技术领域Technical Field
本发明属于抗体组合物领域，具体地讲可用作用于治疗或诊断用途的药物制剂的组合物。The present invention is in the field of antibody compositions, in particular compositions that can be used as pharmaceutical preparations for therapeutic or diagnostic use.
背景技术Background Art
近年来，基于抗体的治疗因为对多种疾病诸如癌症和自身免疫性疾病的有效治疗方案，结合抗体工程和生产技术的许多新发展而走到前沿。In recent years, antibody-based therapeutics have come to the forefront due to effective treatment options for a variety of diseases such as cancer and autoimmune diseases, combined with many new developments in antibody engineering and production technologies.
与抗体制剂和输送相关联的主要挑战之一是维持抗体治疗的稳定性和形式，尤其是对于长期储存和运输而言。抗体，与其它类型的基于蛋白质的治疗相同，容易受到应力条件下物理和化学不稳定性的影响，应力条件诸如来自冻融或运输期间的温度变化、暴露于光、氧气或化学品/溶剂、剪切应力和pH应力。One of the major challenges associated with antibody formulation and delivery is maintaining the stability and form of antibody therapeutics, especially for long-term storage and transport. Antibodies, like other types of protein-based therapeutics, are susceptible to physical and chemical instability under stress conditions such as temperature changes from freeze-thaw or during transport, exposure to light, oxygen or chemicals/solvents, shear stress, and pH stress.
它们可经历变性(例如，丧失三级和/或二级结构)或相互作用以形成聚集体。虽然抗体聚集体可以液体和固体状态出现，但其在液体制剂中，尤其是在高浓度抗体下尤其是有问题的。抗体在相对高剂量下通常是治疗有效的。因此在药物制剂中通常期望高抗体浓度，以使剂量体积最小化并使给药更患者友好(例如，皮下注射而不是静脉滴注、减少注射次数)。In some embodiments, the present invention relates to the antibody of the present invention. Antibody can be used for the treatment of solid or liquid phase. Antibody can be used for the treatment of solid or liquid phase. Antibody can be used for the treatment of solid or liquid phase. They can undergo denaturation (for example, loss of tertiary and/or secondary structure) or interact to form aggregates. Although antibody aggregates can occur in liquid and solid states, they are especially problematic in liquid preparations, especially under high concentration antibodies. Antibodies are typically therapeutically effective at relatively high doses. Therefore, high antibody concentrations are usually desired in pharmaceutical preparations to minimize dosage volume and to make administration more patient-friendly (for example, subcutaneous injection rather than intravenous drip, reducing the number of injections).
实际上，聚集可导致活性抗体治疗失效，从而导致不可靠和无效的给药。更重要的是，聚集还可表现出毒性并触发不可取或严重的免疫反应。导致阻碍流动的大颗粒沉积的聚集对于任一种胃肠外应用均是不可取的。In fact, aggregation can lead to the loss of active antibody therapy, resulting in unreliable and ineffective administration. More importantly, aggregation can also exhibit toxicity and trigger undesirable or severe immune responses. Aggregation leading to the deposition of large particles that hinder flow is undesirable for any parenteral application.
经由化学途径(诸如氧化、脱酰胺、异构化、二硫键形成和其它不可逆交联反应)的抗体改性和降解还可随时间推移进行并导致抗体失活，以及触发聚集。这些反应，连同聚集，常常在升高的温度下加快；因此冷藏几乎经常是必要条件。在许多这些化学反应中，水也诸如在酰胺键的水解裂解中起到重要作用，如作为中介物或作为反应中间体。因此，诸如通过冻干或冷冻干燥排除水以形成固态粉末制剂可以是针对制备抗体治疗的稳定制剂的有效措施。Antibody modification and degradation via chemical pathways (such as oxidation, deamidation, isomerization, disulfide bond formation, and other irreversible cross-linking reactions) can also proceed over time and lead to antibody inactivation, as well as triggering aggregation. These reactions, along with aggregation, are often accelerated at elevated temperatures; therefore, refrigeration is almost always a necessary condition. In many of these chemical reactions, water also plays an important role, such as in the hydrolytic cleavage of amide bonds, as an intermediary or as a reaction intermediate. Therefore, excluding water to form a solid powder formulation, such as by lyophilization or freeze drying, can be an effective measure for preparing a stable formulation for antibody therapy.
