TWI311563B

TWI311563B - Method for production a stable,immunogenic hepatitis b vaccine without trace of thiomersal

Info

Publication number: TWI311563B
Application number: TW090123365A
Authority: TW
Inventors: De Heyder Koen; Schu Peter; Serantoni Michelle; Van Opstal Omer
Original assignee: Smithkline Beecham Biolog
Priority date: 2001-09-21
Filing date: 2001-09-21
Publication date: 2009-07-01

Description

13115631311563

本發明係㈣製造用於治療或預防B型肝炎病毒（感染之B型肝炎疫苗的新類方法。其尚關於可藉由本發新穎方法獲得的HBV疫苗。慢性B型肝炎病毒（HBV)㈣，目前只有有限的治療方法其構成了廣泛的全球公共衛生問題。慢性HBV帶原者估計全球有超過3億人口。冒著發展成爲慢性活化之肝炎、肝硬化和初級肝細胞癌的風險。許多目前可獲得的疫苗需要保存劑以防止破壞。一種經常使用的保存劑是含有汞之化合物，即乙基汞化水楊酸二。某些關於在疫苗中使用汞的顧慮已產生，雖然評論家強調含乙基汞化水楊酸鈉之疫苗可能的害處不應被誇大。 (Offit , P.A. JAMA，第283卷；第16號）。無論如何，找到新的和可能較爲安全之疫苗製備法以替換在製造過程中乙基汞化水楊酸鈉的用途會是較有利的。因此，有必要研發不含乙基汞化水楊酸鈉的疫苗，尤其是B型肝炎疫苗。本發明的第一方面提供一種生產適用於疫苗之B型肝炎抗原的方法，該方法包括於含有自由_SH基之還原劑存在下純化該抗原。本發明較好的是提供一種生產不含微量乙基汞化水楊酸鈉之穩定B型肝炎抗原的方法，該方法包括在具有自由-SH 基之還原劑存在下純化該抗原。該抗原製備物一般而言不含微量的乙基汞化水楊酸鈉，這是在當使用汞的吸收光譜分析法時在純化的抗原產物中測不到乙化水楊酸鈉，該測定法如本説明書中所説明。 -4- 本紙張尺度適用中國國‘家標準(CNS) A4規格(210 X 297公釐) 1311563 A7 B7 五、發明説明（2 該B型肝炎抗原製備物較好是包含少於0.025微克汞/每 20微克蛋白質’可適當地以吸收光譜分析法測量之。較好是純化在不含乙基汞化水楊酸鈉的條件下進行，且經純化的抗原完全不含乙基汞化水楊酸鈉。較好是抗原爲穩定的，本質上適當地穩定_如在有乙基汞化水楊酸鈉存在下所純化的肝炎抗原，例如在本説明書的實知《例1所列示者。較好是肝炎抗原爲致免疫的。較好是還原劑在抗原純化的過程中添加，較好是在能表現抗原的細胞生長之後。較好是該還原劑爲半胱胺酸、二硫蘇糖醇、β-巯基乙醇或穀胱甘肽，而以半胱胺酸爲最佳。因此，本發明較好是提供一種-製備不含微量乙基汞化水楊酸鈉之穩定致免疫B型肝炎抗體的方法，其包括於半胱胺酸存在下純化該抗原。較好是該純化是在半耽胺酸溶液存在下進行。較好是，將半胱胺酸以溶液或粉末的形式在過程中添加至介於1和10 mM的最終濃度，較好是丨至5 mMe更好是半胱胺酸添加至大約2 mM的最終濃度。較好是半胱胺酸爲L-半胱胺酸。六本發明尚提供-種生產不含微量乙基乘化水楊酸納之穩定B型肝炎抗原的方法，其中用膠體滲透層析法、離子交換層析法處理粗製的抗原’並與具有自由.阳基團的還原劑混合。 -5-The present invention is a novel method for the treatment or prevention of hepatitis B virus (infected hepatitis B vaccine. It is also related to the HBV vaccine obtainable by the novel method of the present invention. Chronic hepatitis B virus (HBV) (IV), There are currently only limited treatments that constitute a wide-ranging global public health problem. Chronic HBV carriers are estimated to have more than 300 million people worldwide, risking the development of chronically activated hepatitis, cirrhosis and primary hepatocellular carcinoma. Available vaccines require preservatives to prevent damage. One commonly used preservative is a compound containing mercury, namely ethylmercurylated salicylic acid 2. Some concerns about the use of mercury in vaccines have arisen, although commentators have emphasized The possible harm of a vaccine containing ethylmercury salicylate should not be exaggerated (Offit, PA JAMA, Vol. 283; No. 16). In any case, find new and potentially safe vaccine preparations. It would be advantageous to replace the use of ethylmercury salicylate in the manufacturing process. Therefore, it is necessary to develop a vaccine that does not contain ethylmercuric salicylate, especially type B liver. Inflammatory Vaccine. A first aspect of the invention provides a method of producing a hepatitis B antigen suitable for use in a vaccine, the method comprising purifying the antigen in the presence of a reducing agent comprising a free-SH group. The invention preferably provides a production A method for stabilizing a hepatitis B antigen of a trace amount of ethylmercury salicylate, the method comprising purifying the antigen in the presence of a reducing agent having a free-SH group. The antigen preparation generally does not contain a trace amount of B Mercury-sodium salicylate, which is not detectable in the purified antigen product when using mercury absorption spectroscopy, as described in this specification. This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1311563 A7 B7 V. Description of the invention (2) The hepatitis B antigen preparation preferably contains less than 0.025 micrograms of mercury per 20 micrograms. The protein ' can be suitably measured by absorption spectrometry. Preferably, the purification is carried out in the absence of ethylmercuric sodium salicylate, and the purified antigen is completely free of ethylmercuric sodium salicylate. Preferably the antigen is stable Intrinsically stable - such as a hepatitis antigen purified in the presence of sodium ethylmercuric salicylate, for example, as shown in the description of Example 1 of the present specification. Preferably, the hepatitis antigen is immunogenic. Preferably, the reducing agent is added during the purification of the antigen, preferably after cell growth capable of expressing the antigen. Preferably, the reducing agent is cysteine, dithiothreitol, β-mercaptoethanol or Glutathione, and cysteine is preferred. Therefore, the present invention preferably provides a method for preparing a stable immunogenic hepatitis B antibody containing no traces of ethylmercury salicylate, which comprises The antigen is purified in the presence of cysteine. Preferably, the purification is carried out in the presence of a solution of a semi-proline. Preferably, the cysteine is added to the solution in the form of a solution or a powder. Preferably, the final concentration of 10 mM, preferably 丨 to 5 mMe, is the addition of cysteine to a final concentration of about 2 mM. Preferably, the cysteine is L-cysteine. The present invention provides a method for producing a stable hepatitis B antigen containing no traces of ethylation of sodium salicylate, wherein the crude antigen is treated by colloidal permeation chromatography and ion exchange chromatography. The reducing agent of the cation group is mixed. -5-

1311563 A7 ________B7 _ 五、發明説明（3 ) 較好是該離子交換層析法爲陰離子交換層析法。本發明尚提供可藉由本發明之製造方法獲得而不含乙基乘化水楊酸納之B型肝炎抗原，其中該抗原至少是致免疫的具其抗原性，如同乙基汞化水楊酸鈉存在下所製造者。本發明尚提供一種致免疫的B型肝炎抗原，其具有大於 1 _5之平均ELISA蛋白質比率’以及IC50値至少3倍低於乙基求化水楊酸納存在下，所製造之B型肝炎表面抗原的RF1含量。本發明還有一方面係關於適用於疫苗之肝炎抗原的生產方法’該方法包括純化乙基汞化水揚酸鈉存在下的抗原，並緊接著在含有自由-SH基之還原劑存在下對抗原進行處理。適當地是該處理後，接著進行純化步驟如透析步驟以移除掉乙基汞化水楊酸鈉。較好是該還原劑是半胱胺酸、DTT、穀胱甘肽或2_巯基乙醇。本發明的B型肝炎抗原可用於治療或預防b型肝炎感染 ’尤其是治療或預防例如慢性的B型肝炎感染。本發明尚提供一種包含本發明之B型肝炎抗原與一種佐劑連接之疫苗調配物。較好是該佐劑爲鋁鹽或較佳之TH1 細胞反應刺激物。較好是該抗原爲B型肝炎表面抗原。 B型肝炎表面抗原的製備已有妥善的文件説明。請參考例如Harford等人在《發育生物學標準》，第54期，Η〗頁 -6 - 本紙張尺度適財a g家標ijt(CNS) M規格(21QX撕公着) 1311563 A7 B7 五、發明説明（4 ) (1983年），Gregg等人在《生物技術》，第5期，479頁（1987 年），EP-A4 226 846，ΕΡ-Α-0 299 108以其中的參考資料。如本説明書中使用的「B型肝炎表面抗原」或Γ HBsAg」的表示法包括任何能展現HBV表面抗原之抗原性質的 HBsAg抗原或其片段。吾人需瞭解除了 HBsAg S抗原的226 個胺基酸之序列以外（請參考Tiollais等人《自然》，第317 期，489頁（1985年）及其中的參考資料），本説明書中所説明的HBsAg若需要可包含所有或部位在以上參考文獻和EP-A 0 278 940中所説明的前驅體-S序列。本説明書中所説明的 HBsAg亦可指變異體，例如WO 91/14703號之中説明的「逃逸突變株」。 HBsAg亦可指EP 0 198 474或EP 0 304 578中所説明的多肽。一般而言，HBsAg呈顆粒形式。在一項尤佳的具體實例中，HbsAg本質上係由本説明書以上所提過之HbsAg S-抗原所组成。疫苗可有利地包括醫藥上可接受的賦形劑，如一種適當的佐劑。適當的佐劑可購得。例如，Freund氏的不完全佐劑和完全佐劑（Difco Laboratories出品，位於密西根州的底特律市）；Merck佐劑65(Merck，默克藥廠出品，位於紐澤西州的 Rahway 市）；As-2(SmithKline Beecham 出品，位於賓州費城）；鋁鹽如氫氧化鋁膠體（明礬）或磷酸鋁；鈣、鐵或鋅的鹽；醯化之酪胺酸的不溶性懸浮液；醯化之糖；陽離子或陰離子形成衍生之多醣；聚偶磷氮；生物可分解之微本紙張尺度適用中國國家標準(CNS) A4規格(210X 297公釐) 1311563 A7 B7 五、發明説明（5 ) 球體；單磷醯基脂A和quil A.組織介素，如GM-CSF或介白素-2，-7或-12，亦可用做爲佐劑。在本發明的調配物中，較佳的是該佐劑組合物會謗發主要是TH1型式的免疫反應。高濃度之Thl型组織介素（例如 IFN-γ，TNFcx，IL-2和IL-12)傾向於偏好謗發細胞調節之對於施用抗原的免疫反應。在一項較佳的具體實例中，其中該反應主要是Th-1型的，Th-1型旳组織介素要比Th-2型组織介素增加的程度大。這些組織介素的濃度可以容易地利用標準檢驗法探測。若欲回顧組織介素家族，請參考 Mosmann 和 Coffman，<<免疫學年鑑〉〉，第 7期：145-173 頁，1989年。1311563 A7 ________B7 _ V. DESCRIPTION OF THE INVENTION (3) Preferably, the ion exchange chromatography is anion exchange chromatography. The present invention further provides a hepatitis B antigen obtainable by the production method of the present invention without ethylation of sodium salicylate, wherein the antigen is at least immunogenic and has antigenicity, such as ethylmercuric salicylic acid. Produced in the presence of sodium. The present invention further provides an immunogenic hepatitis B antigen having an average ELISA protein ratio of greater than 1 _5 and an IC50 値 at least 3 times lower than the surface of the hepatitis B produced by the sodium sulphate. The RF1 content of the antigen. Still another aspect of the present invention relates to a method for producing a hepatitis antigen suitable for use in a vaccine comprising: purifying an antigen in the presence of sodium ethylmercury salicylate, and then in the presence of a reducing agent containing a free-SH group. The antigen is processed. Suitably after this treatment, a purification step such as a dialysis step is carried out to remove ethylmercury salicylate. Preferably, the reducing agent is cysteine, DTT, glutathione or 2-mercaptoethanol. The hepatitis B antigen of the present invention can be used for the treatment or prevention of hepatitis B infection, especially for treating or preventing, for example, chronic hepatitis B infection. The invention further provides a vaccine formulation comprising a hepatitis B antigen of the invention linked to an adjuvant. Preferably, the adjuvant is an aluminum salt or preferably a TH1 cell reaction stimulator. Preferably, the antigen is a hepatitis B surface antigen. The preparation of hepatitis B surface antigen has been well documented. Please refer to, for example, Harford et al. in Developmental Biology Standards, No. 54, Η 页 -6 - This paper scale is suitable for ag agged ijt (CNS) M specification (21QX tearing) 1311563 A7 B7 V. Invention Description (4) (1983), Gregg et al., Biotechnology, No. 5, 479 (1987), EP-A4 226 846, ΕΡ-Α-0 299 108, among which are references. The expression "B hepatitis B surface antigen" or Γ HBsAg as used in the present specification includes any HBsAg antigen or a fragment thereof which exhibits the antigenic properties of the HBV surface antigen. We need to know the sequence of 226 amino acids in addition to the HBsAg S antigen (please refer to Tiollais et al., Nature, 317, 489 (1985) and references therein), as described in this specification. The HBsAg may comprise all or part of the precursor-S sequence as described in the above references and in EP-A 0 278 940, if desired. The HBsAg described in the present specification may also be referred to as a variant, such as the "escape mutant" described in WO 91/14703. HBsAg can also refer to the polypeptides described in EP 0 198 474 or EP 0 304 578. In general, HBsAg is in the form of particles. In a particularly preferred embodiment, HbsAg consists essentially of the HbsAg S-antibiotics mentioned above in this specification. The vaccine may advantageously comprise a pharmaceutically acceptable excipient such as a suitable adjuvant. Suitable adjuvants are commercially available. For example, Freund's incomplete adjuvant and complete adjuvant (Difco Laboratories, located in Detroit, Michigan); Merck Adjuvant 65 (Merck, Merck Pharmaceuticals, Rahway, New Jersey); As-2 (SmithKline Beecham, located in Philadelphia, PA); aluminum salts such as aluminum hydroxide colloid (alum) or aluminum phosphate; calcium, iron or zinc salts; insoluble suspension of deuterated tyrosine; Sugar; cation or anion to form derivatized polysaccharide; poly-phosphorus nitrogen; biodegradable micro-paper scale applicable to China National Standard (CNS) A4 specification (210X 297 mm) 1311563 A7 B7 V. Invention description (5) Sphere; Monophosphoryl lipid A and quil A. Interleukins, such as GM-CSF or interleukin-2, -7 or -12, can also be used as adjuvants. In the formulations of the present invention, it is preferred that the adjuvant composition will elicit a predominantly TH1 type of immune response. High concentrations of Thl-type interleukins (e.g., IFN-[gamma], TNFcx, IL-2, and IL-12) tend to favor immune responses to the administration of antigens by hair cell regulation. In a preferred embodiment, wherein the reaction is predominantly of the Th-1 type, the Th-1 type sputum interleukin is increased to a greater extent than the Th-2 type interleukin. The concentration of these interleukins can be readily detected using standard assays. For a review of the interleukin family, please refer to Mosmann and Coffman, <<Immunological Yearbook,  No. 7: 145-173, 1989.

