TWI585101B - Method for preparing antibody f(ab')2 fragments - Google Patents
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本發明係關於一種製備抗體F(ab’)2片段之方法,尤指一種使用陽離子交換樹脂進行純化,且無需使用硫酸銨或硫酸鈉沉澱,以獲得高回收率、高純度之抗體F(ab’)2片段的製備方法。 The present invention relates to a method for preparing an antibody F(ab') 2 fragment, in particular to a method using a cation exchange resin for purification without precipitation with ammonium sulfate or sodium sulfate to obtain a high recovery, high purity antibody F (ab ') Preparation method of 2 fragments.
由於抗體可專一性連結抗原之特性,目前已有許多抗體應用於治療之用途,例如應用於對抗傳染疾病(例如,細菌、病毒、寄生蟲等感染)、癌症、藥物或是其他異物之治療。在抗體之結構中,以IgG為例,經由胃蛋白酶(Pepsin)之水解作用可將抗體分為F(ab’)2及Fc兩部分,F(ab’)2以雙硫鍵結合兩個抗原結合部位,分子量約為110 kDa。 Since antibodies can specifically bind to the characteristics of antigens, many antibodies have been used for therapeutic purposes, such as treatment against infectious diseases (eg, infections such as bacteria, viruses, parasites, etc.), cancer, drugs, or other foreign bodies. In the structure of the antibody to IgG as an example, antibodies can be divided into F (ab ') 2 and Fc portions of two, F (ab') via hydrolysis with pepsin (Pepsin) of 2 disulfide bonds to two antigen The binding site has a molecular weight of approximately 110 kDa.
由於F(ab’)2片段移除Fc部分,故可免除抗體與細胞(如巨噬細胞、樹突狀細胞、中性粒細胞、NK細胞和B細胞)的Fc受體的之間的非專一性結合且由於其分子較小,更能有效地滲透組織,此外,由於移除Fc部分,做為檢測時可不受到抗Fc抗體引起的檢測干擾,應用在檢測上有更高的靈敏度,因此在安全性及效能上較IgG更好。 Since the F(ab') 2 fragment removes the Fc portion, it is possible to eliminate the non-existence between the antibody and the Fc receptor of cells such as macrophages, dendritic cells, neutrophils, NK cells, and B cells. It has a specific sensitivity and is more effective in infiltrating the tissue due to its smaller molecule. In addition, since the Fc portion is removed, it can be detected without interference from the anti-Fc antibody, and the application has higher sensitivity in detection. Better than IgG in terms of safety and efficacy.
生產抗體F(ab’)2片段用於治療用途,往往涉及許多步驟目的在保持其有效性,同時藉由純化以減少副作用的發生率和嚴重性。這些步驟包括硫酸銨或硫酸鈉沉澱、酶消化、熱凝、滲濾,近來更用到離子交換及親和性管柱層析。其中,較精緻的方法為自血清中純化抗體,續經蛋白酶水解產生F(ab’)2片段,再經沉澱法及管柱層析法,將F(ab’)2片段與Fc水解物及蛋白酶分離。也有自血清先以蛋白酶水解再以分段的硫酸銨或硫酸鈉沉澱加上層析管柱分析。這些製備的步驟 可得到有效及安全的F(ab’)2產品,但製備的成本相當高。此外,製程中如用到硫酸銨或硫酸鈉沉澱的步驟,將相當費時且回收率損失相當高,造成製造成本大幅增加。為了達到避免硫酸銨沉澱步驟之目的,已有相關研究應用Q-Sepharose管柱加上切流過濾系統(Tangential flow diafiltration)來純化F(ab’)2,可無需使用硫酸銨沉澱(Jones et al.,Journal of Immunological Methods(2003)275(1-2),239-250)。但此純化方法施作時需監控水解的狀態,使抗血清的水解產物除F(ab’)2外,其他的成分都小於13 kDa才能達到純化的目的。此條件除了容易造成過度水解外,也可能只能在特定血清樣品才能施作,其報告中所用的樣品,IgG為其血清蛋白質的主要成分,含量大於其他蛋白質(含白蛋白)成分的總和。 The production of antibody F(ab') 2 fragments for therapeutic use often involves many steps in maintaining their effectiveness while at the same time reducing the incidence and severity of side effects by purification. These steps include ammonium sulfate or sodium sulfate precipitation, enzymatic digestion, thermocoagulation, diafiltration, and more recently ion exchange and affinity column chromatography. Among them, a more delicate method is to purify the antibody from the serum, continue to be hydrolyzed by protease to produce the F(ab') 2 fragment, and then the F(ab') 2 fragment and the Fc hydrolyzate by precipitation and column chromatography. Protease separation. There are also self-serums which are first hydrolyzed by proteases and then precipitated by fractional ammonium sulfate or sodium sulfate plus chromatography column analysis. These preparative steps result in an effective and safe F(ab') 2 product, but the cost of preparation is quite high. In addition, the step of precipitating ammonium sulfate or sodium sulfate in the process will be quite time consuming and the recovery rate is relatively high, resulting in a substantial increase in manufacturing costs. In order to avoid the ammonium sulfate precipitation step, the related research has applied Q-Sepharose column plus Tangential flow diafiltration to purify F(ab') 2 without the use of ammonium sulfate precipitation (Jones et al . Journal of Immunological Methods (2003) 275 (1-2), 239-250). However, when the purification method is applied, the state of hydrolysis needs to be monitored, so that the hydrolysis product of the antiserum except F(ab') 2 and other components are less than 13 kDa for purification purposes. In addition to being prone to excessive hydrolysis, this condition may only be applied to specific serum samples. The sample used in the report, IgG is the main component of serum protein, and the content is greater than the sum of other protein (albumin-containing) components.
因此,仍需持續改善製備抗體F(ab’)2片段之方法,以縮短製備時間、降低製備成本及提高純化回收率,以獲得高純度及品質之抗體F(ab’)2片段。 Therefore, there is still a need to continuously improve the method of preparing the antibody F(ab') 2 fragment to shorten the preparation time, reduce the preparation cost, and improve the purification recovery rate to obtain a high purity and quality antibody F(ab') 2 fragment.