市售的治疗性抗体或抗体衍生物/片段的制剂中的一些基于冻干粉末制剂，所述制剂需要由受过训练的医疗或医护专业人员在即将给药之前在无菌条件下用水性介质重组。例如，奥马佐单抗(例如由Genentech出售)可作为在一次性使用小瓶中的冻干粉末获得。Some of the commercially available formulations of therapeutic antibodies or antibody derivatives/fragments are based on lyophilized powder formulations that require reconstitution with an aqueous medium under sterile conditions by a trained medical or healthcare professional just prior to administration. For example, omalizumab (e.g., sold by Genentech) is available as a lyophilized powder in single-use vials.
然而，在实际给药之前，作为额外步骤的冻干抗体在无菌水性介质中的重组携带了不正确的处理(例如，振摇)或给药以及污染的风险。如果水性介质的pH和温度未达最佳标准，允许再水合的时间太短或在溶解步骤期间过于剧烈地振摇小瓶，则重组步骤本身可触发聚集。浪费的趋势也更高，因为不能在推荐时间阶段内适当溶解冻干抗体产品常常需要丢弃样品。However, the reconstitution of the lyophilized antibody in a sterile aqueous medium as an additional step before actual administration carries the risk of incorrect handling (e.g., shaking) or administration and contamination. If the pH and temperature of the aqueous medium are not optimal, the time allowed for rehydration is too short, or the vial is shaken too vigorously during the dissolution step, the reconstitution step itself can trigger aggregation. The tendency to waste is also higher, as failure to properly dissolve the lyophilized antibody product within the recommended time period often requires discarding the sample.
应当注意，冻干工艺步骤本身可引发聚集和/或降解。常常将附加的稳定赋形剂诸如糖类或多元醇连同其它赋形剂诸如填充剂一起加入预冻干组合物中。也可要求在冻干之后添加其它赋形剂以便支持较长的抗体储存寿命，从而增加最终制剂中的组分数。It should be noted that the lyophilization process step itself can induce aggregation and/or degradation. Additional stabilizing excipients such as carbohydrates or polyols are often added to the pre-lyophilized composition along with other excipients such as fillers. It may also be desirable to add other excipients after lyophilization in order to support a longer shelf life of the antibody, thereby increasing the number of components in the final formulation.
由于对于给药而言容易制备，所以即用型液体制剂通常是使用者优选的。如果稳定，则液体制剂对于药品制造商而言也具有吸引力，这是由于避免了冻干，所述冻干在药物开发和常规制造两者期间均是费时且昂贵的。实际上，抗体或抗体衍生物的许多市售制剂是基于水的溶液。在含水制剂的情况下，鉴于促进或降低各种降解化学反应的可能性，介质的pH可对抗体的稳定性具有显著影响。因此，常常需要优化的缓冲体系连同其它制剂赋形剂，诸如抗氧化剂自由基清除剂、表面活性剂和其它抗聚集添加剂、或防腐剂以便向抗体提供稳定性并抵消可在含水环境中储存期间随时间推移而出现的各种可能的降解过程。In some embodiments, the present invention relates to the preparation of the present invention.Owing to being easy to prepare for administration, so instant liquid preparation is normally preferred by user.If stable, then liquid preparation also has attraction for pharmaceutical manufacturers, and this is owing to avoiding lyophilization, and described lyophilization is all time-consuming and expensive during pharmaceutical development and conventional manufacturing.In fact, many commercially available preparations of antibody or antibody derivative are solutions based on water.In the case of aqueous preparation, in view of promoting or reducing the possibility of various degradation chemical reactions, the pH of medium can have a significant impact on the stability of antibody.Therefore, usually need the buffer system of optimization together with other formulation excipients, such as antioxidant free radical scavenger, surfactant and other anti-aggregation additive or preservative so that stability is provided to antibody and offset can pass over time and the various possible degradation processes that occur during storage in aqueous environment.