因此，用於引發主要爲Thl-型反應之適當佐劑包括，例如：單磷醯脂A，較好是3-去氧-醯化單磷醯脂A(3D-MPL) 與鋁鹽的組合物。其他能優先誘發ΤΗ 1型免疫反應的已知佐劑包括含有寡核苷酸的CpG。該寡核茹酸的特徵在於CpG 二核#酸是未經甲基化的。此種寡核甞酸己詳知且説明於例如WO 96/02555號。其免疫刺激性之DNA序列亦被説明於例如，Sato等人，《科學》，第273期：352頁（1996年）。另一項較佳的佐劑爲皇素，較好是QS21(Aquila Biopharmaceuticals公司出品，位於麻州的Framingham市），其可單獨使用或與其他佐劑合併使用。例如，一種增進的系統涉及單鱗酿基脂A和皇素衍生物的組合，如Q S 2 1和 30-1^1^之組合，如冒0 94/00153號説明者，或是較不具致反應性的组合物，其中QS2 1乃以膽固醇予以熄滅，如WO -8 - 本紙張尺度適用中國國家標準(CNS) A4規格(210X 297公寶） 1311563 A7 B7 五、發明説明（6 96/33739號所説明者。其他較佳的調配物包括油_水乳化物和生育酚。一種尤其效力之佐劑調配物涉及QS2卜3D-MPL 和生育酚在油-水乳化物中，此係説明於W0 95/17210號之中。一項尤具效力之佐劑組合物涉及QS21，3D-MPL和生育酚在一種油-水乳化物中’係説明於W〇 95/17120號之中且爲一種較佳的調配物。Thus, suitable adjuvants for initiating predominantly Th1-type reactions include, for example, monophosphorus a, preferably 3-deoxy-deuterated monophosphorus A (3D-MPL) in combination with an aluminum salt. Things. Other known adjuvants that preferentially induce a serotype 1 immune response include CpG containing an oligonucleotide. The oligonucleic acid is characterized in that the CpG dinuclear acid is unmethylated. Such oligonucleotides are well known and described, for example, in WO 96/02555. Its immunostimulatory DNA sequence is also described, for example, in Sato et al., Science, 273: 352 (1996). Another preferred adjuvant is Imperial, preferably QS21 (Aquila Biopharmaceuticals, located in Framingham, MA), which may be used alone or in combination with other adjuvants. For example, an improved system involves a combination of a single-flavored adipose A and a primadium derivative, such as a combination of QS 2 1 and 30-1^1^, as described by 0 94/00153, or less A reactive composition in which QS2 1 is quenched with cholesterol, as in WO -8 - This paper scale applies to the Chinese National Standard (CNS) A4 specification (210X 297 Dm) 1311563 A7 B7 V. Description of invention (6 96/33739 Other preferred formulations include oil-water emulsions and tocopherols. A particularly potent adjuvant formulation involves QS2, 3D-MPL and tocopherol in oil-water emulsions, which are described in Among W0 95/17210. A particularly effective adjuvant composition involving QS21, 3D-MPL and tocopherol in an oil-water emulsion' is described in W〇95/17120 and is a A preferred formulation.

裝因而，在本發明的一項具體實例中提供了一種包含本發明之B型肝炎表面抗原的疫苗，其尚包含τη- 1誘發佐劑。一項較佳的具體實例是一種疫苗而其中的ΤΗ-1誘發佐劑係選自於包括以下佐劑的組群：3D-MPL、QS21、QS21與膽固醇的混合物，以及CpG寡核甞酸。另一項較佳的具體實例爲包含B型肝炎表面抗原且佐以單磷醯基脂A或其衍生物，QS21和生育酚在一種油-水乳化物中之疫苗。較好是該疫苗尚包含一種皇素，更好是QS21。另一項尤爲適當的佐劑調配物包括CpG和皇素者説明於w〇 00/09159號，且爲較佳的調配物。最好是在該特殊調配物中之皇素爲QS21。較好是該調配物尚包含一種油_水乳化物與生育酚。本發明尚提供包含本發明之B型肝炎表面抗原與一種佐劑接合且另外包含-種或更多選自以下組群之抗原的疫苗調配物，該抗原组群包含：白喉類毒素⑼、破傷風類毒素 (丁）、非細胞性百曰咳抗原（Pa)、失活的脊髓灰白質炎病毒 (ipv)、流行性感冒嗜血性抗原（Hib)、A型肝炎抗原、單純 -9-Thus, in a specific embodiment of the invention, a vaccine comprising a hepatitis B surface antigen of the invention is provided which further comprises a τη-1 evoked adjuvant. A preferred embodiment is a vaccine wherein the ΤΗ-1 evoked adjuvant is selected from the group consisting of 3D-MPL, QS21, a mixture of QS21 and cholesterol, and CpG oligonucleotide. Another preferred embodiment is a vaccine comprising a hepatitis B surface antigen and a monophosphoryl lipid A or a derivative thereof, QS21 and tocopherol in an oil-water emulsion. Preferably, the vaccine further comprises a royal jelly, more preferably QS21. Another particularly suitable adjuvant formulation, including CpG and Emperor, is described in WO 00/09159 and is a preferred formulation. Preferably, the prime in the special formulation is QS21. Preferably, the formulation further comprises an oil-water emulsion and tocopherol. The present invention further provides a vaccine formulation comprising the hepatitis B surface antigen of the present invention in combination with an adjuvant and additionally comprising one or more antigens selected from the group consisting of diphtheria toxoid (9), tetanus Toxoid (D), acellular cellulite antigen (Pa), inactivated poliovirus (ipv), influenza hemorrhagic antigen (Hib), hepatitis A antigen, simple-9-

1311563 A7 B7 五、發明説明（7 ) 疱疹病毒（HSV)、披衣菌、GSB、HPV、肺炎鏈球菌和奈瑟氏球菌抗原。關於保護以防其他疾病之抗原亦可合併於本發明的疫苗調配物之中。在一項特別的具體實例中，該疫苗調配物包含可得自本發明製造方法的B型肝炎表面抗原與一種佐劑接合，以及失活的脊髓灰白質炎病毒。本發明亦提供一種治療和/或預防B型肝炎病毒感染的方法，其包括對人或動物對象罹患或疑似受到B型肝炎病毒感染者施用安全且有效量之本發明的疫苗以預防和/或治療B 型肝炎。本發明尚提供本發明之B型肝炎表面抗原在製造用以治療受到B型肝炎病毒感染，如慢性B型肝炎病毒感染之病患的醫藥品上之用途。本發明的疫苗包含免疫防護量之抗原且可藉由傳統的技術製備。疫苗製備一般而言説明於《醫藥生物技術》，第61卷， <疫苗設計一次單元體與佐劑方法〉，由Powell和Newman编著，Plenum Press出版（1995年）。<<疫苗新趨勢與發展>> ，由Voller等人編著，University Park Press出版，位於美國馬里蘭州的巴爾地摩市（1978年）。製入微脂粒膠囊的方式説明於例如Fullerton之美國專利案第4,235,877號。將蛋白質接合至巨分子經由例如Likhite在美國專利案第 4,372,945號，Armor等人在美國專利案第4,474,757號之中揭示。Quil A的用途係由Dalsgaard等人在<<Acta Vet -10- 本纸張尺度適用中國國家標準(CNS) A4规格(210 X 297公羡)1311563 A7 B7 V. INSTRUCTIONS (7) Herpesvirus (HSV), chlamydia, GSB, HPV, Streptococcus pneumoniae and Neisseria antigens. Antigens that are protected against other diseases may also be incorporated into the vaccine formulations of the present invention. In a particular embodiment, the vaccine formulation comprises a hepatitis B surface antigen available from the method of the invention for binding to an adjuvant, and an inactivated poliovirus. The invention also provides a method of treating and/or preventing hepatitis B virus infection comprising administering a safe and effective amount of a vaccine of the invention to a human or animal subject suffering from or suspected of being infected with a hepatitis B virus for prevention and/or prevention. Treating hepatitis B. The present invention also provides the use of the hepatitis B surface antigen of the present invention for the manufacture of a medicament for treating a patient infected with a hepatitis B virus infection, such as a chronic hepatitis B virus infection. The vaccine of the present invention comprises an immunologically protective amount of antigen and can be prepared by conventional techniques. Vaccine preparation is generally described in Medical Biotechnology, Vol. 61, < Vaccine Design Primary Units and Adjuvant Methods, edited by Powell and Newman, Plenum Press (1995). <<New Vaccine Trends &Development>>, edited by Voller et al., published by University Park Press, located in Baltimore, Maryland, USA (1978). The method of making a vesicle capsule is described, for example, in U.S. Patent No. 4,235,877 to Fullerton. The splicing of the protein to the macromolecules is disclosed, for example, in U.S. Patent No. 4,372,945 to the disclosure of U.S. Pat. The use of Quil A is based on Dalsgaard et al. <<Acta Vet -10- This paper scale applies Chinese National Standard (CNS) A4 specification (210 X 297 mm)

裝訂Binding

線 1311563 A7 B7 五、發明説明（9 ) 乙基汞化水楊酸鋼。乙基汞化水楊酸鈉被包括在此緩衝液中以控制生物負擔。大多此種乙基汞化水楊酸鈉已在緊接的純化步驟，包括：離子交換層析法、超離心法和去鹽（膠體滲透法）中除去，使得由原先過程製備之經純化的大量抗原製備物包含大約1.2微克和少於2微克乙基汞化水楊酸鈉/每20微克蛋白質。用DEAE基質進行離子交換層析的步驟，並將此匯聚物用铯-梯度超離心在4個預設之不同的氣化铯濃度層中進行處理。抗原顆粒會根據其在梯度中的密度與污染之細胞成份分開，並且在離心過程之末層被沖提出來。然後藉由第二次膠體滲透在Sepharose膠體上將氣化铯從此匯集物中移除。當HBsAg藉由包含乙基汞化水楊酸鈉的4B滲透緩衝液製備時，超過30毫克/毫升的蛋白質濃度即從包含得自CsCl的梯度的部份收集物之匯集的HBsAg中回收，這與藉由購自 Abbott Laboratories 的 AUSZYME套組檢驗所得的 HBsAg濃度相等。Line 1311563 A7 B7 V. Description of invention (9) Ethyl mercuryated salicylic acid steel. Ethyl mercuryated sodium salicylate is included in this buffer to control the biological burden. Most of this ethylmercury salicylate has been removed in the immediate purification steps, including: ion exchange chromatography, ultracentrifugation, and desalting (colloidal permeation) to allow purification from the original process. A large number of antigen preparations contained approximately 1.2 micrograms and less than 2 micrograms of ethyl mercuryated salicylate per 20 micrograms of protein. The ion exchange chromatography step was carried out using a DEAE substrate, and the merpolymer was treated by 铯-gradient ultracentrifugation in four predetermined different vaporization enthalpy concentration layers. The antigen particles are separated from the contaminated cellular components according to their density in the gradient and are flushed out at the end of the centrifugation process. The gasified hydrazine is then removed from the pool by a second colloidal infiltration on the Sepharose colloid. When HBsAg is prepared by 4B permeation buffer containing ethylmercuric sodium salicylate, a protein concentration of more than 30 mg/ml is recovered from the pooled HBsAg containing a fraction of the fraction obtained from CsCl, which The concentrations of HBsAg obtained by testing with the AUSZYME kit purchased from Abbott Laboratories were equal.

CsCl超離心步驟通常能縮減HBsAg製備物中殘留的脂、 DNA和少量蛋白攙雜物。此步驟是以分區離心法在Ti 15離心器（購自Beckman儀器公司，位於加州Fullerton市），以 30,000 rpm進行大約40至60小時。待純化的樣品係施加到具有0.75，1.5，2.5和3.25 M CsCl之最終濃度的CsCl溶液層上。在離心的末尾，將梯度沖提成爲部份收集物。包含有 HBsAg的部份收集物可藉由在280 nm下的UV吸收値，或以 -12- 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐）The CsCl ultracentrifugation step typically reduces residual lipids, DNA, and small amounts of protein impurities in the HBsAg preparation. This step was carried out by zoned centrifugation at a Ti 15 centrifuge (available from Beckman Instruments, Inc., Fullerton, Calif.) at approximately 30,000 rpm for approximately 40 to 60 hours. The sample to be purified was applied to a layer of CsCl solution having a final concentration of 0.75, 1.5, 2.5 and 3.25 M CsCl. At the end of the centrifugation, the gradient was flushed into a partial collection. Some of the collections containing HBsAg can be applied by UV absorption at 280 nm or by Chinese National Standard (CNS) A4 (210X297 mm) on a paper scale of -12-

裝訂 1311563 A7Binding 1311563 A7

S Ζ Υ Μ E套組測試部份收集物的稀釋液鑑認出來。HBsAg 寬帶的密度爲1.17至I·23公克/每立方公分。將含有經純化之HBsAg的溶液消毒過濾，才能用於製造疫苗調配物。 k酵母菌細胞溶解物中進行純化是複雜的，因爲抗原是在細胞中生產且必需使用一系列經設計用以縮減不同型式之（酵母菌）攙雜物的分離技術，以獲得純粹的大量抗原。純化的步驟很重要，因爲待純化的產物是一種含有多數複本之表面抗原多肽的脂蛋白顆粒，且此種結構必須在純化過私中全程保持著。本過程之特殊性在於其所產生的表面抗原顆粒完全具致免疫性，而毋需進一步的化學處理來増進其致免疫性（比較EP0135435)。生產過程之細節進一步説明於歐洲專利案第〇丨99698號。實施例2 以不含乙基汞化水楊酸鈉的方法生產和鑑別得自酵母菌的 HBsAg 1·生產和純化得自酵母菌的HBsAg 1 · 1 ·生產方法之大綱 B型肝炎表面抗原可藉由醱酵適當菌株之酵母菌The dilution of the S Ζ Υ Μ E set test part of the collection was identified. The density of HBsAg broadband is 1.17 to I·23 g/cm3. The solution containing the purified HBsAg is sterilized and filtered to be used in the manufacture of a vaccine formulation. Purification in k yeast cell lysates is complicated because the antigen is produced in cells and a series of separation techniques designed to reduce the different types of (yeast) impurities are obtained to obtain a purely large amount of antigen. The purification step is important because the product to be purified is a lipoprotein particle containing a plurality of copies of the surface antigen polypeptide, and such structure must be maintained throughout the purification process. The particularity of this process is that the surface antigen particles produced by it are completely immunogenic and require further chemical treatment to achieve their immunogenicity (cf. EP 0135435). Details of the production process are further described in European Patent No. 99698. Example 2 Production and Identification of HBsAg from Yeast by Ethyl Mercury-Free Sodium Salicylate Production and Purification of HBsAg 1 · 1 from Yeasts Outline of Production Method Hepatitis B Surface Antigen Yeast by fermenting the appropriate strain