本發明提供製備與純化抗體F(ab’)2片段之方法,以及使用該方法所製備之包含抗體F(ab’)2片段之醫藥組合物。 The present invention provides a method of preparing and purifying an antibody F(ab') 2 fragment, and a pharmaceutical composition comprising the antibody F(ab') 2 fragment prepared using the method.
在一方面,本發明提供一種製備抗體F(ab’)2片段之方法,其包含下列步驟:(1)將抗體以胃蛋白酶(Pepsin)水解以獲得抗體F(ab’)2片段;(2)將抗體F(ab’)2片段以過濾系統進行濃縮並置換緩衝液,其中緩衝液之置換使抗體F(ab’)2片段帶有正電荷;以及(3)將抗體F(ab’)2片段以陽離子交換管柱進行純化,並以鹽類水溶液置換經純化之抗體F(ab’)2片段。其中經純化之抗體F(ab’)2片段不含有血清白蛋白、完整抗體分子及抗體Fc片段。 In one aspect, the invention provides a method of preparing an antibody F(ab') 2 fragment, comprising the steps of: (1) hydrolyzing an antibody with pepsin to obtain an antibody F(ab') 2 fragment; The antibody F(ab') 2 fragment is concentrated in a filtration system and replaced with a buffer wherein the replacement of the buffer causes the antibody F(ab') 2 fragment to have a positive charge; and (3) the antibody F(ab') The 2 fragment was purified by a cation exchange column and the purified antibody F(ab') 2 fragment was replaced with a saline solution. The purified antibody F(ab') 2 fragment does not contain serum albumin, intact antibody molecules and antibody Fc fragments.
較佳地,將經純化之抗體F(ab’)2片段可以第二過濾系統 進行濃縮並依使用需求置換第二緩衝液。 Preferably, the purified antibody F(ab') 2 fragment can be concentrated by a second filtration system and the second buffer replaced with the requirements of use.
在本發明之一具體實施例中,該過濾系統可為切向流過濾系統(Tangential Flow Filtration System)。 In one embodiment of the invention, the filtration system can be a Tangential Flow Filtration System.
在本發明之一具體實施例中,該緩衝液之pH值小於該抗體F(ab’)2片段之等電點(pI)值,例如,該緩衝液可為pH 4.3之醋酸鈉溶液。 In one embodiment of the invention, the pH of the buffer is less than the isoelectric point (pI) value of the F(ab') 2 fragment of the antibody, for example, the buffer may be a sodium acetate solution at pH 4.3.
在本發明之一具體實施例中,該鹽類水溶液可為濃度約0.1 M至約2 M之氯化鈉(NaCl)水溶液。 In one embodiment of the invention, the aqueous salt solution can be a sodium chloride (NaCl) aqueous solution having a concentration of from about 0.1 M to about 2 M.
在本發明之一具體實施例中,該第二緩衝液可為生理食鹽水。 In a specific embodiment of the invention, the second buffer may be physiological saline.
在本發明之一具體實施例中,該第二過濾系統可為切向流過濾系統(Tangential Flow Filtration System)。 In one embodiment of the invention, the second filtration system can be a Tangential Flow Filtration System.
在本發明之一具體實施例中,該抗體可為免疫球蛋白(immunoglobulin),例如,IgG、IgA或ÍgM。 In a particular embodiment of the invention, the antibody can be an immunoglobulin, for example, IgG, IgA or ÍgM.
另一方面,本發明提供一種包含抗體F(ab’)2片段之組合物,其中該抗體F(ab’)2片段係由本發明所述之方法所製得,且該組合物不含有血清白蛋白、完整抗體分子及抗體Fc片段。 In another aspect, the present invention provides a composition comprising an antibody F(ab') 2 fragment, wherein the antibody F(ab') 2 fragment is produced by the method of the present invention, and the composition does not contain serum white Protein, intact antibody molecule and antibody Fc fragment.
又一方面,本發明亦提供一種包含抗體F(ab’)2片段之醫藥組合物,伴隨一或多種藥學上可接受之載劑,其中該抗體F(ab’)2片段係由本發明所述之方法所製得,且該醫藥組合物不含有血清白蛋白、完整抗體分子及抗體Fc片段。 In still another aspect, the present invention also provides a pharmaceutical composition comprising an antibody F(ab') 2 fragment, accompanied by one or more pharmaceutically acceptable carriers, wherein the antibody F(ab') 2 fragment is described in the present invention The method is prepared, and the pharmaceutical composition does not contain serum albumin, intact antibody molecules, and antibody Fc fragments.
本發明之此等及其他態樣將可由下文之較佳具體實例敘述並結合下述圖式時明顯可見,但可在不偏離本揭示內容新穎概念之精神及範圍的情形下對其進行變化及修飾。 These and other aspects of the present invention will be apparent from the following description of the preferred embodiments of the invention and Modification.
除非另外定義,本文中所用之所有技術及科學辭彙具皆有熟習本文所屬技藝者所通常明瞭之相同意義。如有衝突,則以本文件,包括其定義為主。 All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art, unless otherwise defined. In case of conflict, this document, including its definition, is the main one.
當用於此處時,本文所使用冠詞「一」或「該」意指該冠詞文法上之受詞為一或一以上(亦即至少為一)。舉例而言,「一元件」代表一元件或多於一元件。 As used herein, the articles "a" or "the" are used to mean that the grammatically accepted words are one or more (ie, at least one). For example, "an element" means one element or more than one element.
本文所用之術語「大約」、「約」、或「近似」一般而言應意謂指定數值或範圍之20%以內,較佳者為10%以內,且更佳者為5%以內。本文所給予之數量皆為近似值,意謂術語「大約」、「約」、或「近似」如未明確陳述亦可推論。 The terms "about", "about", or "approximately" as used herein shall generally mean within 20% of the specified value or range, preferably within 10%, and more preferably within 5%. The quantities given herein are approximate, meaning that the terms "about", "about", or "approximately" may also be inferred if not explicitly stated.