还已知对冻干和水溶液的可供选择的制剂选项，诸如使用非水性液体作为载体。例如，WO2012/121754描述了非水性高浓度混悬液制剂，其包含疏水性载体诸如芝麻油，和降粘剂诸如油酸乙酯，以及抗-TNFα抗体。这些组合物要求添加完全混溶于载体中的降粘剂，以便使油载体更适用于注射。通常，脂质和油的肠胃外使用可在注射部位处造成疼痛和其它不可取的副作用。这些类型的化合物还可减慢治疗剂的释放，并且在优选快速或立即生物利用时，是不理想的。Also known are alternative formulation options for freeze-dried and aqueous solutions, such as using non-aqueous liquids as carriers. For example, WO2012/121754 describes a non-aqueous high concentration suspension formulation comprising a hydrophobic carrier such as sesame oil, and a viscosity reducer such as ethyl oleate, and an anti-TNFα antibody. These compositions require the addition of a viscosity reducer that is fully miscible in the carrier so that the oil carrier is more suitable for injection. Typically, parenteral administration of lipids and oils can cause pain and other undesirable side effects at the injection site. These types of compounds can also slow down the release of the therapeutic agent and are undesirable when preferably rapidly or immediately bioavailable.
全氟化合物也已经被描述为诸如蛋白质和肽之类生物活性试剂的可能的非水性液体载体。例如，US6458376描述了对于眼科应用(例如，局部施用的滴眼剂)所建议的组合物，其中包括寡肽和蛋白质生长因子的治疗/诊断化合物悬浮于全氟化碳或氟化硅油中或在至少一种表面活性剂存在下悬浮。然而，没有提及包含抗体或抗体片段和衍生物的此类组合物。Perfluorinated compounds have also been described as possible non-aqueous liquid carriers for bioactive agents such as proteins and peptides. For example, US Pat. No. 6,458,376 describes compositions proposed for ophthalmic applications (e.g., topical eye drops) in which therapeutic/diagnostic compounds including oligopeptides and protein growth factors are suspended in perfluorocarbon or fluorinated silicone oil or in the presence of at least one surfactant. However, there is no mention of such compositions comprising antibodies or antibody fragments and derivatives.
US6730328描述了热稳定制剂，其中将非水性、疏水、非反应性载体(诸如矿物油、全氟萘烷、甲氧氟烷、全氟三丁胺和十四烷)用于包含蛋白质、蛋白质化合物和核酸的混悬液组合物。提出用于胃肠外、透皮、粘膜、口腔和肠内给药方法的制剂，及其用于经由可植入装置长期连续给药和输送的用途。然而，未公开抗体或抗体片段或衍生物在此类载体中的混悬液的具体实例，也不具有关于此类组合物在升高的温度下超过三个月时间点的稳定性的任何教导。也未展示这些类型的组合物的实际组织相容性。US6730328 describes a heat-stable formulation in which a non-aqueous, hydrophobic, non-reactive carrier (such as mineral oil, perfluorodecalin, methoxyflurane, perfluorotributylamine, and tetradecane) is used for a suspension composition comprising proteins, protein compounds, and nucleic acids. Formulations for parenteral, transdermal, mucosal, oral, and enteral administration methods are proposed, as well as their use for long-term continuous administration and delivery via an implantable device. However, no specific examples of suspensions of antibodies or antibody fragments or derivatives in such carriers are disclosed, nor is there any teaching on the stability of such compositions at elevated temperatures exceeding a three-month time point. The actual tissue compatibility of these types of compositions is also not demonstrated.
WO 2011/073134公开了环孢素，分子量为1202.31的环状多肽的半氟化烷烃溶液，其任选在助溶剂诸如乙烷存在下。虽然以任选替代方式提及了混悬液和乳液，但没有具体公开此类组合物，或任何包含千道尔顿范围内的高分子量蛋白质物质，诸如抗体或抗体片段或衍生物的组合物。WO 2011/073134 discloses solutions of cyclosporine, a cyclic polypeptide having a molecular weight of 1202.31, in semifluorinated alkanes, optionally in the presence of a cosolvent such as ethane. While suspensions and emulsions are mentioned as optional alternatives, there is no specific disclosure of such compositions, or any compositions comprising high molecular weight proteinaceous materials in the kilodalton range, such as antibodies or antibody fragments or derivatives.
因此本发明的主题是提出新型抗体组合物，其克服了与本领域当前已知的制剂相关联的任何限制和缺点。The subject of the present invention is therefore to propose novel antibody compositions that overcome any limitations and disadvantages associated with the formulations currently known in the art.