Saccharomyces cerevisiae來生產，例如Harford等人所説明者（loc. cit.)。在重組酵母菌株大規模鏺酵之末尾，收獲細胞並且在溫和的界面活性劑如Tween 20存在下將其打破。然後用多步驟萃取和完全如同以上實施例1中的純化程序至第一次在 _ -13- 本紙張尺度適用中國B家樣準(CNS) A4规格(210 X 297公釐） 1311563 A7 B7 五、發明説明（11 )Saccharomyces cerevisiae is produced, for example, by Harford et al. (loc. cit.). At the end of the large-scale fermentation of the recombinant yeast strain, the cells are harvested and disrupted in the presence of a mild surfactant such as Tween 20. Then use multi-step extraction and completely the same as the purification procedure in Example 1 above to apply for the first time in the _ -13- paper scale. China B sample (CNS) A4 specification (210 X 297 mm) 1311563 A7 B7 , invention description (11)

Sephar〇Se4B上進行膠體滲透的步驟，以分離出表面抗原。 1.2·不含乙基汞化水楊酸鈉的純化方法在不含乙基水化水楊酸納的純化方法中，與實施例丨中所説明的方法相較，加入了以下兩項改變。 1. 在4B膠體渗透層析術步驟中的沖提缓衝液不再含乙基汞化水楊酸納。 2. 將半胱胺酸（最終濃度2 mM)添加到得自陰離子交換層析術步驟所得的沖提匯聚物之中。吾人發現從4B膠體滲透緩衝液中省略乙基汞化水楊酸鈉會造成HBsAg顆粒在CsCl密度梯度離心法步驟中沉澱，且損失產物並使回收的抗原集結或成塊。以2 mM的最終濃度將半胱胺酸添加到得自先前陰離子交換層析術步驟所得之沖提匯聚物中能防止CsCM密度離心法進行期間之沉澱和損失抗原。半胱胺酸對於此項處理是較佳的物質，因爲它是天然的胺基酸且可在緊接的去鹽步壞中利用Sepharose 4BCLFF做爲管柱基質而在膠體滲透管柱上移除之。與實施例1中說明的方法比較，該製造過程中沒有其他的改變。該無乙基汞化水楊酸鈉法產生可與實施例丨的方法所得之抗原可相比的純度與性質之大量抗原。 1.2a 添加至50 pg/ml之4B緩衝液的乙基汞化水楊酸鈉被認爲會分解，並且所形成的乙基汞會共價連接到蛋白質的半胱 -14 -A step of colloidal infiltration on Sephar(R) Se4B to separate surface antigens. 1.2·Purification method without ethylmercury salicylate In the purification method without ethyl hydrated salicylate, the following two changes were added as compared with the method described in Example 。. 1. The elution buffer in the 4B colloidal permeation chromatography step no longer contains ethylmercury salicylate. 2. Add cysteine (final concentration 2 mM) to the extracted concentrator from the anion exchange chromatography step. I have found that omitting ethylmercury-sodium salicylate from the 4B colloidal permeation buffer causes the HBsAg particles to precipitate in the CsCl density gradient centrifugation step, and the product is lost and the recovered antigen is aggregated or agglomerated. The addition of cysteine to the rinsed aggregator obtained from the previous anion exchange chromatography step at a final concentration of 2 mM prevents precipitation and loss of antigen during CsCM density centrifugation. Cysteine is preferred for this treatment because it is a natural amino acid and can be removed on the colloidal permeate column using Sepharose 4BCLFF as the column matrix in the immediate desalination step. It. There were no other changes in the manufacturing process as compared to the method described in Example 1. The ethylaluminum free sodium salicylate method produces a large amount of antigen which is comparable in purity and nature to the antigen obtained by the method of the Example. 1.2a Ethyl mercuryated sodium salicylate added to 50 pg/ml of 4B buffer is considered to decompose and the formed ethyl mercury is covalently linked to the protein cysteine -14

13 Π 563 A7 _____ B7 五、發明説明（12 ) 胺酸之自由硫氫基上。該蛋白質包含14個半胱胺酸殘基，而其中7個位於第1〇1和第150個位置之間。這個蛋白質區域被認爲是位在顆粒表面，且包含HBsAg 的主要抗原區，包括支配免疫的生區和RF1單株抗體的辨識位置（Waters J.等人，《Postgrad. Med. J.>>，1987年，第 63期（補充 2): 51-56 頁。和 Ashton-Rickardt之 Murray<<醫用病毒學雜誌》，1989年，第29期：196頁）。以乙基汞化水楊酸鈉存在於4B膠體滲透法缓衝液中所純化出來的抗原在純化過程末層包含大約0.5-0.6微克汞《此種汞無法完全以簡易的透析法除去。在一項實驗中，在大量抗原製備中測出0_56微克汞/每20 微克蛋白質。此種製備物在室溫下對抗150 mM NaCl，10 mMNaP04缓衝液，pH6.9透析16小時。在透析末尾時，測量到0.33微克汞/每20微克蛋白質的濃度。相對地，在有還原劑如L-半胱胺酸在0.1至5.0毫克/毫升，DTT在50 mM或2-巯基乙醇在0.5 Μ存在下進行透析，接著用第二次透;析除去還原劑，則會使抗原製備物之汞含量降低至少於0.025微克汞/每20微克蛋白質。這是本方法之偵測最底限。汞含量是藉由吸收光譜分析法測定的。抗原經稀釋在含有0.01%(重量/體積）之重鉻酸鉀（K2Cr2〇7)和5%(體積/體積）的硝酸中。標準溶液係以乙基汞化水楊酸鈉製備’做爲汞的來源。樣品與標準溶液的原子吸收則是在蒸氣產生器中蒸發後，以特定於汞的陰極在253.7 nm波長下測定的。稀 -15- ___ 本紙張尺度適用中a圉家搮準(CNS) A4规格(210 X 297公釐） A7 B7 1311563 五、發明説明（14 ) 三批大量抗原，且經鑑認爲HEF001，HEF002，HEF003 ° 將這些抗原與先前方法（如實施例1説明者）在乙基采化水楊酸鈉存在下所製備的一批大量抗原（HEP2055)做比較。 2.1.2對於大查抗原之試驗與檢驗對於用無乙基杀·化水楊酸鈉法生產的三批大量抗原進行試驗，且其結果摘錄於表2之中。蛋白質含量則是以L〇wry等人的方法（<<生物化學雜德>〉，第193期：265頁，1951年）測量。内毒素的含量是以Limulus膠禮凝塊技術利用可購自 Cape Cod Associates(繪魚角協會），美國麻州Falmouth市 Main. St. 704號02540之套組進行測量》該試劑係以美國藥物内毒素參考標準（US Pharm. Endotoxin Reference Standard)予以標準化。13 Π 563 A7 _____ B7 V. INSTRUCTIONS (12) On the free sulfhydryl group of the amine acid. The protein contains 14 cysteine residues, 7 of which are located between the 1st and 150th positions. This protein region is thought to be located on the surface of the particle and contains the major antigenic region of HBsAg, including the immunogenic region and the identified position of the RF1 monoclonal antibody (Waters J. et al., Postgrad. Med. J.>>, 1987, No. 63 (Supplement 2): 51-56. and Ashray-Rickardt's Murray<<Journal of Medical Virology, 1989, No. 29: 196). The antigen purified by the presence of ethylmercury-sodium salicylate in 4B colloidal osmosis buffer contains approximately 0.5-0.6 micrograms of mercury at the end of the purification process. This mercury cannot be completely removed by simple dialysis. In one experiment, 0-56 micrograms of mercury per 20 micrograms of protein was measured in the preparation of large amounts of antigen. This preparation was dialyzed against 150 mM NaCl, 10 mM NaP04 buffer, pH 6.9 for 16 hours at room temperature. At the end of the dialysis, a concentration of 0.33 micrograms of mercury per 20 micrograms of protein was measured. In contrast, in the presence of a reducing agent such as L-cysteine at 0.1 to 5.0 mg/ml, DTT is dialyzed in 50 mM or 2-mercaptoethanol in the presence of 0.5 Torr, followed by a second pass through to remove the reducing agent. Will reduce the mercury content of the antigen preparation by at least 0.025 micrograms of mercury per 20 micrograms of protein. This is the detection limit of this method. The mercury content is determined by absorption spectroscopy. The antigen was diluted in nitric acid containing 0.01% (w/v) potassium dichromate (K2Cr2?7) and 5% (vol/vol). The standard solution was prepared as ethyl mercury-sodium salicylate as a source of mercury. The atomic absorption of the sample and the standard solution was determined at a wavelength of 253.7 nm with a mercury-specific cathode after evaporation in a vapor generator. Rare -15- ___ This paper size is applicable to a 圉搮搮 (CNS) A4 specification (210 X 297 mm) A7 B7 1311563 V. Invention description (14) Three batches of large antigens, and it is considered that HEF001, HEF002 HEF003 ° These antigens were compared to a batch of large antigens (HEP2055) prepared in the presence of sodium acetylated salicylate in a previous method (as described in Example 1). 2.1.2 Tests and tests for large-spectrum antigens Three batches of antigens produced by the method of sodium-free sodium salicylate were tested, and the results are summarized in Table 2. The protein content is measured by the method of L〇wry et al. (<<>> Biochemical Hydra >> 193: 265, 1951). The endotoxin content is measured using the Limulus gelatin clot technique using a kit available from Cape Cod Associates, Main. St. 704, 04540, Falmouth, MA, USA. The endotoxin reference standard (US Pharm. Endotoxin Reference Standard) is standardized.