抗體,亦稱為免疫球蛋白,其係存在於個體之血清中並為免疫系統之一部分,藉由與特定抗原之結合與中和,並誘導後續之免疫反應以達到保護個體免於外來物質,例如病原體,入侵之功能。由於抗原與抗體之間特異性結合之特性,目前在產業上已有各種應用,例如,檢驗、偵測、預防及治療疾病等。在目前已知的抗體中,例如,IgG、ÍgM、IgA、IgE,以IgG在血液中的含量最高,且由於IgG為成熟免疫反應之產物因此應用廣泛。 An antibody, also known as an immunoglobulin, which is present in the serum of an individual and is part of the immune system, by binding to a specific antigen and neutralizing it, and inducing a subsequent immune response to protect the individual from foreign substances, For example, pathogens, invasive functions. Due to the specific binding properties between antigen and antibody, there are various applications in the industry, such as testing, detecting, preventing and treating diseases. Among the currently known antibodies, for example, IgG, ÍgM, IgA, IgE, IgG is the highest in blood, and IgG is widely used as a product of a mature immune reaction.
當以酵素分解IgG時,依據使用之酵素可獲得不同之產物,例如,當使用木瓜酵素(papain)時,IgG會分解成一個Fc片段(crystallizing fragment)及兩個Fab片段(antigen-binding fragment),若使用胃蛋白酶(pepsin)時,則可獲得一個F(ab’)2片段及一個Fc片段。Fab片段及F(ab’)2片段仍保留可特異性結合抗原的特性,而F(ab’)2片段更具有沉澱抗原之功能。相對於此,Fc片段則扮演訊號標記的角色,吸引並活化巨噬細胞、嗜中性白血球等而辨識並吞噬抗原抗體複合物。此外,當異種之抗體進入個體中時,Fc片段因其抗原決定部位之特定序列會被視為外來物質,故誘發嚴重免 疫反應而造成副作用,因此目前在產業利用上多將抗體以酵素處理以去除Fc片段、保留Fab片段及F(ab’)2片段,且由於Fab片段及F(ab’)2片段之分子較小,更能進入有效地滲入組織提高功效。 When IgG is decomposed by an enzyme, different products can be obtained depending on the enzyme used. For example, when using papain, IgG is decomposed into an Fc fragment (crystallizing fragment) and two Fab fragments (antigen-binding fragment). If pepsin is used, an F(ab') 2 fragment and an Fc fragment can be obtained. The Fab fragment and the F(ab') 2 fragment still retain the property of specifically binding to the antigen, while the F(ab') 2 fragment has a function of precipitating the antigen. In contrast, the Fc fragment plays the role of a signal marker, attracting and activating macrophages, neutrophils, etc. to recognize and phagocytose antigen-antibody complexes. In addition, when a heterologous antibody enters an individual, the Fc fragment is regarded as a foreign substance due to a specific sequence of its epitope, thereby inducing a serious immune reaction and causing side effects. Therefore, the antibody is currently treated with an enzyme in industrial utilization. The Fc fragment, the Fab fragment and the F(ab') 2 fragment are removed, and since the molecules of the Fab fragment and the F(ab') 2 fragment are smaller, it is more effective to infiltrate into the tissue to improve the efficacy.
目前產業上針對F(ab’)2片段製備方法,大部分是先純化抗體後再以蛋白酶水解將Fc切除,再以親和性管柱或其他特性的層析法將產物分離。此類的方法操作的風險較低,但過程較長且花費驚人,一般只用在研究實驗室的小量製備。而應用於大量製備的製程時,多以胃蛋白酶直水解血清或血漿,再以硫酸銨沉澱將F(ab’)2片段和其他的水解產物做粗分離,但此步驟是個耗時的工作,一般沉澱、固液分離及沉澱物的再溶解透析,最少須2個工作天,再者固液分離在大量操作時需要的設備和濾膜耗材也是很大的花費。而且硫酸銨沉澱中F(ab’)2片段的純度和回收率是個兩難的抉擇,工業上常用的13%硫酸銨可得到較純的F(ab’)2片段但其回收率低於50%,45%硫酸銨可將F(ab’)2片段幾乎都沉澱下來但無法與其餘蛋白質分離。此外,以硫酸銨沉澱做粗分離,仍需要經陰離子交換樹脂做進一步的純化。 At present, most of the preparation methods for F(ab') 2 fragments in the industry are to first purify the antibody and then excise the Fc by protease hydrolysis, and then separate the product by affinity column or other characteristic chromatography. Such methods operate at a lower risk, but the process is longer and costly and is generally only used in small batches in research laboratories. When applied to a large-scale preparation process, the p(ab') 2 fragment and other hydrolysates are mostly separated by pepsin, and the F(ab') 2 fragment is coarsely separated by ammonium sulfate precipitation, but this step is a time-consuming task. Generally, precipitation, solid-liquid separation and re-dissolution dialysis of sediments take at least 2 working days, and the equipment and filter consumables required for large-scale operation in solid-liquid separation are also very expensive. Moreover, the purity and recovery of the F(ab') 2 fragment in ammonium sulfate precipitation is a dilemma. The 13% ammonium sulfate commonly used in the industry can obtain a relatively pure F(ab') 2 fragment but the recovery rate is less than 50%. 45% ammonium sulfate precipitates the F(ab') 2 fragment almost but cannot be separated from the rest of the protein. In addition, the crude separation with ammonium sulfate precipitation still requires further purification by anion exchange resin.
為了克服硫酸銨沉澱的兩難困境,Jones和Landon已報導將血清或血漿中蛋白質水解為小分子胜肽,再以透析的方式分離F(ab’)2片段(Jones et al.,Journal of Immunological Methods(2003)275(1-2),239-250)。然於此方法中,需持續監控胃蛋白酶之水解反應,並具有兩個反應終點要觀察,其一為抗體需水解為F(ab’)2,但過度的水解會使F(ab’)2產率降低甚至失去活性;另一需控制血清或血漿中的其他蛋白質都水解為小分子胜肽(13 kDa),這兩個反應終點很難同時達到,因此在產業上使用仍有其侷限性。 In order to overcome the dilemma of ammonium sulfate precipitation, Jones and Landon have reported the hydrolysis of protein in serum or plasma to small peptides, and then dialysis to isolate F(ab') 2 fragments (Jones et al., Journal of Immunological Methods) (2003) 275 (1-2), 239-250). However, in this method, it is necessary to continuously monitor the hydrolysis reaction of pepsin, and have two reaction endpoints to be observed, one of which is that the antibody needs to be hydrolyzed to F(ab') 2 , but excessive hydrolysis causes F(ab') 2 The yield is reduced or even inactivated; the other is to control the hydrolysis of other proteins in serum or plasma into small peptides (13 kDa), which are difficult to achieve at the same time, so there are still limitations in industrial use. .