发明内容Summary of the Invention
在第一方面，本发明提出抗原结合的多肽或蛋白质和液体载体的新型组合物，所述液体载体包含式RFRH的半氟化烷烃，其中RF为具有4至12个碳原子的直链全氟化烃片段，并且其中RH为具有4至8个碳原子的直链烷基基团。抗原结合的多肽或蛋白质掺入组合物中从而形成分散体或混悬液。In a first aspect, the present invention provides novel compositions of an antigen-binding polypeptide or protein and a liquid carrier comprising a semifluorinated alkane of the formula RFRH, wherein RF is a linear perfluorinated hydrocarbon fragment having 4 to 12 carbon atoms, and wherein RH is a linear alkyl group having 4 to 8 carbon atoms. The antigen-binding polypeptide or protein is incorporated into the composition to form a dispersion or suspension.
在另一方面，抗原结合的多肽或蛋白质可选自单克隆抗体、抗体片段、多克隆抗体、包含抗体片段的融合蛋白、或抗体-药物偶联物。In another aspect, the antigen-binding polypeptide or protein can be selected from a monoclonal antibody, an antibody fragment, a polyclonal antibody, a fusion protein comprising an antibody fragment, or an antibody-drug conjugate.
在另一方面，本发明提供用于治疗、预防或诊断有需要的患者的疾病或病症的方法，所述方法包括给药组合物的步骤，所述组合物包含抗原结合的多肽或蛋白质，以及液体载体，所述液体载体包含式RFRH的半氟化烷烃，其中RF为具有4至12个碳原子的直链全氟化烃片段，并且其中RH为具有4至8个碳原子的直链烷基基团，并且其中抗原结合的多肽或蛋白质掺入组合物中从而形成分散体或混悬液。In another aspect, the present invention provides a method for treating, preventing, or diagnosing a disease or condition in a patient in need thereof, the method comprising the step of administering a composition comprising an antigen-binding polypeptide or protein and a liquid carrier comprising a semifluorinated alkane of the formula RFRH, wherein RF is a linear perfluorinated hydrocarbon fragment having 4 to 12 carbon atoms, and wherein RH is a linear alkyl group having 4 to 8 carbon atoms, and wherein the antigen-binding polypeptide or protein is incorporated into the composition to form a dispersion or suspension.
在另一个实施方式中，本发明提供稳定抗原结合的多肽或蛋白质的方法，所述方法包括将抗原结合的多肽或蛋白质与液体载体混合以形成分散体或混悬液的步骤，所述液体载体包含式RFRH的半氟化烷烃，其中RF为具有4至12个碳原子的直链全氟化烃片段，并且其中RH为具有4至8个碳原子的直链烷基基团。In another embodiment, the present invention provides a method for stabilizing an antigen-binding polypeptide or protein, the method comprising the step of mixing the antigen-binding polypeptide or protein with a liquid carrier to form a dispersion or suspension, the liquid carrier comprising a semifluorinated alkane of the formula RFRH, wherein RF is a linear perfluorinated hydrocarbon segment having 4 to 12 carbon atoms, and wherein RH is a linear alkyl group having 4 to 8 carbon atoms.
具体实施方式DETAILED DESCRIPTION
在第一方面，本发明提供组合物，所述组合物包含抗原结合的多肽或蛋白质以及液体载体，所述液体载体包含式RFRH的半氟化烷烃，其中RF为具有4至12个碳原子的直链全氟化烃片段，并且其中RH为具有4至8个碳原子的直链烷基基团。另外，抗原结合的多肽或蛋白质掺入组合物中从而形成分散体或混悬液；即抗原结合的多肽或蛋白质分散或悬浮于液体载体中。In a first aspect, the present invention provides a composition comprising an antigen-binding polypeptide or protein and a liquid carrier comprising a semifluorinated alkane of the formula RFRH, wherein RF is a linear perfluorinated hydrocarbon segment having 4 to 12 carbon atoms, and wherein RH is a linear alkyl group having 4 to 8 carbon atoms. Furthermore, the antigen-binding polypeptide or protein is incorporated into the composition to form a dispersion or suspension; that is, the antigen-binding polypeptide or protein is dispersed or suspended in the liquid carrier.