Tween 20則是以Huddleston和Allred的方法進行測量（<< 美國油類化學會雜誌》，第42期：983頁，1965年）。 HBsAg含量是以購自Abbott Laboratories(位於美國伊利諾州 Abbott Park市之 Abbott Park路 IL 60064)的 AusZYEM 套組進行測量。以包含乙基汞化水楊酸鈉之方法純化的一批大量抗原被用做標準物以建立劑量反應曲線。以Dubois等人的方法（《分析化學雜誌》，第28期：350 頁，1956年）測量多醣。利用可購自 E. Merck公司（B.P. 4119，Darmstad D-6100 ，德國）的套組（Merkotest完整脂類3321)測量脂類。利用可構自 Molecular Devices公司（位於Gutenbergstrape -17- 本紙張尺度適用中國國家襟準(CNS) A4规格(210X297公釐) 1311563 A7 ----- -B7 五、發明説明（17 ) 表2:純化之無乙基汞化水楊酸鈉之大量抗原的試驗和檢驗結果試驗結果 HEF001 HEF002 HEF003 HEP2055 PH 6.8 6.8 6.8 6.8 以Lowry法測蛋白質含量 1312 pg/ml 888 pg/ml 913 pg/ml 995 pg/ml 内毒素含量 < 0.25 EU < 0.25 EU < 0.25 EU < 0.25 EU Tween 20 含量 7·1 μΒ 6.6 pg 7.4 pg 5.8 pg 用ELISA測定抗原活性 2957 pg/ml 1505 pg/ml 1486 pg/ml 1128 pg/ml ELISA/蛋白質比率 2.25 1.69 1.63 1.13 多醣含量 0.33 pg 0.35 μξ 0.33 μξ 0.34 pg 脂質含量 13.7 pg 12.8 pg 12.9 pg 11.8 μ§ 用門摇·法測定DNA含量 <ipg <ipg <ipg <ipg 2.1.4其他生化試驗和檢驗 2·1·4·1 DNA 含量三種大量抗原批的DNA含量係以門摇法（Molecular Devices公司）進行測量。所測得的量少於10xi〇 i2公克（即 10 pg)DNA/每20微克蛋白質（表2);相同程度的DNA含量也在以目前改進的方法所生產之大量抗原中觀察到。 2.1.4.2胺基酸組成三種HEF大量抗原批之胺基酸組成是在以6N HC1進行酸水解後藉著在離子交換管柱上進行層析而用管柱層析後之 -20- 本纸張尺度適用中國國家標準(CNS) A4規格(210X297公釐） 1311563 A7 B7 五、發明説明（18 ) 水合第三酮（ninhydrin)偵測來決定的。浦胺酸和色胺酸則未經測定。結果顯示於表3。所發現到的組成與在HEP2055測得者在良好吻合並且具有得自DNA序列之期望組成。雖然由HEP2055測得之甘胺酸殘基數目接近所期望的組成，16至17個殘基的値則較常在大量抗原製備物中測到。半胱胺酸殘基的平均數目經發現爲所期望的14個，顯示並未因爲CsCi梯度步驟之處理而造成多餘的半胱胺酸結合至顆粒上。 2.1.4.3自由半胱胺酸的定量根據説明的方法所得到的大量抗原製備物中出現的自由半胱胺酸之量是以過甲酸不經先前酸水解即將顆粒氧化之後測量的。氧化之自由半胱胺酸殘基則在離子交換管柱上進行分離，而以水合第三酮進行管柱層析後的偵測。用這種方法探測半胱胺酸的限制是丨微克/每毫升。在表2所示的起始蛋白質濃度測試時，在3種HEF抗原製備物中未能測到任何自由的半胱胺酸。該技術是測量出現在緩衝液中的半胱胺酸殘基，以及與 HBsAg蛋白質以二硫鏈連接，但並未形成多肽序列之部份的半胱胺酸。 2.1.4.4 N-端序列分析可之蛋白質攙雜物的存在與否及以修改過之方法生產的三種大量抗原批中的分解產物係根據Edman氏降解法以 N-端序列分析探測。HBsAg蛋白質的N端序列menits.. 經偵測而沒有其他序列干擾。N•端的甲硫胺酸亦經確認有 __ -21 - 本纸張尺歧λ t s s家料(cl^) A4規格(21QX 297公爱j --------- 1311563 A7 B7 五、發明説明（19 ) 60-75%被乙醯化所封阻。正如先前以例行方法，生產之 HBsAg多肽所觀察到的結果。表3 : HBsAg的胺基酸組成胺基酸 HEF001 HEF002 HEF003 平均組成 HEP2055 期望組成 Asp 11.3 11.3 11.3 11.3 11.5 10 Thr 17.5 17.4 17.2 17.4 17.8 17 Ser 21.4 21.6 21.4 21.5 20.9 23 Glu 11 11 11 11.0 10.5 9 Pro nd nd nd nd 24 Gly 17.1 16.8 16.7 16.9 14.6 14 Ala 7.5 7.4 7.4 7.4 7.2 6 Cys 12.3 14.95 14.9 14.1 13.2 14 Val 10.9 11 10.9 10.9 10.7 11 Met 6.8 6.7 7.1 6.9 7.1 6 lie 12.3 12.4 12.5 12.4 12.2 16 Leu 26.3 26.6 26.2 26.4 26.7 33 Tyr 6.8 6.8 6.8 6.8 7 6 Phe 13.8 13.9 13.8 13.8 13.9 15 His 3 2.8 3.3 3.0 3.3 1 Lys 4 4 3.9 4.0 4.2 3 Arg 5.7 5.8 5.7 5.7 6.1 5 Trp nd nd nd nd 13 -22- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公爱) 1311563 A7 B7 五、發明説明（2〇 ) 2.1.4.5雷射光散射分析利用修改方法製造的HBsAg顆粒與HEP2055參考批之間的顆杻大小比較是以雷射光散射來達成的（表4)。測得之平均分子量顯示製備物之間有良好的一致性。表4 :以雷射光散射測定HBsAg顆粒之分子量抗原批號分子量（道爾呑） HEF001 3.07 X106 HEF002 2.76 X 106 HEF003 2.76 X106 HEP2055 3.34 X 106 2.1.4.6電子顯微術大量抗原製備物在固定和用醋酸鈾醯染色後以電子顯微術進行檢驗。所觀察到的顆粒在所有樣品中均相似，且構形爲±20 nm 次球體或類似鵝卵石的典型HBsAg顆粒。在三種HEF批中觀察到的顆粒與HEP2055無法區辨。 2.1.5免疫學的分析 2.1.5.1與RF 1單株抗體的反應活性藉由ELISA抑制檢驗利用RF 1單株抗體測試三種大量抗原製備物的反應活性。RF 1單株抗體已顯示能保護猩猩對抗HBV的挑戰，且被認爲能辨識HBsAg顆粒上具保護構形的抗原決定部位。（I war son S等人，1985年，<< 醫用病毒學雜誌》，第16期：89-96頁）。 -23- 本紙張尺度適用中國國家標準(CNS) A4規格(210X 297公釐〉 1311563 A7 B7 五、發明説明（21 ) RF1融合瘤可在BalbC鼠的腹腔中或在組織培養中增生。以1/50000稀釋於飽和緩衝液（PBS含有1% BSA，0.1% Tween 20)之中的腹水與各種待測試之HBsAg樣品在PBS中的稀釋物以1 : 1混合（最終濃度範圍介於100微克和〇_〇5微克/每毫升之間）。將混合物培育在Nunc.免疫盤（96U)之中在37°C經1小時，然後在37°C經1小時轉移到用HBsAg標準製備物包覆的盤上。該標準的HBsAg製備物是一批大量抗原（Hep 286)，經含乙基果化楊酸納的過程純化者：在以含有0.1 % Tween 20的PBS清洗之步驟之後，添加稀釋成1/1000在飽和緩衝液中的生物素連接之羊的抗老鼠IgG並且在3 7Ό培育1小時。在沖洗步驟之後，將鏈抗生物素蛋白-生物素連接的過氧化酶複合物稀釋1 /1 〇〇〇於飽和緩衝液者添加到同一孔之中並且在37°C培育30分鐘。沖洗盤子並與OPDA 0.04%，H2〇2 0.03%溶於0.1 Μ擰檬酸鹽緩衝液pH 4.5的溶液一起培育在室溫下經20分鐘。用2 N H2SOJt反應停止並在490/630 nm 波長下測量光密度（O.D.)並予作圖。 IC50定義爲能抑制50%抗體結合至被覆蓋之HBsAg的抗原濃度（抑制劑濃度），係以4種參數的公式計算並且用ng/ml 爲單位表示。一系列HEP抗原批包括HEP2055亦被測試，而以簡單型疱疹gD抗原做爲負的對照組。該檢驗測量每一種試驗抗原抑制RF 1與結合至微滴盤上之標準抗原製備物（HEP286)的能力。 -24- 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐）Tween 20 is measured by Huddleston and Allred (<<" American Journal of Oil Chemistry, No. 42: 983, 1965). The HBsAg content was measured using an AusZYEM kit purchased from Abbott Laboratories (Abbott Park Road IL 60064, Abbott Park, Ill.). A batch of large antigens purified by the method comprising ethylmercury salicylate was used as a standard to establish a dose response curve. The polysaccharide was measured by the method of Dubois et al. (Journal of Analytical Chemistry, No. 28: 350 pages, 1956). Lipids were measured using a kit (Merkotest intact lipid 3321) available from E. Merck (B.P. 4119, Darmstad D-6100, Germany). The use can be constructed from Molecular Devices (located at Gutenbergstrape -17- This paper scale applies to China National Standard (CNS) A4 specification (210X297 mm) 1311563 A7 ----- -B7 V. Invention description (17) Table 2: Test results of a large number of purified antigens without ethylmercury salicylate. Test results HEF001 HEF002 HEF003 HEP2055 PH 6.8 6.8 6.8 6.8 Protein content measured by Lowry method 1312 pg/ml 888 pg/ml 913 pg/ml 995 pg /ml endotoxin content < 0.25 EU < 0.25 EU < 0.25 EU < 0.25 EU Tween 20 Content 7.1 μΒ 6.6 pg 7.4 pg 5.8 pg Determination of antigen activity by ELISA 2957 pg/ml 1505 pg/ml 1486 pg/ Ml 1128 pg/ml ELISA/protein ratio 2.25 1.69 1.63 1.13 Polysaccharide content 0.33 pg 0.35 μξ 0.33 μξ 0.34 pg Lipid content 13.7 pg 12.8 pg 12.9 pg 11.8 μ§ Determination of DNA content by gate shaking method <ipg <ipg < Ipg <ipg 2.1.4 Other biochemical tests and tests 2·1·4·1 DNA content The DNA content of the three large antigen batches was measured by the gate method (Molecular Devices). The measured amount was less than 10 xi〇. I2 g (ie 10 pg) DNA per 20 micrograms of protein (Table 2); the same level of DNA content was also observed in the large number of antigens produced by the currently improved method. 2.1.4.2 Amino acid composition The amino acid composition of the three HEF mass antigen batches was at 6N. HC1 is acid-hydrolyzed and chromatographed on an ion exchange column and chromatographed with column chromatography. -20- This paper scale is applicable to China National Standard (CNS) A4 specification (210X297 mm) 1311563 A7 B7 V. Description of the invention (18) Determination of ninhydrin by hydration. Puramide and tryptophan were not determined. The results are shown in Table 3. The composition found is in good agreement with those measured in HEP2055. And having the desired composition derived from the DNA sequence. Although the number of glycine residues measured by HEP 2055 is close to the desired composition, 16 to 17 residues of hydrazine are more commonly detected in a large number of antigen preparations. The average number of cysteine residues was found to be 14 as expected, indicating that no excess cysteine was bound to the particles due to the CsCi gradient step treatment. 2.1.4.3 Quantification of free cysteine The amount of free cysteine present in the large number of antigen preparations obtained according to the methods described was measured after the peroxycarboxylic acid was oxidized by the previous acid hydrolysis. The oxidized free cysteine residue is separated on the ion exchange column and detected by column chromatography with hydrated third ketone. The limit for detecting cysteine in this way is 丨μg/ml. At the initial protein concentration test shown in Table 2, no free cysteine was detected in the three HEF antigen preparations. This technique measures the cysteine residues present in the buffer and the cysteine which is linked to the HBsAg protein as a disulfide chain but does not form part of the polypeptide sequence. 2.1.4.4 N-terminal sequence analysis The presence or absence of proteinaceous impurities and the decomposition products of the three large antigen batches produced by the modified method are detected by N-terminal sequence analysis according to Edman's degradation method. The N-terminal sequence of the HBsAg protein menits.. was detected without other sequence interference. N• terminal methionine has also been confirmed to have __ -21 - paper size difference λ tss home material (cl^) A4 specifications (21QX 297 public love j --------- 1311563 A7 B7 five Inventive Note (19) 60-75% is blocked by acetylation. As observed by the conventional method, the observed results of the HBsAg polypeptide produced. Table 3: Amino acid composition of HBsAg Amino acid HEF002 HEF002 HEF003 Average composition HEP2055 Expected composition Asp 11.3 11.3 11.3 11.3 11.5 10 Thr 17.5 17.4 17.2 17.4 17.8 17 Ser 21.4 21.6 21.4 21.5 20.9 23 Glu 11 11 11 11.0 10.5 9 Pro nd nd nd nd 24 Gly 17.1 16.8 16.7 16.9 14.6 14 Ala 7.5 7.4 7.4 7.4 7.2 6 Cys 12.3 14.95 14.9 14.1 13.2 14 Val 10.9 11 10.9 10.9 10.7 11 Met 6.8 6.7 7.1 6.9 7.1 6 lie 12.3 12.4 12.5 12.4 12.2 16 Leu 26.3 26.6 26.2 26.4 26.7 33 Tyr 6.8 6.8 6.8 6.8 7 6 Phe 13.8 13.9 13.8 13.8 13.9 15 His 3 2.8 3.3 3.0 3.3 1 Lys 4 4 3.9 4.0 4.2 3 Arg 5.7 5.8 5.7 5.7 6.1 5 Trp nd nd nd nd 13 -22- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 public) 1311563 A7 B7 V. Description of invention (2〇) 2.1.4 .5 Laser Light Scattering Analysis The size comparison between the HBsAg particles produced by the modified method and the HEP2055 reference batch is achieved by laser light scattering (Table 4). The average molecular weight measured shows good between the preparations. Consistency. Table 4: Determination of molecular weight of HBsAg particles by laser light scattering. Molecular weight of the batch (Dow) HEF001 3.07 X106 HEF002 2.76 X 106 HEF003 2.76 X106 HEP2055 3.34 X 106 2.1.4.6 Electron microscopy Large amount of antigen preparation is fixed After staining with uranyl acetate, it was examined by electron microscopy. The observed particles were similar in all samples and were configured as ±20 nm subspheres or typical HBsAg particles resembling pebbles. The particles observed in the three HEF batches were indistinguishable from HEP 2055. 2.1.5 Analysis of Immunology 2.1.5.1 Reactivity with RF 1 monoclonal antibody The reactivity of three large antigen preparations was tested by RF 1 monoclonal antibody by ELISA inhibition assay. RF 1 monoclonal antibodies have been shown to protect orangutans from the challenge of anti-HBV and are believed to recognize epitopes with protective profiles on HBsAg particles. (I war son S et al., 1985, <<Journal of Medical Virology, No. 16: 89-96). -23- This paper scale applies to Chinese National Standard (CNS) A4 specification (210X 297 mm) 1311563 A7 B7 V. INSTRUCTIONS (21) RF1 fusion tumor can proliferate in the abdominal cavity of BalbC mice or in tissue culture. /50000 diluted ascites in saturated buffer (PBS containing 1% BSA, 0.1% Tween 20) mixed with various dilutions of HBsAg sample in PBS in 1:1 (final concentration range between 100 μg and 〇_〇5 μg/ml.) The mixture was incubated in Nunc. immunoplate (96 U) at 37 ° C for 1 hour, then transferred at 37 ° C for 1 hour to coat with HBsAg standard preparation. The standard HBsAg preparation is a batch of large antigen (Hep 286), purified by a process containing sodium oleate sodium sulphate: after washing with PBS containing 0.1% Tween 20, add dilution 1/1000 anti-mouse IgG of biotin-conjugated sheep in saturated buffer and incubated for 1 hour at 37. After the rinsing step, the streptavidin-biotin-linked peroxidase complex was diluted 1 /1 is added to the same hole in the saturation buffer and Incubate for 30 minutes at 37 ° C. Rinse the plate and incubate with OPDA 0.04%, H 2 〇 2 0.03% in 0.1 Μ citrate buffer pH 4.5 for 20 minutes at room temperature. React with 2 N H 2 SOJt Stop and measure the optical density (OD) at 490/630 nm and plot it. IC50 is defined as the antigen concentration (inhibitor concentration) that inhibits 50% antibody binding to the covered HBsAg, using four parameters. Calculated and expressed in ng/ml. A series of HEP antigens including HEP2055 were also tested, while the simple herpes gD antigen was used as a negative control group. This test measures each test antigen to inhibit RF 1 and bind to droplets. The ability of the standard antigen preparation (HEP286) on the plate. -24- This paper size applies to the Chinese National Standard (CNS) A4 specification (210X297 mm)

裝訂 1311563 A7 B7 五、發明説明（22 ) 表5提出所發現能抑制50% RF1結合至固定之抗原的每一種抗原的濃度。表5 : RF1單株抗體與HBsAg結合之抑制大量抗原 IC50 (ng/ml)* HEP286 3834 HEP673 3437 HEP720 3150 HEP2055 2384 HEF001 468 HEF002 574 HEF003 540 *IC50=抑制50% RF1與固定抗原結合之抗原的濃度（ng/ml) 該結果顯示少於4至7倍的HEF抗原需用於抑制RFI結合（表5)。這顯示以修改方式製備的抗原比HEP大量抗原具有增加出現之RF 1抗原決定部位。吾人進行同樣型式的抑制檢態，但利用得自Engerix BTM 疫苗之人的血清替代RF 1 mAb，而並未顯露在HEP抗原抑和HEF抗原之間有差異。 2.1.5.2對於單株RF1的結合親合力 RF1單株抗體結合至3種HEF抗原批和HEP2055之動力學參數係利用購自Amersham Pharmacia Biotech公司，位於 -25- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐） 1311563 A7 B7 五、發明説明（23 )Binding 1311563 A7 B7 V. INSTRUCTIONS (22) Table 5 presents the concentration of each antigen found to inhibit 50% of RF1 binding to immobilized antigen. Table 5: Inhibition of RF1 monoclonal antibody binding to HBsAg A large number of antigens IC50 (ng/ml)* HEP286 3834 HEP673 3437 HEP720 3150 HEP2055 2384 HEF001 468 HEF002 574 HEF003 540 *IC50=Inhibition of 50% RF1 binding to immobilized antigen (ng/ml) This result shows that less than 4 to 7 times the HEF antigen is required to inhibit RFI binding (Table 5). This shows that the antigen prepared in a modified manner has an increased appearance of the RF 1 epitope than the HEP large antigen. We performed the same type of inhibition assay, but replaced the RF 1 mAb with the serum from the Engerix BTM vaccine, but did not reveal a difference between the HEP antigen and the HEF antigen. 2.1.5.2 Binding affinity for single RF1 RF1 monoclonal antibody binding to three HEF antigen batches and HEP2055 kinetic parameters were purchased from Amersham Pharmacia Biotech, located at -25- This paper scale applies to Chinese national standards (CNS) A4 size (210 X 297 mm) 1311563 A7 B7 V. Description of invention (23)

Amersham Place，Little Chalfont，Bucks，英國之Biacore 2000裝置以表面質粒基因組共振測量之。所測量的動力學參數爲： ka :結合速率常數（M_1S’ kd :解離速率常數（S’Amersham Place, Little Chalfont, Bucks, UK Biacore 2000 device was measured by surface plasmid genomic resonance. The measured kinetic parameters are: ka : binding rate constant (M_1S' kd : dissociation rate constant (S'

Ka :平衡或親和力常數（ΓνΓ1)Ka: balance or affinity constant (ΓνΓ1)