有鑑於此,本發明提供一種製備抗體F(ab’)2片段之方法,其包含下列步驟:(1)將抗體以胃蛋白酶(Pepsin)水解 以獲得抗體F(ab’)2片段;(2)將抗體F(ab’)2片段以過濾系統進行濃縮並置換緩衝液,其中緩衝液之置換使抗體F(ab’)2片段帶有正電荷;以及(3)將抗體F(ab’)2片段以陽離子交換管柱進行純化,並以鹽類水溶液置換經純化之抗體F(ab’)2片段。其中經純化之抗體F(ab’)2片段不含有血清白蛋白、完整抗體分子及抗體Fc片段。較佳地,經純化之抗體F(ab’)2片段可以第二過濾系統進行濃縮並依使用需求置換第二緩衝液。如此,藉由上述方法不但可克服硫酸銨沉澱步驟所造成之低回收率或低純度,更可縮短製備時間、降低製備成本及提高純化回收率,進而獲得高純度及品質之抗體F(ab’)2片段。 In view of the above, the present invention provides a method for preparing an antibody F(ab') 2 fragment, which comprises the steps of: (1) hydrolyzing an antibody with pepsin to obtain an antibody F(ab') 2 fragment; The antibody F(ab') 2 fragment is concentrated in a filtration system and replaced with a buffer wherein the replacement of the buffer causes the antibody F(ab') 2 fragment to have a positive charge; and (3) the antibody F(ab') The 2 fragment was purified by a cation exchange column and the purified antibody F(ab') 2 fragment was replaced with a saline solution. The purified antibody F(ab') 2 fragment does not contain serum albumin, intact antibody molecules and antibody Fc fragments. Preferably, the purified antibody F(ab') 2 fragment can be concentrated by a second filtration system and replaced with a second buffer as needed. Thus, the above method can not only overcome the low recovery rate or low purity caused by the ammonium sulfate precipitation step, but also shorten the preparation time, reduce the preparation cost, and improve the purification recovery rate, thereby obtaining the high purity and quality antibody F(ab'. ) 2 fragments.
本文所用之術語「抗體」係指完整抗體,「抗體片段」係指完整抗體中包含F(ab’)2片段之抗原結合片段,其仍保有與抗原特異性結合之功能而可替代完整抗體之功能。在一實施例中,抗體與抗體片段可由重組DNA技術製得,在另一實施例中,抗體與抗體片段可由自然來源獲得,其中抗體片段亦可由完整抗體經化學或酵素作用而獲得。 The term "antibody" as used herein refers to an intact antibody, and "antibody fragment" refers to an antigen-binding fragment comprising an F(ab') 2 fragment in an intact antibody, which still retains the function of specifically binding to an antigen and can replace the intact antibody. Features. In one embodiment, the antibody and antibody fragments can be made by recombinant DNA techniques. In another embodiment, the antibody and antibody fragments can be obtained from natural sources, wherein the antibody fragments can also be obtained by chemical or enzymatic action of intact antibodies.
使抗體水解而獲得F(ab’)2片段之胃蛋白酶有效量可由所屬技術領域中具有通常知識者依所需水解之抗體含量、胃蛋白酶之活性加以決定,在本發明之具體實施例中,胃蛋白酶之最終濃度為0.05%(w/v)。 The pepsin effective amount obtained by hydrolyzing the antibody to obtain the F(ab') 2 fragment can be determined by the amount of the antibody which is hydrolyzed by a person having ordinary knowledge in the art, and the activity of pepsin. In a specific embodiment of the present invention, The final concentration of pepsin was 0.05% (w/v).
在本發明之具體實施例中,使抗體F(ab’)2片段帶有正電荷之緩衝液係為酸性,例如,具有介於約2.0至6.0之pH值,較佳為介於3.0至5.0,所屬技術領域中具有通常知識者可理解任何可使抗體F(ab’)2片段帶有正電荷之緩衝系統皆可應用,該緩衝系統包含但不限於,醋酸、檸檬酸、磷酸、2-嗎啉乙烷磺酸(2-morpholinoethanesulfonic acid,MES)。 In a particular embodiment of the invention, the buffer having a positive charge on the F(ab') 2 fragment of the antibody is acidic, for example, having a pH of between about 2.0 and 6.0, preferably between 3.0 and 5.0. Anyone skilled in the art will appreciate that any buffer system that can positively charge an F(ab') 2 fragment of an antibody can be used, including but not limited to acetic acid, citric acid, phosphoric acid, 2- 2-morpholinoethanesulfonic acid (MES).
另一方面,本發明提供一種包含抗體F(ab’)2片段之組合物,其中該抗體F(ab’)2片段係由本發明所述之方法所製得, 且該組合物不含有血清白蛋白、完整抗體分子及抗體Fc片段。 In another aspect, the present invention provides a composition comprising an antibody F(ab') 2 fragment, wherein the antibody F(ab') 2 fragment is produced by the method of the present invention, and the composition does not contain serum white Protein, intact antibody molecule and antibody Fc fragment.
其中該組合物更可包含使抗體F(ab’)2片段半衰期延長之載體,例如但不限於線型高分子聚合物(如,PEG)、支鏈高分子聚合物、脂質、膽固醇、醣類、寡糖、以及合成或自然生成之蛋白質、聚肽、胜肽。 Wherein the composition may further comprise a carrier for prolonging the half-life of the F(ab') 2 fragment of the antibody, such as but not limited to a linear polymer (eg, PEG), a branched polymer, a lipid, cholesterol, a saccharide, Oligosaccharides, as well as synthetic or naturally occurring proteins, peptides, peptides.