从药物角度来看，半氟化烷烃提供了多种优点。它们基本上是非毒性的并且发现在局部或胃肠外给药时被各种类型的人或动物组织很好地耐受。此外，它们是化学惰性的并且通常与药物制剂中的活性或非活性成分相容。它们还能够溶解大范围的化合物，诸如小分子活性成分和许多常用的药学上可接受的赋形剂，这可能是由于它们固有的两亲性。另外，半氟化烷烃在充当不溶或难溶化合物(诸如抗原结合蛋白质或多肽)的载体时，形成具有非常有用的物理或药物特性的分散体或混悬液，即具有很少的或不具有形成固体的不可分散的沉淀物的趋势。From a pharmaceutical perspective, semifluorinated alkanes offer a variety of advantages. They are essentially non-toxic and have been found to be well tolerated by various types of human or animal tissues when administered topically or parenterally. In addition, they are chemically inert and generally compatible with active or inactive ingredients in pharmaceutical formulations. They are also capable of dissolving a wide range of compounds, such as small molecule active ingredients and many commonly used pharmaceutically acceptable excipients, likely due to their inherent amphipathic nature. Additionally, semifluorinated alkanes, when acting as carriers for insoluble or poorly soluble compounds (such as antigen-binding proteins or polypeptides), form dispersions or suspensions with very useful physical or pharmaceutical properties, i.e., have little or no tendency to form solid, non-dispersible precipitates.
本发明人已经发现半氟化烷烃作为液体载体存在于包含抗原结合的蛋白质或多肽的组合物中具有对这些组分的显著的稳定效应。具体地讲，包含作为液体载体的半氟化烷烃的组合物能够基本上防止或抑制其在室温和甚至在较高的温度诸如40℃下在相当长的时间段内聚集并减少化学降解，但不丧失生物活性。The present inventors have discovered that the presence of a semifluorinated alkane as a liquid carrier in a composition comprising an antigen-binding protein or polypeptide has a significant stabilizing effect on these components. Specifically, compositions comprising a semifluorinated alkane as a liquid carrier are able to substantially prevent or inhibit their aggregation and reduce chemical degradation at room temperature and even at elevated temperatures such as 40° C. for a substantial period of time without loss of biological activity.
还发现抗原结合蛋白质在半氟化烷烃中的分散体或混悬液表现出显著的物理稳定性程度。漂浮或沉淀的发生缓慢进行，留出足够的时间用于在将具有分散体或混悬液的容器(例如小瓶)温和振摇或涡旋之后取出剂量。半氟化烷烃中的抗原结合蛋白质颗粒呈现出在很大程度上保留其原始粒度分布，并且容易再分散；差再分散性的聚集体看起来未形成。重要的是，从精度和重复性来看，这提供了更高的给药精度水平。It was also found that dispersions or suspensions of antigen-binding proteins in semifluorinated alkanes exhibited a significant degree of physical stability. Floating or precipitation occurred slowly, leaving enough time for the dose to be removed after the container (e.g., vial) with the dispersion or suspension was gently shaken or vortexed. The antigen-binding protein particles in the semifluorinated alkanes appeared to retain their original size distribution to a large extent and were easily redispersed; aggregates with poor redispersibility did not appear to form. Importantly, this provided a higher level of dosing accuracy in terms of precision and repeatability.
相反，在其它化学惰性载体中的混悬液或分散体趋于不稳定，导致形成致密且再分散性差的聚集体，并且使得精确给药具有挑战性，或在一些情况下不可能，诸如导致通常用于皮下注射的细规格针堵塞。聚集的蛋白质颗粒还存在引发不良免疫反应的高风险。In contrast, suspensions or dispersions in other chemically inert carriers tend to be unstable, leading to the formation of dense, poorly redispersible aggregates and making precise dosing challenging or, in some cases, impossible, such as by clogging fine-gauge needles commonly used for subcutaneous injections. Aggregated protein particles also present a high risk of triggering adverse immune responses.
取决于分散相和连续相的相对密度，不稳定的混悬液或分散体趋于通过分散相的漂浮，或通过其沉淀而快速分离。此类行为通常通过快速形成致密且再分散性差的聚集体来完成。如果不是不可能，快速漂浮或沉淀也使得精确和可重复的给药具有挑战性。例如，如果振摇之后可注射或眼部混悬液快速沉降，并且如果振摇之后不立即从充满的容器中取出第一剂量，则取出的剂量会包含低于预期数的药物颗粒(或如果容器保持倒置，则将分配大于预期剂量)。然后，稍后从相同容器中取出的剂量也将包含太高或太低的药物剂量每体积。旨在将再分散性差的聚集体再分散的抗原结合多肽和蛋白质的剧烈振摇也会进一步引发其进一步的聚集和变质。In some embodiments, the present invention relates to a suspension or dispersion of a pharmaceutical composition comprising a plurality of pharmaceutical compositions ...