Jrn 其中Ka=竺 kd 所發現的數値提供於表6之中。表6 : RF1結合至HBsAg的親和力常數大量抗原 ka (X 1〇-3) kd (X 105) Ka (X 10·7) HEF001 6.81 3.21 21.97 HEF002 6.89 3.73 18.83 HEF003 7.39 4.67 15.80 HEP2055 3.31 6.30 5.31 三種HEF抗原批提出相似的結合/解離常數和結合親和力値。相對地，HEP2055對於結合至RF 1具微弱的親和力。這與得自ELISA抑制檢驗的結果一致，該檢驗顯示以無乙基汞化水楊酸鈉之方法製備的抗原其有增加的RF1抗原決定部位。 2.2.對於用修改方式生產之抗原所調配的疫苗之試驗與檢驗三種HEF抗原批經吸附在氫氧化鋁上並根據表1所示的 -26- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐） 1311563 A7 B7 五、發明説明（24 ) 組成予以調配成疫苗。該表顯示的是在小管子中的成人劑量（20 ng抗原蛋白質在1毫升中）。該批係以DENS001A4， DENS002A4和 DENS003A4鑑認之0 疫苗的效力係利用Abbott Laboratories出品的AUSZYME ELISA套組藉由體外抗原含量檢驗測量，並以與50 ng/ml 乙基汞化水楊酸鈉調配之傳統疫苗批做爲標準物。疫苗效力則是利用在《PharmaEuropa特刊Bi〇97-2>>(1997年12月出刊）中説明的方法B測量。三種HEF批顯示高的抗原含量値，幾乎是所申明之20微克抗原蛋白質的兩倍。 2.2.1 DENS與RF1單株抗體的反應活性吸附之疫苗的抗原性在一項與RF1單株抗體之抑制檢驗中進一步被試驗。該檢驗係測量疫苗樣品抑制RF丨結合至固定之大量抗原（HEP286)的能力。將以1/50000稀釋於/飽和緩衝液（卩38含1% BSA，〇 1〇/ Tween 20)之腹水與各種待試驗之疫苗樣品在ρΒδ中的稀釋液以1 : 1混.合（濃度範圍介於20微克和〇·〇5微克/毫升）。將混合物培育在Nunc免疫盤中（96U)於37"Cm2小時，並予攪拌，而後轉移到有HBsAg包覆於上的盤中。用於包覆的HBsAg製備物是一批以含有乙基汞化水楊酸鈉純化的大量抗原（Hep286)。用含有0.1% Tween 20的PBS進行;中洗步躁之後’將1/1000稀釋於飽和缓衝液之與生物素連接的抗老鼠IgG添加進去’並且在37χ培育丨小時，在沖洗步躁^ 後，將以1/1 〇〇〇稀釋於飽和緩衝液的鏈抗生物素蛋白-生物素連接的過氧化酶複合物添加到孔槽中，並且在3<rc拉金 -27-Jrn where the number found by Ka=竺 kd is provided in Table 6. Table 6: Affinity constants of RF1 binding to HBsAg A large number of antigens ka (X 1〇-3) kd (X 105) Ka (X 10·7) HEF001 6.81 3.21 21.97 HEF002 6.89 3.73 18.83 HEF003 7.39 4.67 15.80 HEP2055 3.31 6.30 5.31 Three HEFs The antigen batches presented similar binding/dissociation constants and binding affinities. In contrast, HEP2055 has a weak affinity for binding to RF1. This is consistent with the results obtained from the ELISA inhibition assay, which showed that the antigen prepared by the method without ethylmercury salicylate has an increased RF1 epitope. 2.2. Testing and testing of vaccines formulated with modified antigens The three HEF antigens were adsorbed onto aluminum hydroxide and applied to the Chinese National Standard (CNS) A4 specifications according to the -26- paper scale shown in Table 1. (210 X 297 mm) 1311563 A7 B7 V. INSTRUCTIONS (24) The composition is formulated into a vaccine. The table shows the adult dose in a small tube (20 ng of antigenic protein in 1 ml). The efficacy of the batch of vaccines identified by DENS001A4, DENS002A4 and DENS003A4 was measured by in vitro antigen content assay using the AUSZYME ELISA kit from Abbott Laboratories and formulated with 50 ng/ml ethylmercuric salicylate. The traditional vaccine batch is used as a standard. Vaccine efficacy was measured using Method B as described in Pharma Europa Special Issue Bi〇97-2>> (published in December 1997). The three HEF batches showed a high antigen content of 値, almost twice the 20 micrograms of antigenic protein claimed. 2.2.1 Reactivity of DENS and RF1 monoclonal antibodies The antigenicity of the adsorbed vaccine was further tested in a inhibition assay with RF1 monoclonal antibodies. This test measures the ability of a vaccine sample to inhibit the binding of RF丨 to a fixed mass of antigen (HEP286). Diluted ascites diluted 1/50,000 in /saturated buffer (卩38 with 1% BSA, 〇1〇/Tween 20) and various vaccine samples to be tested in ρΒδ in a ratio of 1:1 (concentration) The range is between 20 μg and 〇·〇 5 μg/ml). The mixture was incubated in a Nunc immunoplate (96 U) at 37 "Cm for 2 hours, and stirred, and then transferred to a tray coated with HBsAg. The HBsAg preparation used for coating is a batch of a large amount of antigen (Hep286) purified by the use of ethylmercury salicylate. It was carried out with PBS containing 0.1% Tween 20; after washing the step, '1/1000 diluted biotin-conjugated anti-mouse IgG was added to the saturated buffer and incubated at 37 丨 for a few hours, after washing step 躁 ^ Adding a streptavidin-biotin-linked peroxidase complex diluted in 1/1 饱和 in a saturated buffer to the well, and at 3 <rc Lajin-27-

裝訂 t 1311563 A7 B7 五、發明説明（25 ) 30分鐘。沖洗盤子並且與含有OPDA 0.04%，H2〇2 0.03%於 0.1 Μ檸檬酸鹽緩衝液，pH 4.5的溶液在室溫下培育20分鐘。用2N H2S〇4使反應終止，並且在490/630 nm波長下測量光密度（O.D.)並予作圖。 IC50定義爲能抑制50%抗體結合至所包覆之HBsAg的抗原（抑制劑）濃度，其乃利用含4個參數的公式計算並以ng/ml 表示之。將以修改過之方法生產之大量抗原所製備的疫苗與從傳統HEP大量抗原調製且不含乙基汞化水楊酸鈉做保存劑之 Engerix B™疫苗做比較0 該檢驗以三個重複試樣進行。表7顯示結果並顯示大約有半數之DENS疫苗與不含保存劑之Engerix B™疫苗做比較，其需要達到50%的RF1結合受抑制。這反應了 HEF/DENS抗原上有增加的RF1抗原決定部份，並且這與RF1抗體在經純化的大量抗原上進行的試驗 —致0 表7以調配之疫苗抑制RF 1結合疫苗批號 IC-50 (ng/ml)(1) 實驗平均 1 2 3 DENS001A4 913 662 603 726 DENS002A4 888 715 521 708 DENS003A4 817 685 582 695 ENG5100A2 1606 1514 1481 1534 ENG3199B9 1329 1170 1286 1262 ENG3328A9 1417 1194 1334 1315 -28- 本紙張尺度適用中國國家標準(CNS) A4規格(210X 297公釐) 1311563 A7 B7 五、發明説明（26 ) (1)能抑制50% RF1抗體結合至固定之抗原的疫苗濃度 2..2..2 DENS疫苗在老鼠中的致免疫性在Balb/C老鼠身上進行一項研究以求對三種DENS—致性的抗原批之致免疫性與根據目前之抗原製造方法生產且以乙基汞化水楊酸納調配之Engerix BTM做比較。以下的抗原批被試驗： # DENS001A4 # DENS002A4 # DENS003A4 # ENG2953A4/Q做爲參考簡言之，以二週之間隔用相當於成人劑量之1/10 (2 pg) 或1/50 (0.4 pg)的疫苗劑量以肌内免疫方式對12隻老鼠的組群進行處理，從第28曰取樣之血清偵測其對HBsAg的抗體反應和由疫苗接種所謗發之異種型式的結果描述。實驗設計將含有12隻Balb/C老鼠的組群於雙腿進行肌内免疫注射 (2 X 50 μΐ)，在第0天和第15天施予下列疫苗劑量： -29- 本紙張尺度適用中國國家標準(CNS) Α4規格(210X 297公釐)Binding t 1311563 A7 B7 V. Description of invention (25) 30 minutes. The plates were rinsed and incubated for 20 minutes at room temperature with a solution containing OPDA 0.04%, H2〇2 0.03% in 0.1 Μ citrate buffer, pH 4.5. The reaction was terminated with 2N H 2 S 〇 4 and the optical density (O.D.) was measured at a wavelength of 490 / 630 nm and plotted. IC50 is defined as the concentration of an inhibitor (inhibitor) that inhibits 50% of antibody binding to the coated HBsAg, which is calculated using a formula containing 4 parameters and expressed in ng/ml. A vaccine prepared from a large number of antigens produced by a modified method is compared with an Engerix BTM vaccine prepared from a conventional HEP large antigen and containing no ethylmercured salicylate as a preservative. Sample. Table 7 shows the results and shows that approximately half of the DENS vaccines are compared to the non-preservative Engerix BTM vaccine, which requires 50% inhibition of RF1 binding. This reflects an increased RF1 epitope on the HEF/DENS antigen, and this is tested with the RF1 antibody on a purified large amount of antigen - 0 Table 7 with formulated vaccines to inhibit RF 1 binding vaccine batch IC-50 (ng/ml)(1) Experimental average 1 2 3 DENS001A4 913 662 603 726 DENS002A4 888 715 521 708 DENS003A4 817 685 582 695 ENG5100A2 1606 1514 1481 1534 ENG3199B9 1329 1170 1286 1262 ENG3328A9 1417 1194 1334 1315 -28- This paper size applies Chinese National Standard (CNS) A4 Specification (210X 297 mm) 1311563 A7 B7 V. Description of Invention (26) (1) Vaccine concentration that inhibits 50% of RF1 antibody binding to immobilized antigen 2..2..2 DENS vaccine Immunization in mice A study was conducted in Balb/C mice to obtain immunogenicity against three DENS-like antigenic batches and to produce mercury-sodium salicylate according to current antigen manufacturing methods. The blended Engerix BTM is compared. The following antigen batches were tested: # DENS001A4 # DENS002A4 # DENS003A4 # ENG2953A4/Q For short reference, use 1/10 (2 pg) or 1/50 (0.4 pg) of adult dose at intervals of two weeks. The vaccine dose was treated intramuscularly to a group of 12 mice, and the serum sampled from the 28th sputum was tested for its antibody response to HBsAg and the results of the heterologous pattern emanating from vaccination. Experimental Design A group of 12 Balb/C mice was intramuscularly injected (2 X 50 μΐ) on both legs, and the following vaccine doses were administered on days 0 and 15: -29- This paper scale applies to China National Standard (CNS) Α4 Specifications (210X 297 mm)

裝訂Binding

1311563 A7 B7 五、發明説明（27 表8 :組群和疫苗劑量組別疫苗體積抗原劑量 1 2 DENS001A4 —稀釋5倍在P〇4/NaCl中 100 μΐ 100 μΐ 2 Mg 0.4 pg 3 4 DENS002A4 ->稀釋5倍在P〇4/NaCl中 100 μΐ 100 μΐ 0.4 pg 5 6 DENS003A4 —稀釋5倍在P〇4/NaCl中 100 μΐ 100 μΐ 2 Kg 0.4 7 8 ENG2953A4/Q —稀釋5倍在P〇4/NaCl中 100 μΐ 100 μΐ 0.4 pg 在第15天（第一次注射後2週）和第28天（第二次注射後2週 )從後眼巢竇採血。爲了設計這項實驗（4種調配物X 2種劑量而以每組12隻老鼠進行），吾人以PASS統計程式先計算其效力。PASS(效力及樣品大小）統計程式係得自NCSS，329 North 1000 East ’猶他州Kays ville市，84037。對於兩種差異分析方法而言，應當偵測到兩種調配物之間有2.5倍的GMT差異，而a-誤差爲5%，而其效力>90%。結果血清學：藉由丑1：18八檢驗利用1^3八§(1^卩286)做爲覆被抗原並使用生物素連接之抗老鼠抗體體液免疫反應以透露抗-HBS 抗體之結合情形。只有第二次注射後（postII)的血清被分析》表9顯示在post II之後二週個別血清所測得之平均値與 -30- 本紙張尺度適用中國國家標準(CNS) A4规格(210X297公釐)1311563 A7 B7 V. INSTRUCTIONS (27 Table 8: Group and vaccine dose group vaccine volume antigen dose 1 2 DENS001A4 - diluted 5 times in P〇4/NaCl 100 μΐ 100 μΐ 2 Mg 0.4 pg 3 4 DENS002A4 -&gt Diluted 5 times in P〇4/NaCl 100 μΐ 100 μΐ 0.4 pg 5 6 DENS003A4 — diluted 5 times in P〇4/NaCl 100 μΐ 100 μΐ 2 Kg 0.4 7 8 ENG2953A4/Q — diluted 5 times in P〇 100 μΐ 100 μΐ 0.4 pg in 4/NaCl Blood was collected from the posterior sinus of the posterior eye on day 15 (2 weeks after the first injection) and day 28 (2 weeks after the second injection). Formulation X doses were administered in groups of 12 mice. We calculated the potency first by PASS. The PASS (effectiveness and sample size) statistical program was obtained from NCSS, 329 North 1000 East 'Kaysville, Utah City, 84037. For the two differential analysis methods, a 2.5-fold difference in GMT between the two formulations should be detected with an a-error of 5% and an efficacy of >90%. Results Serology: Use 1^3 八§(1^卩286) as the coating antigen and connect with biotin by ugly 1:18 The mouse antibody humoral immune response revealed the binding of anti-HBS antibodies. Only after the second injection (post II) serum was analyzed. Table 9 shows the mean 値 and -30- measured by individual sera two weeks after post II. This paper scale applies to the Chinese National Standard (CNS) A4 specification (210X297 mm)