又一方面,本發明亦提供一種包含抗體F(ab’)2片段之醫藥組合物,伴隨一或多種藥學上可接受之載劑,其中該抗體F(ab’)2片段係由本發明所述之方法所製得,且該醫藥組合物不含有血清白蛋白、完整抗體分子及抗體Fc片段。 In still another aspect, the present invention also provides a pharmaceutical composition comprising an antibody F(ab') 2 fragment, accompanied by one or more pharmaceutically acceptable carriers, wherein the antibody F(ab') 2 fragment is described in the present invention The method is prepared, and the pharmaceutical composition does not contain serum albumin, intact antibody molecules, and antibody Fc fragments.
在預防及治療之應用上,本發明之抗體F(ab’)2片段或其衍生物可與藥學上可接受載劑調配成醫藥組合物。此處所使用之「藥學上可接受」意指該載劑係與包含於該組合物中之活性成分相容,較佳能穩定該活性成分而不會對投予該醫藥組合物之對象產生傷害。該載劑可為該活性成分之稀釋劑、載體、賦形劑或介質。適合載劑之實例包含生理相容緩衝液,如漢克氏溶液、林格氏溶液、生理食鹽水緩衝液、乳糖、右旋葡萄糖、蔗糖、山梨醇、甘露醇、澱粉、阿拉伯膠、磷酸鈣、海藻膠、黃耆膠、明膠、矽酸鈣、微晶型纖維素、聚乙烯咯啶酮、纖維素、無菌水、糖漿及甲基纖維素。該醫藥組合物可額外包含潤滑劑,例如,滑石、硬脂酸鎂及礦物油;潤濕劑;乳化與懸浮劑;保存劑,例如,甲基-及丙基-羥基苯甲酸鹽;甜味劑;以及調味劑。 For use in prophylaxis and therapy, the F(ab') 2 fragment of the invention or a derivative thereof can be formulated into a pharmaceutical composition with a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" as used herein means that the carrier is compatible with the active ingredient contained in the composition, preferably to stabilize the active ingredient without causing harm to the subject to which the pharmaceutical composition is administered. The carrier can be a diluent, carrier, excipient or vehicle for the active ingredient. Examples of suitable carriers include physiologically compatible buffers such as Hank's solution, Ringer's solution, physiological saline buffer, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate , seaweed gum, tragacanth, gelatin, calcium citrate, microcrystalline cellulose, polyvinyl bromo ketone, cellulose, sterile water, syrup and methyl cellulose. The pharmaceutical composition may additionally comprise a lubricant, for example, talc, magnesium stearate and mineral oil; a wetting agent; an emulsifying and suspending agent; a preservative, for example, methyl- and propyl-hydroxybenzoate; sweet Flavor; and flavoring agent.
根據本發明之醫藥組合物可為片狀、藥丸、粉末、錠劑、囊袋、藥包、藥酒、懸浮液、乳化液、溶液、糖漿、軟明膠膠囊與硬明膠膠囊、栓劑、無菌注射溶液及經包裝之粉末之形式。 The pharmaceutical composition according to the present invention may be in the form of tablets, pills, powders, tablets, pouches, sachets, medicinal liquors, suspensions, emulsions, solutions, syrups, soft gelatin capsules and hard gelatin capsules, suppositories, sterile injectable solutions. And the form of the packaged powder.
本發明之醫藥組合物可經由任何生理可接受途徑傳送。此些途徑包含但不限於非經口服投藥、系統性投藥、口服投 藥、鼻腔投藥、直腸投藥、腹腔注射、血管注射、皮下注射、經皮投藥、吸入投藥及肌肉注射。 The pharmaceutical compositions of the invention can be delivered via any physiologically acceptable route. Such routes include, but are not limited to, non-oral administration, systemic administration, oral administration Medicine, nasal administration, rectal administration, intraperitoneal injection, vascular injection, subcutaneous injection, transdermal administration, inhalation administration, and intramuscular injection.
本發明係藉由下列範例進一步說明,此僅提供而用於展現而非限制之目的。由於本揭露,本領域具有通常知識者應可理解所揭露之特定具體實施例,並對該些具體實施例進行諸多修改而獲得相似或類似的結果而仍未脫離本發明之精神與範疇。 The invention is further illustrated by the following examples, which are provided for purposes of illustration and not limitation. It will be apparent to those skilled in the art that <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt;
以犬瘟熱病毒(Canine distemper virus,CDV)免疫豬隻,並藉由增強(boost)免疫提高豬隻血液之中和抗體力價。採集豬隻血液樣品,若需求為血漿,將血液樣品緩慢注入至含3.2%檸檬酸鈉溶液之採血袋至最終體積比為9:1,輕搖數分鐘使血液與抗凝血劑混合。若需求為血清,則將血液樣品注入無菌試管內,傾斜放置10-30分鐘,俟血液凝固後收集以免溶血影響後續分析。將上述樣品經離心以分離上清液與固形物,分別獲得之上清液為血漿樣品或血清樣品。 Pigs were immunized with Canine distemper virus (CDV) and boosted by the booster immunization of the pigs. Pig blood samples were collected. If the plasma was required, the blood sample was slowly injected into a blood collection bag containing 3.2% sodium citrate solution to a final volume ratio of 9:1, and the blood was mixed with the anticoagulant by shaking for several minutes. If the demand is serum, the blood sample is injected into a sterile test tube, placed obliquely for 10-30 minutes, and the blood is coagulated and collected to avoid hemolysis affecting subsequent analysis. The above sample was centrifuged to separate the supernatant and the solid, and the supernatant was obtained as a plasma sample or a serum sample, respectively.
經由上述步驟所獲得之含有抗CDV抗體之血清或血漿先於58℃加熱30分鐘以使病毒去活化,接著進行胃蛋白酶水解或-80℃冷凍保存備用。 The serum or plasma containing the anti-CDV antibody obtained through the above procedure was heated at 58 ° C for 30 minutes to deactivate the virus, followed by pepsin hydrolysis or cryopreservation at -80 ° C until use.
