hepatitis A virus, hepatitis B disease race Type 〇 .. Hepatitis virus, Ferrella. (V-ar-i zella) Gangue vesicular osmosis-radon, megalovirus, respiratory syncytial virus, parvovirus B19, simple measles virus 1 and 2 Type, rabies virus, and potential human anti -... Original CD2, CD3, CD4, CD5, CD28, CD4l, CD72., ICAM, LPA-1, LFA-3, PNA and phospholipids. 11. The method as described in item 1 of Shen Lili, in which the ligand is modified by a chemical method or chemical or physical method. 12. The method according to any one of items 1 to η in the Shen Fan Patent Park, wherein the so-called high-potency immunoglobulin is made-like JT is composed of immunoglobulin G group or A group or M group or Any combination. 13. If applying for the dog method of any one of items 1 to 11 of Gao Li Fan Yuan, the obtained Gaoli 俪 immunoglobulin _ is subjected to virus deactivation treatment, and if necessary, a stabilizer such as albumin is added. , Amino acids, or ammonium compounds to stabilize and / or freeze-dry the product. 14. The method according to any one of items 1 to 11 of the application of Lilifan, wherein the obtained high potency immunoglobulin The preparation is converted into a "drug-acceptable" product, such as intravenously, intramuscularly, or topically. -3-This paper is a standard for the Chinese and Korean National Kneading Rate (CNS) A4 (210x297), please read the note f on the back and save 1 ^ this-set · page)? · _