裝訂Binding

1311563 A7 B7 五、發明説明（28 ) GMT抗-HBs Ig抗體反應。可比較的抗體反應係由DENS和傳統的B型肝炎調配物所誘發一 GMT範圍介於2304和3976 EU/毫升對於DNES批相較於2882 EU/毫升對於SB Biologicals B型肝炎單價疫苗 (Engerix B™)，以2微克劑量得到的結果；且GMT範圍介於 696和1182 EU/毫升對於DENS批相較於627 EU/毫升對於 SB Biologicals B型肝炎單價疫苗（Engerix B™)，在0.4微克劑量所得到的結果。 •如吾人預期的，吾人發現對於所有調配物在2微克和0.4 微克劑量有清楚的抗原劑量範圍，而其GMT有3到6倍的差異。 •有四隻未反應老鼠（滴定量<50 EU/毫升）被觀察到與注射之抗原劑量或批號沒有清楚的關連（第1，2，3和8組，每組1隻）。根據統計分析（Grubbs試驗），這些老鼠被摒除在進一步分析之外。表9在第28天（post II二週）老鼠的抗體反應組別疫苗劑量數量 ELISA 滴平均定度(lg) GMT 1 DENS001A4 2 μξ 11 3466 2971 2 0.4 pg 11 1283 1182 3 DENS002A4 2 Mg 11 2436 2304 4 0.4 pg 12 984 786 . 5 DENS003A4 2 12 4583 3976 6 0.4 pg 12 997 696 7 ENG2953A4/Q 2 μξ 12 3999 2882 0.4 μξ 11 737 627 -31 - 本紙張尺度適用中國國家標準(CNS) A4規格(210X 297公釐) 1311563 A7 B7 五、發明説明（29 ) 統計分析：在對數轉換post II數據之後對於抗-HBs滴定度進行差異之雙向分析，利用疫苗（4種批號）和抗原劑量（2微克和0.4微克）做爲因子。這項分析確定了在兩種抗原劑量之間可觀察到顯著的差異（P値<〇.〇〇1)，而未顯示在疫苗批號間有任何顯著的差異（P値=0.2674)。如先前所提到的該效力先經估計，並且該實驗經設計使得效力>90%的兩種調配物之間可偵測到2.5倍的GMT差異而ex誤差爲5%。異種型式的結果描述：表10顯示異種型式的再區分（IgGl，IgG2a和IgG2b)由第二次注射後（post II)之匯集血清分析而得者。 •如吾人預期的，清楚的TH2反應是由這些以礬爲基底的疫苗所謗發的，而主要是IgGl抗體被發現。就異種型式的結果描述而言，DENS批或SB Biologicals B 型肝炎單株疫苗之間未觀察到任何差異。表10第28天之匯集血清中IgG異種型式之再區分組別疫苗劑量 IgGl 異種型式(％: IgG2a IgG2b 1 DENS001A4 2 Kg 91 4 5 2 0.4 μξ 87 8 5 3 DENS002A4 97 2 1 4 0.4 pg 87 6 7 5 DENS003A4 98 1 1 6 0.4 pg 93 4 3 7 ENG2953A4/Q 2pg 88 8 4 0.4 pg 88 9 3 -32- 本紙張尺度適用中國國家標準(CNS) A4規格(210X 297公釐) 1311563 A7 B7 五、發明説明（3〇 ) 實施例3調配合併之疫苗本發明之大量抗原尤其適用於調配包含IP V之合併的疫苗。對於起始批之合併的DTPa-HBV-IPV疫苗所進行的穩定度研究指出IPV成份之效力下降，尤其是第1型的脊髓灰白質炎抗原，此係當吾人使用體外免疫檢驗（用ELISA測定D-抗原的含量）和體内老鼠效力試驗所得之結果。對於第3型抗原未觀察到任何效力減損。對於第2型抗原而言，效力減損是在吾人所期望的範圍以内（每年貯存不超過10%的減損）。吾人開始研究以測定在該合併的DTPa-HBV-IPV疫苗中效力減損的原因。從吾人對於IPV在SB Biologicals公司的 DTPa-IPV疫苗中之穩定度令人滿意之觀察來看每年儲存期其不高於10%抗原含量之損失，吾人作此結論：HBV成份很可能負責IPV在DTPa-HBV-IPV疫苗之不穩定性。用於起始DTPa-HBV-IPV調配物之HBV成份是純化過的 r_DNA，得自酵母菌的HBsAg亦用於製造SB Biologicls的B 型肝炎單價疫苗，且其如實施例1所説明的方法製備。由於第一次試圖測定哪一種HBV成份中的元素會對IPV 有害，因而分析HBsAg大量製備物是否含有乙基汞化水楊酸納。先前已發現（Davisson等人，1956年，<<臨床醫學雜誌》，第47期：8-19頁）乙基汞化水楊酸鈉用做爲DTP疫苗中之保存劑會對於DTP-IPV組合物中之「脊髓灰白質炎病毒造成損害」。這項發現受到業將乙基汞化水楊酸鈉以其他保存劑替代以調配其含IPV疫苗之疫苗製造者的考量。更 -33- 本纸張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐） 1311563 A7 B7 五、發明説明（31 ) 爲晚近，乙基汞化水楊酸鈉在長期貯在於+4°C之條件下對於IP V效力之影響再被研究。經報導第1型脊髓灰白質炎病毒抗原之效力在4-6月之後喪失到不可偵測的程度（Sawyer， L.A.等人，1994年，《疫苗》，第 12期：85 1-856頁）。利用原子吸收光譜法，大約有0.5微克汞（Hg)/每20毫克 HBsAg在根據實施例1所純化之抗原中被偵測出來。此量的录（以乙基杀化水楊酸納和氣化乙基杀乙基录化水楊酸鈉之分解產物）可降低至無法偵測的程度，在ELISA 對於培育在37°C，7天之IPV大量濃縮物中之D-抗原第1型含量的反應上是如此。已有一種方法被建立以釋出存在於HBsAg大量製備物中的汞。吾人假定汞可以結合至HBsAg顆粒上的硫醇基，因而可在還原劑存在下被釋出。當與其他還原劑進行實驗之後，L-半胱胺酸經選出做爲將汞從HBsAg顆粒釋出之用劑。在將HBsAg大量製備物對含有5.7毫莫耳濃度之L-半胱胺酸進行透析之後，未有任何汞在存留物中被測到（該試驗方法的偵定極限爲：25 ng Hg/20 pg HBsAg)。將透析過的抗原與IPV大量濃縮物混合，而第1型病毒之穩定度則藉由測量培育在37°C，7天之後的D-抗原含量未得知。以未與經半胱胺酸處理之HBsAg混合和與之混合的IPV大量濃縮物做爲對照組。參考用之ELISA滴定度係得自貯存於+2°C至+8 °C，7天的樣品。其結果摘錄於表11之中： -34- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐) 1311563 A7 B7 五、發明説明（32 ) 表11 樣品 _—— D-抗原含量(第1型)(υ 損失 7 天/4。。 7 天/37Ό IPV (未經混合的） 31.6 24.2 23% IPV+未經處理的HBsAg 31.1 18.1 42% IPV+以半胱胺酸處理的HBsAg 31.4 27.6 12% IPV+乙基果化水楊酸納(1 30.5 11.0 74% (1)以D-抗原單位（DU)表示得自這些實驗至製備物的數據清楚地證實了第1型脊髓灰白質炎病毒的穩定性會顯著增進，若HBsAg在與ιρν混合之前經半胱胺酸處理以除去殘留的求。以上所示的數據顯示在37°C培育7天以後參考用的IPV製備物損失了 23%的D-抗原含量。還確認了第1型Mahoney脊髓灰白質炎病毒固有的不穩定性，如先前所報導（|§aWyer ’1^人等人（1994年）’《疫苗>>，第12期：85 1-856頁）。雖然商業上之DTPa-HBV-IPV和 DTPa-HBV-IPV/Hib疫苗批之製備使用了透析法以5.7 mM L-半胱胺酸除去殘留的汞並保留了 IP V的穩定性，該透析法並不適用於大規模生產並且涉及一系列增補的步驟以製備不含乙基汞化水楊酸鈉或汞的HBsAg »相對的，本發明之以無乙基汞化水楊酸鈉方式製備的HBsAg可直接用於調配合併的疫苗，尤其是含有IPV者。 -35- 本纸張尺度適用中國@家棣準(CNS) A4規格(210 X 297公釐)1311563 A7 B7 V. INSTRUCTIONS (28) GMT anti-HBs Ig antibody response. Comparable antibody responses were induced by DENS and traditional hepatitis B formulations with a GMT range of 2304 and 3976 EU/ml for DNES batches compared to 2882 EU/ml for SB Biologicals hepatitis B monovalent vaccine (Engerix B) TM), results obtained at 2 μg dose; and GMT range between 696 and 1182 EU/ml for DENS batch compared to 627 EU/ml for SB Biologicals hepatitis B monovalent vaccine (Engerix BTM) at 0.4 μg dose The result obtained. • As I expected, we found a clear range of antigen doses for all formulations at 2 and 0.4 micrograms, and a 3 to 6 fold difference in GMT. • Four unreacted mice (titration <50 EU/ml) were observed to be unclearly related to the injected antigen dose or lot number (Groups 1, 2, 3 and 8, 1 in each group). According to statistical analysis (Grubbs test), these mice were excluded from further analysis. Table 9 Antibody response group dose on the 28th day (post II two weeks). Vaccine dose number ELISA Drop average (lg) GMT 1 DENS001A4 2 μξ 11 3466 2971 2 0.4 pg 11 1283 1182 3 DENS002A4 2 Mg 11 2436 2304 4 0.4 pg 12 984 786 . 5 DENS003A4 2 12 4583 3976 6 0.4 pg 12 997 696 7 ENG2953A4/Q 2 μξ 12 3999 2882 0.4 μξ 11 737 627 -31 - This paper size applies to the Chinese National Standard (CNS) A4 specification (210X 297 mm) 1311563 A7 B7 V. INSTRUCTIONS (29) Statistical analysis: Two-way analysis of differences in anti-HBs titers after logarithmically converted post II data, using vaccine (4 batches) and antigen dose (2 μg and 0.4 micrograms as a factor. This analysis determined that a significant difference was observed between the two antigen doses (P値<〇.〇〇1), and did not show any significant difference between vaccine batch numbers (P値=0.2674). This efficacy was first estimated as previously mentioned, and the experiment was designed such that a potency of >90% of the two formulations could detect a 2.5 fold difference in GMT with an ex error of 5%. Description of results for heterotypic patterns: Table 10 shows the reclassification of heterogeneous patterns (IgGl, IgG2a and IgG2b) from pooled serum analysis after post-injection (post II). • As I expected, the clear TH2 response was elicited by these sputum-based vaccines, and mainly IgG1 antibodies were found. No differences were observed between the DENS batch or the SB Biologicals Hepatitis B single vaccine for the heterologous version of the results. Table 10 Day 28 pooled serum IgG heterotypic repopulation group vaccine dose IgGl xenotype (%: IgG2a IgG2b 1 DENS001A4 2 Kg 91 4 5 2 0.4 μξ 87 8 5 3 DENS002A4 97 2 1 4 0.4 pg 87 6 7 5 DENS003A4 98 1 1 6 0.4 pg 93 4 3 7 ENG2953A4/Q 2pg 88 8 4 0.4 pg 88 9 3 -32- This paper scale applies to Chinese National Standard (CNS) A4 specification (210X 297 mm) 1311563 A7 B7 V BRIEF DESCRIPTION OF THE INVENTION (3〇) Example 3 Modulated Vaccines The large number of antigens of the present invention are particularly useful for formulating a vaccine comprising a combination of IP V. Stability studies for the combined DTPa-HBV-IPV vaccine in the initial batch It is pointed out that the efficacy of IPV components is degraded, especially the type 1 poliovirus antigen, which is the result of in vitro immunoassay (D-antigen determination by ELISA) and in vivo rat efficacy test. No potency loss was observed for the type of antigen. For type 2 antigens, the potency loss was within the range expected by us (no more than 10% impairment per year). We started the study to determine the DTP in the pool. The reason for the detriment of efficacy in the a-HBV-IPV vaccine. From the observation that the stability of IPV in SB Biologicals' DTPa-IPV vaccine is satisfactory, the annual storage period is not higher than the loss of 10% antigen content. We conclude that the HBV component is likely responsible for the instability of IPV in the DTPa-HBV-IPV vaccine. The HBV component used to initiate the DTPa-HBV-IPV formulation is purified r_DNA, which is also derived from yeast HBsAg. A hepatitis B monovalent vaccine for the manufacture of SB Biologicls, and which was prepared as described in Example 1. Since the first attempt was made to determine which element of the HBV component would be detrimental to IPV, analysis of whether the HBsAg bulk preparation contained Ethyl mercuryated salicylate. Previously discovered (Davisson et al., 1956, <<<>> Journal of Clinical Medicine, 47: 8-19) ethylmercury salicylate is used as DTP The preservative in the vaccine will cause damage to the poliovirus in the DTP-IPV composition. This finding has been replaced by the use of other inhibitors to formulate ethylmercury salicylate with other preservatives to formulate IPV-containing vaccines. Vaccine makers' considerations. More -33 - This paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1311563 A7 B7 V. Description of invention (31) For late, ethylmercury salicylate is stored at +4 °C for a long time. The effect on the effectiveness of IP V was investigated again. The efficacy of type 1 poliovirus antigens has been reported to be undetectable after April-June (Sawyer, LA et al., 1994, Vaccines, No. 12: 85 1-856) . About 0.5 micrograms of mercury (Hg) per 20 milligrams of HBsAg were detected in the antigen purified according to Example 1 by atomic absorption spectroscopy. The amount of this record (decomposed products of sodium thiosalicylate and gasified ethyl-ethyl-ethylated salicylate) can be reduced to an undetectable extent, in ELISA for incubation at 37 ° C, 7 This is the case for the D-antigen type 1 content in the large IPV concentrate. A method has been established to release mercury present in a large amount of HBsAg preparation. It is assumed that mercury can bind to the thiol group on the HBsAg particles and thus can be released in the presence of a reducing agent. After the experiment with other reducing agents, L-cysteine was selected as an agent for releasing mercury from the HBsAg particles. After dialysis of a large amount of HBsAg preparation against L-cysteine containing 5.7 millimolar concentration, no mercury was detected in the retentate (the detection limit of this test method is: 25 ng Hg/20) Pg HBsAg). The dialyzed antigen was mixed with a large amount of IPV concentrate, and the stability of the type 1 virus was measured by incubation at 37 ° C, and the D-antigen content after 7 days was not known. A large amount of IPV concentrate which was not mixed with and mixed with the cysteine-treated HBsAg was used as a control group. The reference ELISA titer was obtained from a sample stored at +2 ° C to +8 ° C for 7 days. The results are summarized in Table 11: -34- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1311563 A7 B7 V. Description of invention (32) Table 11 Sample _—— D-antigen Content (Type 1) (υ Loss 7 days / 4 days. 7 days / 37 Ό IPV (unmixed) 31.6 24.2 23% IPV + untreated HBsAg 31.1 18.1 42% IPV + cysteine treated HBsAg 31.4 27.6 12% IPV + sodium ethylated salicylate (1 30.5 11.0 74% (1) D-antigen unit (DU) data from these experiments to preparations clearly confirmed type 1 poliovirus The stability of the virus is significantly enhanced if the HBsAg is treated with cysteine prior to mixing with ιρν to remove residuals. The data shown above shows that the reference IPV preparation lost 7 days after incubation at 37 °C. % D-antigen content. The inherent instability of type 1 Mahoney poliovirus was also confirmed, as previously reported (|§aWyer '1^人等人(1994)'"Vaccine>> , No. 12: 85 1-856). Although commercial preparation of DTPa-HBV-IPV and DTPa-HBV-IPV/Hib vaccine batches The dialysis method was used to remove residual mercury with 5.7 mM L-cysteine and retained the stability of IP V. This dialysis method is not suitable for large-scale production and involves a series of supplementary steps to prepare ethyl mercury free. HBsAg of sodium salicylate or mercury. In contrast, the HBsAg prepared by the method of sodium ethylmercury-free salicylate in the present invention can be directly used for the formulation of vaccines, especially those containing IPV. -35- Paper Zhang scale applies to China@家棣准(CNS) A4 specification (210 X 297 mm)