於進行胃蛋白酶水解反應前,取1 L含有抗CDV抗體之血清或血漿加入等體積之超純水進行稀釋,接著加入預先溶於0.1 N HCl之10%胃蛋白酶(SIGMA-ALDRICH)至最終濃度為0.05%(w/v),隨後以5N HCl緩緩注入上述溶液中以調 整pH值至約pH 3.2±0.1。將含有胃蛋白酶之豬血清(漿)溶液置於37℃下反應2小時進行水解反應,而後將溶液移置4℃冰箱中以中止反應待後續透析。 Before performing the pepsin hydrolysis reaction, 1 L of serum or plasma containing anti-CDV antibody is added to an equal volume of ultrapure water for dilution, followed by 10% pepsin (SIGMA-ALDRICH) previously dissolved in 0.1 N HCl to the final concentration. 0.05% (w/v), then slowly infused with 5N HCl into the above solution to adjust The pH is adjusted to about pH 3.2 ± 0.1. The pepsin-containing pig serum (pulp) solution was subjected to a hydrolysis reaction at 37 ° C for 2 hours, and then the solution was placed in a refrigerator at 4 ° C to terminate the reaction for subsequent dialysis.
經胃蛋白酶水解之豬血清(漿)溶液接著以切向流過濾系統(Tangential Flow Filtration Systems)進行濃縮及緩衝液置換,切向流過濾系統於此處使用孔徑為50 kD,膜面積為0.11 m2的膜匣(GE Healthcare Life Sciences;Kvick Lab Cassette-UFELA0050010ST)。豬血清(漿)溶液經濃縮至50%體積後,接續以10倍體積的50 mM醋酸鈉溶液(pH4.3)進行透析,持續監控透析後的血清(漿)溶液樣品,至導電度與50 mM醋酸鈉溶液相近(低於5 ms/cm)後停止。收集的溶液接著以0.22 μm濾膜進行無菌過濾。 The pepsin-hydrolyzed porcine serum (plasma) solution is then concentrated and buffer-replaced using a Tangential Flow Filtration System, where the tangential flow filtration system uses a pore size of 50 kD and a membrane area of 0.11 m. Membrane of 2 (GE Healthcare Life Sciences; Kvick Lab Cassette-UFELA0050010ST). The porcine serum (pulp) solution was concentrated to 50% by volume, and then dialyzed against 10 volumes of 50 mM sodium acetate solution (pH 4.3), and the serum (plasma) solution sample after dialysis was continuously monitored until the conductivity was 50. Stop after mM sodium acetate solution is similar (less than 5 ms/cm). The collected solution was then sterile filtered through a 0.22 μm filter.
陽離子交換管柱SP-Sepharose FF(50 x 125 mm)先以50 mM醋酸鈉溶液(pH 4.3)平衡,將前述經胃蛋白酶水解並經透析以濃縮與置換緩衝液之豬血清(漿)溶液通入管柱,接續以50mM醋酸鈉溶液(pH4.3)沖洗管柱以洗出未結合物,持續監測洗出液於280 nm之吸光值,待接近管柱平衡時之吸光值作為終止判定。再分別以含0.2 M NaCl之50mM醋酸鈉溶液(pH4.3)沖洗出所要的F(ab’)2部分和以含1 M NaCl之50mM醋酸鈉溶液(pH4.3)沖洗出雜蛋白,管柱最終以0.1M的NaOH作在位清洗(CIP)以重複使用。 The cation exchange column SP-Sepharose FF (50 x 125 mm) was first equilibrated with 50 mM sodium acetate solution (pH 4.3), and the aforementioned pepsin was hydrolyzed and dialyzed to concentrate the porcine serum (plasma) solution with the replacement buffer. After entering the column, the column was washed with 50 mM sodium acetate solution (pH 4.3) to wash out the unbound, and the absorbance of the eluate at 280 nm was continuously monitored, and the absorbance near the column balance was judged as termination. Then, the desired F(ab') 2 fraction was washed out with a 50 mM sodium acetate solution (pH 4.3) containing 0.2 M NaCl, and the heteroprotein was washed out with a 50 mM sodium acetate solution (pH 4.3) containing 1 M NaCl. The column was finally re-used with 0.1 M NaOH for in situ cleaning (CIP).
收集陽離子交換管柱0.2 M NaCl之洗出溶液,接著以切向流過濾系統(Tangential Flow Filtration Systems)進行濃縮與置換緩衝液,切向流過濾系統於此處使用孔徑為50 kD,膜面積為0.11 m2的膜匣(GE Healthcare Life Sciences;Kvick Lab Cassette-UFELA0050010ST)。0.2 M NaCl洗出液經濃縮至50%體積後,接續以10倍體積的注射用生理食鹽水進行透析。收集的溶液接著以0.22 μm濾膜進行無菌過濾。 The cation exchange column 0.2 M NaCl elution solution was collected, followed by a tangential flow filtration system (Tangential Flow Filtration Systems) for concentration and displacement buffer. The tangential flow filtration system used here a pore size of 50 kD, and the membrane area was 0.11 m 2 of membrane enthalpy (GE Healthcare Life Sciences; Kvick Lab Cassette-UFELA 0050010ST). After the 0.2 M NaCl eluate was concentrated to 50% by volume, it was dialyzed against 10 volumes of physiological saline for injection. The collected solution was then sterile filtered through a 0.22 μm filter.
為測定抗體F(ab’)2片段中和病毒之力價,以50 μl的CDV病毒(約200 TCID50)與50 μl不同稀釋倍數的樣品混合反應1小時,再將混合液加入預先培養的B95-8細胞株,觀察是否產生細胞病變(CPE),以不產生細胞病變的最高稀釋倍數作為樣品的中和抗體力價。樣品為血清或血漿時需先置於56℃反應30分鐘使補體去活化後再進行試驗。 To determine the antibody valence of the antibody F(ab') 2 fragment, 50 μl of CDV virus (about 200 TCID 50 ) was mixed with 50 μl of different dilutions for 1 hour, and the mixture was added to the preculture. B95-8 cell line was observed for cytopathic effect (CPE), and the highest dilution factor that did not produce cytopathic effects was used as the neutralizing antibody titer of the sample. When the sample is serum or plasma, it needs to be placed at 56 ° C for 30 minutes to deactivate the complement before testing.