本发明的主要优点由在组合物中存在用作液体载体的半氟化烷烃所带来。基于半氟化烷烃的混悬液的有利特性导致优异的药物质量和性能特征，并且还增加患者和/或医疗保健提供者的使用便利性。The main advantage of the present invention is brought about by the presence of a semifluorinated alkane used as a liquid carrier in the composition. The favorable properties of semifluorinated alkane-based suspensions result in excellent drug quality and performance characteristics and also increase the convenience of use for patients and/or healthcare providers.
半氟化烷烃是直链或支化的烷烃，其氢原子中的一些被氟取代。在用于本发明的半氟化烷烃(SFA)中，存在一个直链非氟化烃片段和一个直链全氟化烃片段。这些化合物因此符合通式F(CF2)n(CH2)mH。根据本发明，n选自4至12的范围，并且m选自4至8的范围。Semifluorinated alkanes are linear or branched alkanes in which some of the hydrogen atoms are substituted by fluorine. In the semifluorinated alkanes (SFA) used in the present invention, there is one linear non-fluorinated hydrocarbon segment and one linear perfluorinated hydrocarbon segment. These compounds therefore conform to the general formula F(CF2 )n (CH2 )mH . According to the present invention, n is selected from the range of 4 to 12, and m is selected from the range of 4 to 8.
经常用于半氟化烷烃的命名法将全氟化烃片段命名为RF，并且将非氟化片段命名为RH。另选地，化合物可分别称为FnHm和FnHm，其中F是指全氟化烃片段，H指示非氟化片段，并且n和m限定各个片段中的碳原子数。例如F3H3用于全氟丙基丙烷，F(CF2)3(CH2)3H。另外，这种命名法通常用于具有直链片段的化合物。因此，除非另外指明，否则应当设想F3H3是指1-全氟丙基丙烷，而不是2-全氟丙基丙烷、1-全氟异丙基丙烷或2全氟异丙基丙烷。The nomenclature often used for semifluorinated alkanes designates the perfluorinated hydrocarbon segment as RF and the non-fluorinated segment as RH. Alternatively, the compounds may be referred to as FnHm and FnHm, respectively, where F refers to the perfluorinated hydrocarbon segment, H indicates the non-fluorinated segment, and n and m define the number of carbon atoms in each segment. For example, F3H3 is used for perfluoropropylpropane, F(CF2 )3 (CH2 )3H . In addition, this nomenclature is commonly used for compounds with straight-chain segments. Therefore, unless otherwise indicated, F3H3 should be assumed to refer to 1-perfluoropropylpropane, rather than 2-perfluoropropylpropane, 1-perfluoroisopropylpropane, or 2-perfluoroisopropylpropane.
优选的半氟化烷烃具体包括化合物F4H5、F4H6、F4H8、F6H4、F6H6、F6H8和F6H10。特别优选用于进行本发明的是F4H5、F4H6、F6H6和F6H8。在另一个特别优选的实施方式中，本发明的组合物包括F6H8。Preferred semifluorinated alkanes specifically include compounds F4H5, F4H6, F4H8, F6H4, F6H6, F6H8 and F6H10. Particularly preferred for use in the practice of the present invention are F4H5, F4H6, F6H6 and F6H8. In another particularly preferred embodiment, the composition of the present invention includes F6H8.
任选地，组合物可包含多于一个SFA。例如，为了实现特定目标特性诸如一定密度或粘度，将SFA混合会是有用的。如果使用SFA的混合物，则还优选混合物包含下列物质中的至少一种：F4H5、F4H6、F6H4、F6H6、F6H8和F6H10，并且特别是下列物质中的一种：F4H5、F4H6、F6H6和F6H8。在另一个实施方式中，混合物包含选自下列物质的至少两个成员：F4H5、F4H6、F6H4、F6H6、F6H8和F6H10，并且具体地讲选自下列物质的至少两个成员：F4H5、F6H6和F6H8。Optionally, the composition may contain more than one SFA. For example, in order to achieve a specific target property such as a certain density or viscosity, it may be useful to mix the SFAs. If a mixture of SFAs is used, it is also preferred that the mixture contains at least one of the following: F4H5, F4H6, F6H4, F6H6, F6H8 and F6H10, and in particular one of the following: F4H5, F4H6, F6H6 and F6H8. In another embodiment, the mixture contains at least two members selected from the following: F4H5, F4H6, F6H4, F6H6, F6H8 and F6H10, and in particular at least two members selected from the following: F4H5, F6H6 and F6H8.