裝訂Binding

1311563 A7 B7 五、發明説明（33 ) 4.摘要先前用於純化得自酵母菌之表面抗原的方法含有膠體滲透步驟，其中含汞的抗微生物化合物乙基汞化水揚酸鈉係包含於沖提緩衝液中以調控生物負擔。乙基汞化水楊酸納並未在接下來之方法步骤中完全清除 ’因此在純化的大量抗原中大約有1.2微克乙基汞化水楊酸鈉/每20微克蛋白質存在。爲了生產完全不含乙基汞化水楊酸鈉（汞）的大量抗原，純化方法在兩個步骤中經過更改。 •在4B膠體滲透的步驟中，將乙基汞化水楊酸鈉從沖提緩衝液中删除。 •將半胱胺酸（2 mM最終濃度）添加到得自陰離子交換層析術步驟之沖提匯集物中。同樣可以防止抗原在CsC1密度梯度離心術中發生沉殿。在生產過程中未有其他改變。以修改過之方法生產的大量抗原業經特性鑑認。物理_ 化學試驗與檢驗顯示該不含乙基汞化水楊酸鈉的抗原在性質上與先前使用之方法所生產的抗原無法區分。該抗原顆粒具有相同的組成。 HBsAg多肽之本身與完整性並不受到修改過之方法所影響’這是經由SDS-PAGE分析，使用多株抗HBsAg抗體之西方墨點法，N-端序列分析和胺基酸组成未判斷的。電子顯微術和雷射光散射分析顯示該顆粒具有吾人對得自酵母菌之HBsAg所預期的典型形式和大小。用抗酵母菌蛋白質血 -36- 本纸張尺度適用中g國家標準(CNS) A4規格(210 X 297公釐)1311563 A7 B7 V. INSTRUCTIONS (33) 4. Abstract The method previously used to purify surface antigens derived from yeast contains a colloidal infiltration step in which the mercury-containing antimicrobial compound ethylmercury salicylate is contained in the rush. Lift the buffer to regulate the biological burden. Ethyl mercuryated sodium salicylate was not completely eliminated in the next method step. Thus, approximately 1.2 micrograms of ethylmercurylated sodium salicylate per 20 micrograms of protein was present in the purified bulk antigen. In order to produce a large amount of antigen completely free of ethylmercuric salicylate (mercury), the purification method was modified in two steps. • In the 4B colloidal infiltration step, ethyl mercuryated salicylate is removed from the flushing buffer. • Add cysteine (2 mM final concentration) to the pooled fraction from the anion exchange chromatography step. It is also possible to prevent antigen from occurring in the CsC1 density gradient centrifugation. There have been no other changes in the production process. A large number of antigens produced by the modified method are characterized by characterization. Physics _ Chemical tests and tests have shown that the antigen containing no ethylmercury salicylate is indistinguishable from the antigen produced by the previously used method. The antigen particles have the same composition. The identity and integrity of the HBsAg polypeptide is not affected by the modified method. This is a Western blot method using multiple anti-HBsAg antibodies by SDS-PAGE analysis. N-terminal sequence analysis and amino acid composition are not judged. . Electron microscopy and laser light scattering analysis showed that the particles had the typical form and size expected by ours for HBsAg from yeast. Anti-yeast protein blood -36- This paper size applies to the national standard (CNS) A4 specification (210 X 297 mm)

裝訂 1311563 A7 B7 五、發明説明（34 ) 清進行西方墨點分析顯示以不含乙基汞化水楊酸鈉之方法所生產的抗原具有與非攙雜之酵母菌蛋白質相似的形式，然而，在23K移動之攙雜寬帶的量則在3種以修改過之方法生產的HBsAg批中大幅減少。免疫學分析顯示不含乙基汞化水楊酸鈉之顆粒具有增加的抗原性。該顆粒對於Abbott公司出品之AUZYME套组（含有多株抗體）更具反應性，產生之ELISA/蛋白質比率爲1.6 至2.25。此種增加之抗原性亦顯示於與保護之RF 1單株抗體的作用。大約4至7倍少量之不含乙基汞化水楊酸鈉的抗原需用於抑制RF 1結合至標準之固定抗原。無乙基汞化水楊酸鈉之抗原與傳統抗原之抑制結合曲線落在兩個截然不同的家族。這項差異也由利用表面質粒基因組合共振測量 RF1之結合親和力常數顯示出來。不含乙基汞化水楊酸鈉之製備物的結合親和力與傳統大量抗原批相較高出3倍到4 倍。該大量抗原製備物係藉由吸附到氫氧化鋁上以調配成疫苗且其不含保存劑。利用Abbott公司出品的AUZYME ELISA套套測試體外效力並且用包含乙基汞化水楊酸鈉的SB Biologicals B型肝炎單價疫苗做爲標準物，顯示其可獲得高的體外效力値。以此試驗所測得之抗原含量幾乎是所申明之20 pg蛋白質/每毫升之値的雙倍。在抑制檢驗中亦可見到由不含乙基汞化水楊酸鈉之抗原所製備之疫苗有增加的反應活性，該檢驗係以RF 1單株抗 -37- 本紙張尺度適用中國画家標準(CNS) A4規格(210X 297公釐） 1311563 A7 B7 五、發明説明（35 ) 體與固定的抗原結合。與先前使用方法純化且不含保存劑的方式調製之抗原比較，大約只需要一半量之無乙基汞化水楊酸鈉之疫苗用以產生50% RF 1與固定抗原結合的抑制作用。該不含乙基汞化水楊酸鈉之疫苗相對於RF 1之增進的抗原性與體外效力試驗（抗原含量）及在大量抗原製備物上所進行的RF 1抗體試驗結果一致。老鼠的致免疫性試驗是利用初次與相隔兩週的加強接種，並使用2微克和0.4微克的抗原進行的。在第28天，即加強接種後的第14天對老鼠採血。分析該血清之抗體滴定度及異種型式的組成。吾人觀察到對於所施用之兩種劑量有清楚的抗原劑量效應，但是就抗體滴定度（GMT)而言，不含乙基汞化水楊酸鈉和不含保持劑的疫苗之前並沒有統計學上顯著的反應差異。在異種型式的結果描述方面未觀察到重大的差異。 -38- 本紙張尺度適用中國國家標準(CNS) A4規格(210X 297公釐) 1311563Binding 1311563 A7 B7 V. Description of invention (34) Western blot analysis shows that the antigen produced by the method without ethylmercury salicylate has a similar form to the non-noisy yeast protein, however, The amount of noisy broadband for 23K mobile is significantly reduced in three batches of HBsAg produced by modified methods. Immunological analysis showed that particles containing no ethylmercury salicylate had increased antigenicity. The granules are more responsive to Abbott's AUZYME kit (containing multiple antibodies) and produce an ELISA/protein ratio of 1.6 to 2.25. This increased antigenicity is also shown to interact with the protected RF 1 monoclonal antibody. Approximately 4 to 7 times the small amount of antigen containing no ethylmercury salicylate is required to inhibit RF 1 binding to standard immobilized antigens. The inhibition binding curves of anti-ethylmercured sodium salicylate antigen to traditional antigens fall in two distinct families. This difference was also revealed by the binding affinity constant of RF1 measured by the surface plasmid gene combination resonance. The binding affinities of the preparations containing no ethylmercury salicylate are three to four times higher than the conventional bulk antigen phase. The bulk antigen preparation is formulated into a vaccine by adsorption onto aluminum hydroxide and is free of preservatives. The in vitro potency was tested using the AUZYME ELISA kit from Abbott and the SB Biologicals Hepatitis B monovalent vaccine containing ethylmercury salicylate was used as a standard and showed high in vitro potency. The antigen content measured by this test is almost double that of the stated 20 pg protein/ml. In the inhibition test, it was also observed that the vaccine prepared from the antigen containing no ethylmercury-sodium salicylate had an increased reactivity. The test was applied to the Chinese painter standard on the scale of RF 1 monoclonal anti-37- paper ( CNS) A4 size (210X 297 mm) 1311563 A7 B7 V. Description of the invention (35) The body binds to the immobilized antigen. Approximately half of the vaccine containing no ethylmercuric salicylate is required to produce a 50% inhibition of the binding of RF 1 to the immobilized antigen as compared to the antigen prepared by the previously purified method without the preservative. The enhanced toxicity of the vaccine containing no ethylmercury salicylate relative to RF 1 is consistent with the in vitro potency test (antigen content) and the RF 1 antibody test results performed on a large number of antigen preparations. The mouse immunogenicity test was performed using a booster vaccination for the first time and two weeks apart, using 2 micrograms and 0.4 micrograms of antigen. On day 28, the mice were bled on day 14 after the booster vaccination. The antibody titer of the serum and the composition of the heterotypic pattern were analyzed. I have observed a clear antigenic dose effect for the two doses administered, but in terms of antibody titer (GMT), there is no statistical analysis before the ethanol-free sodium salicylate and the vaccine without the retainer. Significant differences in response. No significant differences were observed in the description of the results of the heterogeneous patterns. -38- This paper size is applicable to China National Standard (CNS) A4 specification (210X 297 mm) 1311563

97.10. 〇2 申請曰期 ———_ 090123365 案號 90.09.21 類別〇ηκ'%^ 以上各搁由本局填註）、$|_名稱 "A4 C4 中文說明書替換頁(97年1〇月）專利説明書文中 ---—, 英文97.10. 〇2 Application deadline -__ 090123365 Case number 90.09.21 Category 〇ηκ'%^ Each of these is filled by this Bureau), $|_Name"A4 C4 Chinese manual replacement page (97 years 1 month) ) Patent Description ---, English

製備不含微量乙基汞化水楊酸納之安定免疫性B型肝炎疫苗的方法 METHOD FOR PRODUCING A STABLE, IMMUNOGENIC HEPATITIS B VACCINE WITHOUT TRACE OF THIOMERSAL 1·寇恩笛海德 KOEN DE HEYDER 2.彼德斯索 PETER SCHU 3.米奇爾斯雷敦尼 MICHELLE SERANTONI 4.歐姆斯汎歐普斯塔 1.3.4.均比利時 2.德國 OMERVANOPSTAL 均比利時利克森沙特市第一學院路89號參裝訂國籍比利時商史密斯克萊美占生物公司 SMITHKLINE BEECHAM BIOLOGICALS S. A. 比利時線申請人 f事務居$ 比利時利克森沙特市第一學院路89號詹恩史帝芬 JEAN STEPHENNE 72889-971002.doc 297公釐）本紙張尺歧财弟UW 123:5W领;寻利甲請茶 13 1 1 5β®文說明書替換頁(98年2月）五、發明説明（8 9a 2. is A7 年月R> B7 . 補充Method for preparing a stable immunological hepatitis B vaccine containing no traces of ethylmercury salicylate METHOD FOR PRODUCING A STABLE, IMMUNOGENIC HEPATITIS B VACCINE WITHOUT TRACE OF THIOMERSAL 1. 寇恩笛海德KOEN DE HEYDER 2. Petersso PETER SCHU 3. Mitchell Leeden MICHELLE SERANTONI 4. Oms Pan Opusta 1.3.4. Both Belgium 2. Germany OMERVANOPSTAL All of the first college road in Lexen, Belgium, No. 89, ginseng, nationality, Belgian, Smith, Clay Accounting company SMITHKLINE BEECHAM BIOLOGICALS SA Belgian line applicant f affairs resides at No. 89, First College Road, Lexen, Saudi Arabia, JEAN STEPHENNE 72889-971002.doc 297 mm) This paper ruler is UW 123:5W Collar; seek for Lijia please tea 13 1 1 5β® manual replacement page (February 1998) V. Invention description (8 9a 2. is A7 year R> B7. Supplement

Scand»，第1 8期：3 49頁（1977年）之中揭示。3D-MPL可得自Ribi immunochem，位於美國，並且揭示於英國專利申請案第222021 1號及美國專利案第49 12094號。QS21係揭示於美國專利案第5057540號。本發明由以下實例解說，但並不受限於彼，其中：圖1說明Engerix B™之無乙基汞化水揚酸鈉生產法；圖2說明大量抗原批之SDS-PAGE分析（銀采：每一樣品1微克蛋白質）；且圖3說明在以無乙基汞化水楊酸鈉生產之大量抗原化之中的殘留酵母菌蛋白質（以免子抗母菌蛋白質血清進行西方墨點法）。實施例1 : g采化水攝_^存在下之B型肝炎袅面抗原生產法將SB Bi〇l〇gicals公司出品的B型肝炎單價疫苗（Engerix B™)之B型肝炎表面抗原（HBsAg)在酵母菌 cerevisiae中表現為重組蛋白質（請參考Harf〇rd等人_ £11，）。24 kD之蛋白質在細胞内表現並且累積在重組的酵母菌細胞中。在醱酵末層，將酵母菌細胞予以收獲並且在溫和的表面活性劑如Tween 20存在下將之打破以釋出所需的蛋白質。緊接著將含有可溶性表面抗原顆粒的細胞均質物以一系列沉澱予以錢化，然後經由超過濾法予以濃縮。重組抗原之進-步純化是在緊接的層析法分離之中進行 :二f第“ 一步驟中將粗製的抗原濃縮物以膠體滲透層析法在Sepharose 4B基質上處理。乙基汞化 4B膠體滲透層析步驟的沖摇物吸奶仔在於以下"、，乂液中。該冲提緩衝液具有乂下成伤.iomMTris，5%乙二醇，pH7〇, 5〇毫克/公升 72889.980213.doc -11-Scand», Issue 18: 3, 49 pages (1977). 3D-MPL is available from Ribi immunochem, in the United States, and is disclosed in British Patent Application No. 2,221,021 and U.S. Patent No. 49,129,094. QS21 is disclosed in U.S. Patent No. 5,057,540. The present invention is illustrated by the following examples, but is not limited thereto, wherein: Figure 1 illustrates the Engerix BTM-free ethylmercury salicylate production process; Figure 2 illustrates the SDS-PAGE analysis of a large number of antigenic batches (silver mining) : 1 μg of protein per sample); and Figure 3 illustrates residual yeast protein in a large amount of antigenization produced by sodium-free mercury-sodium salicylate (to prevent Western anti-fungal protein serum from Western blotting) . Example 1: Production of Hepatitis B Surface Antigen in the Presence of g-Capitalized Water _^ Hepatitis B Surface Antigen (HBsAg) of Hepatitis B Monovalent Vaccine (Engerix BTM) produced by SB Bi〇l〇gicals ) expressed as a recombinant protein in the yeast cerevisiae (please refer to Harf〇rd et al. _ £11,). The 24 kD protein is expressed intracellularly and accumulated in recombinant yeast cells. At the end of the fermentation layer, the yeast cells are harvested and disrupted in the presence of a mild surfactant such as Tween 20 to release the desired protein. The cell homogenate containing the soluble surface antigen particles is then subjected to a series of precipitation and then concentrated by ultrafiltration. The further step-by-step purification of the recombinant antigen is carried out in the next chromatographic separation: "f" "The crude antigen concentrate is treated in a step by colloidal permeation chromatography on a Sepharose 4B substrate. Ethylmercury. The 4B colloidal osmosis chromatography step of the irrigating sucker is in the following ", sputum. The rinsing buffer has underarm injury. iomMTris, 5% ethylene glycol, pH 7 〇, 5 〇 mg / liter 72889.980213.doc -11-