將含有豬隻抗CDV抗體經胃蛋白酶水解以獲得F(ab’)2片段,並經切向流過濾系統透析以濃縮與置換緩衝液後,帶正電之F(ab’)片段進一步以陽離子交換樹脂進行分離與純化,其分離純化層析圖如圖1所示,其中標示為unbound之波峰代表未與管柱結合之物質,而後分別以0.2 M與1.0之NaCl沖洗管柱以將結合管柱之物質置換洗出,最後以0.1 M NaOH清洗管柱。 The porcine anti-CDV antibody is hydrolyzed by pepsin to obtain the F(ab') 2 fragment, and after dialysis by a tangential flow filtration system to concentrate and displace the buffer, the positively charged F(ab') fragment is further cationized. The exchange resin is separated and purified. The chromatogram of the separation and purification is shown in Fig. 1. The peak marked as unbound represents the substance not combined with the column, and then the column is washed with 0.2 M and 1.0 NaCl respectively to bond the tube. The column material was washed out and finally the column was washed with 0.1 M NaOH.
收集上述各階段之洗出液並以SDS-PAGE進行分析。SDS-PAGE經考馬斯藍染色後之結果如圖2所示,第1道顯示未經水解與純化之含有豬抗CDV抗體之血漿,其中PSA代表豬血清白蛋白,IgG抗體之分子量約為135 kDa;第2道係為前述血漿經胃蛋白酶水解後之產物,各蛋白質之分子量顯著降低;第3道係為前述水解產物經50 kDa超過濾膜透析置換 緩衝液之結果,相較於使用超過濾膜透析前,蛋白質組成分布與第2道並無差異,顯示超過濾膜透析並無法獲得分離純化之效果;第4道則係為陽離子交換管柱之過濾液;最後則為陽離子交換管柱經0.2 M NaCl沖洗管柱之洗出物,可觀察到經純化之抗體F(ab’)2片段,蛋白質純化程度顯著增加。 The eluate of each of the above stages was collected and analyzed by SDS-PAGE. The results of SDS-PAGE staining with Coomassie blue are shown in Fig. 2. The first lane shows the unhydrolyzed and purified plasma containing porcine anti-CDV antibody, wherein PSA represents porcine serum albumin, and the molecular weight of IgG antibody is about 135 kDa; the second channel is the product of the above-mentioned plasma pepsin hydrolysis, the molecular weight of each protein is significantly reduced; the third channel is the result of the above hydrolysis product through 50 kDa ultrafiltration membrane dialysis displacement buffer, compared with the use Before the ultrafiltration membrane was dialyzed, the protein composition distribution did not differ from that of the second lane, indicating that the ultrafiltration membrane dialysis could not obtain the separation and purification effect; the fourth lane was the cation exchange column filter; the last was the cation exchange column. After washing the column eluted with 0.2 M NaCl, the purified antibody F(ab') 2 fragment was observed, and the degree of protein purification was significantly increased.
接著以西方墨點分析確認純化物,此處使用兔子抗豬隻IgG F(ab’)2片段之抗體(購自NOVUS)偵測,其結果如圖3所示,其中第1道為含有豬抗CDV抗體之血漿經胃蛋白酶水解之產物,第2道為前述經水解之血漿以陽離子交換樹脂分離純化後以0.2 M NaCl沖提之產物,第3道則為以陽離子交換樹脂分離純化後以0.5 M NaCl沖提之產物。三者均於100 kDa處測得訊號,與預期IgG抗體F(ab’)2片段之分子量相當,。 The purified material was then confirmed by Western blot analysis, and the rabbit anti-porcine IgG F(ab') 2 fragment antibody (purchased from NOVUS) was used here, and the results are shown in Fig. 3, wherein the first lane contains pigs. The anti-CDV antibody plasma is hydrolyzed by pepsin. The second channel is the product of the above hydrolyzed plasma which is separated and purified by cation exchange resin and then extracted with 0.2 M NaCl, and the third channel is separated and purified by cation exchange resin to 0.5. The product eluted by M NaCl. All three measured the signal at 100 kDa, which is equivalent to the molecular weight of the expected IgG antibody F(ab') 2 fragment.
進一步計算F(ab’)2片段產物之回收率,於純化前以1000 ml、中和力價為2700倍之血漿為原料,經上述F(ab’)2片段產製過程後,可製得380 ml、蛋白質濃度7.9 mg/ml、中和力價大於6400倍之F(ab’)2產物,以中和力價計算其回收率大於90%。 Further calculating the recovery rate of the F(ab') 2 fragment product, which is obtained by using 1000 ml of plasma with a neutralization price of 2700 times before purification, and the above F(ab') 2 fragment production process can be obtained. The 380 ml, protein concentration 7.9 mg/ml, and the F(ab') 2 product with a neutralization price greater than 6400 times, the recovery rate is greater than 90% at the neutralization price.
豬隻因個體差異及飼養環境的不同,造成免疫反應亦不相同,以CDV感染豬隻為例,將3批次免疫血清之樣品以SDS-PAGE進行分析並以考馬斯藍(commassive brilliant blue)染色,其結果如圖4所示。其中雖然注入分析之總蛋白質含量相等,但可觀察到豬血清白蛋白(PSA)之含量差異不大,但抗體(IgG)之含量則有很大差異。然而,本發明之製備方法可適用於不同濃度之樣品,如圖5所示,經免疫之豬隻血清以及經本發明之方法純化之產物以SDS-PAGE進行分析並以考馬斯藍(commassive brilliant blue)染色,其中第1道所 示之IgG含量比例雖然遠高於第2圖之第1道所示之IgG含量比例,然而於高濃度IgG血清(血漿)樣品中,抗體F(ab’)2片段之回收率仍可大於90%。 Pigs have different immune responses due to individual differences and feeding environment. Taking CDV-infected pigs as an example, samples of 3 batches of immune serum were analyzed by SDS-PAGE and Comass blue (commassive brilliant blue) ) Dyeing, the results of which are shown in Figure 4. Although the total protein content of the injection analysis was equal, it was observed that the content of porcine serum albumin (PSA) was not much different, but the content of antibody (IgG) was greatly different. However, the preparation method of the present invention can be applied to samples of different concentrations, as shown in Fig. 5, the immunized pig serum and the product purified by the method of the present invention are analyzed by SDS-PAGE and coomass blue (commassive brilliant) Blue) staining, in which the ratio of IgG content shown in the first lane is much higher than the ratio of IgG content shown in the first lane of Fig. 2, but in the high concentration IgG serum (plasma) sample, the antibody F(ab') The recovery of 2 fragments can still be greater than 90%.