A7 年 B7A7 years B7

牙rUyUlZJJODm*寻孑1J甲I育系 1311568文說明書替換頁(98年2月）五、發明説明（13 ) 釋液體的原子吸收經測量做為空白組。樣品的汞含量則經由得自標準溶液的校正曲線予以計算。結果則以微克汞/每 20微克蛋白質表示。 1.3不含乙基汞化水楊酸鈉之大量抗原製造大量抗原之純化過程的步驟顯示於圖1之中。 1.4不含己基汞化水楊酸鈉之條件下調配之疫苗組成無保存劑且從不含乙基汞化水楊酸鈉之過程製備的抗原所調配出來的B型肝炎疫苗之典型定量組成提供於表1之中。表1 : 成份每毫升中含量活性成份-至少95%為HBsAg的蛋白質 20微克氫氧化鋁(吸附劑） 0.95毫克 (以ai2o3表示）氯化納 9.0毫克(最多）磷酸二鈉鹽之二水合物 0.98毫克礙酸二氫鈉鹽之二水合物 0.71毫克注射用水q.s. ad. 1.0毫升該組成可藉由添加3D-MP.L和/或其他佐劑而予變化。 2.以無乙基汞化水楊酸鈉過程生產之大量抗原與疫苗的特徵鑑認 2.1.純化之大量抗原的試驗與檢驗 2.1.1比較之基礎根據本實施例（實施例1.2)以無乙基汞化水楊酸鈉法製備 72889-980213.doc - 16 - 本紙張尺度適用中國國家標準(CNS) A4規格(210X 297公釐) 98. 2. 13 A7 年月日 B7 修正補充弗uyuiz：ub：>现寻利靖茶 131 156®文說明書替換頁(98年2月）五、發明説明（15 ) 10’ 1酿ning，德國慕尼黑市)的褒置和試劑以門檻法測量 DNA含量。在試驗與核驗中發現的值係在利用乙基汞化水楊酸納於 Sepharose 4B膠體滲透步驟之汁提緩衝液中所製造的大量抗原批所見到的範圍内’以EUSA測得之抗原活性則除外。三項HEF製備物的這項測量值（1 63_2 25)要高於對大量抗原批HEP2055所觀察到者，後者之eusa/蛋白質比率為 1.13。用AUSZYME套組對含有乙基汞化水楊酸納之大量抗原批進行測量所得的ELISA/蛋白質比率一般而言約為1〇 ’且在0.8-1.2的範圍.内’絕少超過1,4。 2.1.3 SDS-PAGE膠體分析大量抗原之製備物是在還原條件下以SDS_pAGE檢驗並以Coomassieblue予以染色。所有樣品均顯示在24κ有一主要寬帶而帶有少量r聚體蛋白冑。該樣品經麟具有高純度（>99%純度），此乃以沒有可見之挽雜蛋白質之寬帶而得知。大量抗原製備物的樣品（1微克）係以8〇81>八(3£在還原和非還原條件下進行分析並予銀染（辨_圖2) ^在還原條件下，該樣品顯示在24K移動的深密寬帶，其帶有少量的二聚物和多聚物形式。該膠體形式與做為比較者的HEP2〇55之形式無法區別。該樣品亦在非還原條件下檢驗。在這些條件下較少物質在24K移動，而移動在二聚體和多聚體位置的多肽量則增加。不含乙基汞化水揚酸鈉之大量抗原批顯示其較之比較者HEP2055批具有略為高度的聚合作用。 72889-980213.doc _ 18 _ ^紙張尺度適用中國國家揉準(CMS) A4規格(21〇X 297公董] —--- 弟WU123365鈮寻利曱請茱弟WU123365鈮寻利曱請茱 A7 年月 B7Tooth rUyUlZJJODm* search for 1J A I breeding system 1311568 manual replacement page (February 1998) V. Description of invention (13) The atomic absorption of liquid is measured as a blank group. The mercury content of the sample is calculated from the calibration curve obtained from the standard solution. The results are expressed in micrograms of mercury per 20 micrograms of protein. 1.3 Preparation of a large amount of antigen without ethylmercury salicylate The steps of the purification process for a large number of antigens are shown in Fig. 1. 1.4 Vaccine composition without hexylmercuric sodium salicylate The typical quantitative composition of the hepatitis B vaccine formulated without the preservative and prepared from the antigen prepared without ethylmercury salicylate In Table 1. Table 1: Ingredients per ml of active ingredient - at least 95% protein of HBsAg 20 micrograms of aluminum hydroxide (adsorbent) 0.95 mg (expressed as ai2o3) sodium chloride 9.0 mg (maximum) diammonium phosphate dihydrate 0.98 mg dihydrogen sodium dihydrochloride salt dihydrate 0.71 mg water for injection qs ad. 1.0 ml This composition can be varied by adding 3D-MP.L and/or other adjuvants. 2. Identification of a large number of antigens and vaccines produced by the process of ethyl-free mercury-sodium salicylate 2.1. Testing and testing of purified large amounts of antigens 2.1.1 Basis of comparison According to this example (Example 1.2) Preparation of ethylmercury salicylate 72889-980213.doc - 16 - This paper scale is applicable to China National Standard (CNS) A4 specification (210X 297 mm) 98. 2. 13 A7 day and day B7 amendment supplement Fuyuiz :ub:> Now looking for Lijing tea 131 156® manual replacement page (February 1998) V. Invention description (15) 10' 1 brewing ning, Munich, Germany) The measurement and reagents are measured by the threshold method. content. The values found in the tests and tests are within the range seen by a large number of antigen batches prepared by using ethylmercury salicylic acid in the juice extraction buffer of the Sepharose 4B colloidal permeation step. 'Antigen activity measured by EUSA. Except for. This measurement of the three HEF preparations (1 63_2 25) was higher than that observed for the large batch of anti-HEP2055, which had an eusa/protein ratio of 1.13. The ELISA/protein ratio measured with a large number of antigenic batches containing ethylmercurylated salicylate in the AUSZYME kit is generally about 1 〇' and in the range of 0.8-1.2. It is rarely more than 1,4. . 2.1.3 SDS-PAGE Colloidal Analysis A large amount of antigen preparation was tested by SDS_pAGE under reducing conditions and stained with Coomassieblue. All samples showed a major broadband at 24 k with a small amount of r-mer peptone. The sample has a high degree of purity (> 99% purity), which is known as the broad band of no visible complexed proteins. A large amount of antigen preparation sample (1 μg) was analyzed by 8〇81>8 (3 £ under reducing and non-reducing conditions and silver stained (discrimination_Fig. 2) ^ Under reducing conditions, the sample was shown at 24K Moving deep dense broadband with a small amount of dimer and polymer form. The colloidal form is indistinguishable from the comparative HEP2〇55 form. The sample is also tested under non-reducing conditions. Less material moves at 24K, while the amount of polypeptide that moves at the dimer and multimer sites increases. A large number of antigen batches that do not contain ethylmercury salicylate show a slight height compared to the comparator HEP2055 batch. 72889-980213.doc _ 18 _ ^ Paper scale applies to China National Standard (CMS) A4 specifications (21〇X 297 DON)]---- Brother WU123365 铌曱曱曱 W 123 123 123 123茱Please 茱A7 year B7

1311563文說明書替換頁(98年2月）五、發明説明（16 ) 24K多肽的身份以Coomassie blue或銀染透露者係採用兔子抗原生質HBsAg培養的多株抗體進行西方墨點法來確認。該大量抗原製備物顯示在24K有一主要寬帶，以及帶有二聚體和三聚體的形式。該技術透露少數微量之表面抗原蛋白質的分解產物。大量之以無乙基汞化水楊酸鈉過程製備的抗原與HEP2055批無異。殘留之酵母菌蛋白質的存在與否係以SDS-PAGE分析在還原條件下，並以兔子對抗酵母菌蛋白質培育之多株抗血清進行西方墨點術來檢驗（倒-圖3)。該技術是定性的且不容許用來定量雜質。三種以無乙基汞化水楊酸鈉製備的大量抗原批顯示一種固定的寬帶形式，而HEP205 5批則例外。在HEP2055大量抗原中出現於±23K的重染寬帶在三種 HEF製備物中實質上不存在。西方墨點法顯示無乙基汞化水楊酸鈉之純化過程能產生較純的抗原產物。本紙張尺度適用中國國家標準(CNS) Α4規格(210 X 297公釐)1311563 Instruction Manual Replacement Page (February 1998) V. INSTRUCTIONS (16) The identity of the 24K polypeptide was identified by Coomassie blue or silver staining using a plurality of antibodies cultured in rabbit antigen-producing HBsAg for Western blotting. The bulk antigen preparation showed a major broad band at 24K, as well as a form with dimers and trimers. This technology reveals a small amount of a breakdown of surface antigenic proteins. A large number of antigens prepared by the process of ethyl-free mercury-sodium salicylate are no different from HEP 2055 batches. The presence or absence of residual yeast protein was examined by SDS-PAGE under reducing conditions and Western blotting was performed on rabbit anti-serum against yeast protein culture (pour-Fig. 3). This technique is qualitative and should not be used to quantify impurities. Three large batches of antigen prepared with ethyl-free mercuryated salicylate showed a fixed broadband form, with the exception of HEP205 5 batches. The heavily stained broadband appearing at ±23K in the HEP 2055 bulk antigen was essentially absent in the three HEF preparations. Western dot method shows no ethylmercury. The purification process of sodium salicylate produces a relatively pure antigenic product. This paper scale applies to the Chinese National Standard (CNS) Α4 specification (210 X 297 mm)

Claims

13 1 15系(3〇123365號專利申請案j、-·、中文申請專利範圍替換本(9^Τδ1Τ 申請專利範固 V8 38 V8 D8 1. -，製備不含微量乙基汞化水楊酸鈉之安定免疫性B型肝炎疫苗的方法，該方法包含·· ⑷在酵母菌SaeeharGmyees㈣⑽心中表現B型肝炎表面抗原之重組蛋白； ⑻處理該酵母細胞以產结製抗原製備物；膠體滲透層析處理該粗製抗原製備物，其中於該谬體滲透層析中使収冲提衫含乙基汞化水楊酸納；物⑷以離子交換層析處理步驟⑷所得含有抗原之冲提 ⑷將半胱胺酸加人步驟⑷所得含有抗原之冲提物中. (f)使步驟（e)所得製備物超離心；及 =)使純化之B型肝炎表面抗原與醫藥上可接受賦形劑、-Ό σ，以產生安定免疫性B型肝炎疫苗；其中，所得疫苗中不加入乙基汞化水楊酸納。 2.根據申請專利範圍第1頂&古、1 祀固弟1項的方法，其中該半胱胺酸係添加至"於1-10 mM的最終濃度。 3·根據申請專利範圍筮9 n μ 士、i «. 5 “。方法，其中該半胱胺酸係添加至大約2 mM的最終濃度。 4·根據申請專利範圍第1岑2^ 化铯超離心。項的方法’其中該超離心係氣 5. =申請專利範圍第_項的方法，其中該離子交換層析為陰離子交換層析。 6. 根據申請專利範圍第1啖2 佐劑併用。或2項的方法’其中該疫苗另與一種 72889-971002.doc ' 1 - I紙張尺度適财S _準_;格(21GX^^ 、申請專利範圍的方法，其中該佐劑是一種鋁鹽的方法，其中該佐劑為氫氧化鋁根據申請專利範圍第6項〇根據申請專利範圍第7項或磷酸銘。 9. 根據申睛專利範圍第佐劑。 6項的方法，其中該佐劑為TH-1誘發 Μ艮據中β專利範圍第9項的方法’丨中該誘發佐劑為 3D MPL ’ QS21 ’ 3D-MPL 和 QS21，或 CpG寡核嘗酸。 U·根據申請專利範圍第9項的方法，其中該佐劑為3D_mpl 及一種鋁鹽。 -2 - 72889-971002.doc 本紙張尺度適用中國國家標準(CNS) A4规格(210X297夂D13 1 15 series (3〇123365 Patent Application j,-·, Chinese Patent Application Substitution Replacement (9^Τδ1Τ Patent Application Fan Gu V8 38 V8 D8 1. -, Preparation of Trace Alumino-Free Mercury Salicylic Acid A method for neutralizing an immunological hepatitis B vaccine comprising: (4) expressing a recombinant protein of hepatitis B surface antigen in the heart of yeast Saeehar Gmyees (4) (10); (8) treating the yeast cell to produce a prepared antigen preparation; colloidal permeation chromatography Treating the crude antigen preparation, wherein in the corpuscular permeation chromatography, the thief containing the ethylmercury salicylate is contained; the substance (4) is subjected to the ion exchange chromatography step (4), and the antigen-containing scouring (4) is half The cystine is added to the extract containing the antigen obtained in the step (4). (f) the ultracentrifugation of the preparation obtained in the step (e); and =) the purified hepatitis B surface antigen and the pharmaceutically acceptable excipient, - Ό σ to produce a stable immune hepatitis B vaccine; wherein, the obtained vaccine does not contain ethylmercury salicylate. 2. According to the scope of the patent application, the first top & ancient, 1 祀固弟1 Method wherein the cysteine is added " at a final concentration of 1-10 mM. 3. According to the patent application scope 筮9 n μ, i «. 5". The method wherein the cysteine is added to a final concentration of about 2 mM. Patent application No. 1 岑 2 铯铯铯。。。。其中其中其中其中其中其中其中其中其中其中超超超超超超超超超超超超超超超超超超超超超超超超超超超Patent application No. 1 啖 2 adjuvants used together. or 2 methods 'The vaccine is another with a 72889-971002.doc ' 1 - I paper scale suitable for S _ quasi- _; grid (21GX ^ ^, patent application scope The method wherein the adjuvant is an aluminum salt method, wherein the adjuvant is aluminum hydroxide according to item 6 of the patent application scope, according to item 7 of the patent application or phosphoric acid. 9. According to the scope of the patent application The method of the sixth item, wherein the adjuvant is a method of the third aspect of the beta patent range in the TH-1-induced sputum, wherein the evoked adjuvant is 3D MPL 'QS21 '3D-MPL and QS21, or CpG oligo Nuclear acid. U. According to the method of claim 9 of the patent scope, The adjuvant is an aluminum salt 3D_mpl and -2 -. 72889-971002.doc this paper scale applicable Chinese National Standard (CNS) A4 size (210X297 Fan D