F(ab’)2片段產物經序列稀释後進行中和抗體力價測試,在稀釋6400倍的樣品中觀察不到細胞病變,而在12800倍稀释的樣品中觀察到細胞病變,將此樣品的中和抗體力價訂為6400倍。 The F(ab') 2 fragment product was subjected to serial dilution and subjected to neutralization antibody titer test. No cell lesion was observed in the diluted 6400-fold sample, and cytopathic effect was observed in the 12800-fold diluted sample. The neutralizing antibody price was set at 6400 times.
咸信本發明所屬技藝中具一般知識者基於本文之敘述,無須進一步之例示即可將本發明應用至其最廣泛之範圍。因此,應了解此處所提供之敘述及申請專利範圍係供例示目的而非以任何方式限制本發明之範疇。 The present invention may be applied to its broadest scope without further elaboration, based on the description of the present invention. Therefore, it is to be understood that the claims and claims are not intended to limit the scope of the invention.
圖1顯示以陽離子交換樹脂進行抗體F(ab’)2片段之分離與純化的層析圖,其中標示為unbound之波峰代表未與管柱結合之物質,而後分別以0.2 M與1.0之NaCl,以及0.1 M NaOH沖提管柱。。 Figure 1 shows a chromatogram of the separation and purification of antibody F(ab') 2 fragments with a cation exchange resin, wherein the unlabeled peaks represent substances that are not bound to the column, and then 0.2 M and 1.0 NaCl, respectively. And 0.1 M NaOH flushing column. .
圖2顯示抗體F(ab’)2片段製備過程中各階段之產物以SDS-PAGE進行分析並以考馬斯藍(commassive brilliant blue)染色之結果圖。M:分子量標記;1:未經水解與純化之含有豬抗CDV抗體之血漿,其中PSA代表豬血清白蛋白,IgG抗體之分子量約為135 kDa;2:前述血漿經胃蛋白酶水解後之產物;3:前述水解產物經50 kDa超過濾膜進行透析、置換緩衝液之產物,相較於使用超過濾膜透析前,蛋白質組成分布並無差異,顯示超過濾膜透析並無法獲得分離純化之效果;4:陽離子交換管柱之過濾液;5:陽離子交換管柱經0.2 M NaCl沖提管柱之洗出物,可觀察到經分離純化之抗體F(ab’)2片段。 Figure 2 is a graph showing the results of SDS-PAGE analysis of products at various stages in the preparation of antibody F(ab') 2 fragments and staining with comass blue. M : molecular weight marker; 1 : unhydrolyzed and purified plasma containing porcine anti-CDV antibody, wherein PSA represents porcine serum albumin, IgG antibody has a molecular weight of about 135 kDa; 2 : product of the aforementioned plasma by pepsin hydrolysis; 3 : The hydrolyzate was subjected to dialysis and displacement of the buffer product through a 50 kDa ultrafiltration membrane. There was no difference in protein composition distribution before dialysis using an ultrafiltration membrane, indicating that the ultrafiltration membrane dialysis could not obtain the effect of separation and purification; 4 : Filter of the cation exchange column; 5 : The cation exchange column was washed with 0.2 M NaCl to elute the column, and the isolated and purified antibody F(ab') 2 fragment was observed.
圖3顯示以西方墨點分析確認抗體F(ab’)2片段之存在與純化程度。此處使用兔子抗豬隻IgG F(ab’)2片段之抗體(NOVUS)為初級抗體用以偵測。1:含有豬抗CDV抗體之血漿經胃蛋白酶水解之產物;2:前述經水解之血漿以陽離子交換樹脂純化後0.2 M NaCl之沖提產物;3:前述經水解之血漿以陽離子交換樹脂純化後0.5 M NaCl之沖提產物。 Figure 3 shows the presence and degree of purification of antibody F(ab') 2 fragments by Western blot analysis. Here, a rabbit anti-porcine IgG F(ab') 2 fragment antibody (NOVUS) was used as a primary antibody for detection. 1 : a product containing pepsin hydrolyzed by a plasma containing a porcine anti-CDV antibody; 2 : a product of the above-mentioned hydrolyzed plasma purified by a cation exchange resin and 0.2 M NaCl; 3 : after the above-mentioned hydrolyzed plasma is purified by a cation exchange resin The product was eluted with 0.5 M NaCl.
圖4顯示經CDV感染之不同批次豬隻的血清樣品分析結果,將總蛋白質含量相等之血清樣品以SDS-PAGE進行分析並以考馬斯藍(commassive brilliant blue)染色。M:分子量標記;1、2、3:不同批次豬隻的血清樣品。 Figure 4 shows the results of serum sample analysis of different batches of pigs infected with CDV. Serum samples with equal total protein content were analyzed by SDS-PAGE and stained with comassive brilliant blue. M : molecular weight marker; 1, 2, 3 : serum samples from different batches of pigs.
圖5顯示本發明之抗體F(ab’)2片段製備方法可適用於高濃度IgG之血清樣品。經免疫之豬隻血清以及經純化之產物以SDS-PAGE進行分析並以考馬斯藍(commassive brilliant blue)染色。M:分子量標記;1:未經水解與純化之含有豬抗CDV抗體之血漿,其中PSA代表豬血清白蛋白,IgG抗體之分子量約為135 kDa;2:前述血漿經胃蛋白酶水解及陽離子交換樹脂純化後之產物。於高濃度IgG血清(血漿)樣品中,抗體F(ab’)2片段之回收率仍可大於90%。 Figure 5 shows that the preparation method of the antibody F(ab') 2 fragment of the present invention can be applied to serum samples of high concentration IgG. The immunized pig serum and the purified product were analyzed by SDS-PAGE and stained with comassive brilliant blue. M : molecular weight marker; 1 : unhydrolyzed and purified plasma containing porcine anti-CDV antibody, wherein PSA represents porcine serum albumin, IgG antibody has a molecular weight of about 135 kDa; 2 : the aforementioned plasma is subjected to pepsin hydrolysis and cation exchange resin The product after purification. The recovery of antibody F(ab') 2 fragments can still be greater than 90% in high concentration IgG serum (plasma) samples.
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