液体SFA是化学和生理惰性、无色且稳定的。它们的典型密度在1.1至1.7g/cm3的范围内，并且其表面张力可以低至19mN/m。RFRH型的SFA不溶于水而且在一定程度上是两亲的，其中亲脂性增加与非氟化片段的大小增加相关。Liquid SFAs are chemically and physiologically inert, colorless, and stable. Their typical density ranges from 1.1 to 1.7 g/cm³ , and their surface tension can be as low as 19 mN/m. RFRH-type SFAs are insoluble in water and are somewhat amphiphilic, with increasing lipophilicity correlating with increasing size of the non-fluorinated segment.
RFRH型的液体SFA在商业上用于视网膜的展开和重新应用，用于长期填塞作为玻璃体替代物(H.Meinert等人，European Journal of Ophthalmology，第10(3)卷，第189-197页，2000年)，以及作为玻璃体-视网膜手术后的残余硅油的洗出溶液。在实验上，它们也被用作血液替代物(H.Meinert等人，Biomaterials，Artificial Cells，andImmobilization Biotechnology，第21(5)卷，第583-95页，1993年)。这些应用已经将SFA构造成生理上良好耐受的化合物。在另一方面，截至今天SFA未在批准的药物产品中用作赋形剂。Liquid SFAs of the RFRH type are commercially used for the unfolding and reapplication of the retina, for long-term tamponade as vitreous substitutes (H. Meinert et al., European Journal of Ophthalmology, Vol. 10(3), pp. 189-197, 2000), and as washout solutions for residual silicone oil after vitreoretinal surgery. Experimentally, they have also been used as blood substitutes (H. Meinert et al., Biomaterials, Artificial Cells, and Immobilization Biotechnology, Vol. 21(5), pp. 583-95, 1993). These applications have made SFAs physiologically well-tolerated compounds. On the other hand, SFAs are not used as excipients in approved pharmaceutical products to date.
本发明的组合物包含抗原结合的多肽或蛋白质。多肽和蛋白质通常表示具有彼此由肽键连接的氨基酸单元的聚合物。因为常用于区分多肽和蛋白质的尺寸界限在一定程度上是任意的，所以用于这些分子的两种表述在本发明的上下文中不应当被理解为是相互排斥的：多肽可也可被称为蛋白质，反之亦然。通常，术语“多肽”仅是指单个聚合物链，然而，表述“蛋白质”还可是指通过非共价键彼此连接的两个或多个多肽链。The compositions of the present invention comprise antigen-binding polypeptides or proteins. Polypeptides and proteins generally refer to polymers comprising amino acid units linked to each other by peptide bonds. Because the size boundaries commonly used to distinguish between polypeptides and proteins are somewhat arbitrary, the two terms used for these molecules should not be understood as mutually exclusive in the context of the present invention: a polypeptide may also be referred to as a protein, and vice versa. Generally, the term "polypeptide" refers only to a single polymer chain, however, the term "protein" may also refer to two or more polypeptide chains linked to each other by non-covalent bonds.
更具体地，并且如本发明上下文中所使用的，抗原结合的多肽或蛋白质是指呈其单体或聚合物形式的全长或整个抗体(也被称为免疫球蛋白)，和衍生自全长抗体的能够特异性结合到抗原的任何片段、链、结构域或任何改性物。抗原结合的多肽或蛋白质可属于IgG、IgA、IgD、IgE或IgM免疫球蛋白同型或类中的任一种。包含能够特异性结合到抗原的抗体片段的融合蛋白和抗体-药物偶联物也在如本文所用的抗原结合的多肽或蛋白质的定义内。More specifically, and as used in the context of the present invention, an antigen-binding polypeptide or protein refers to a full-length or entire antibody (also referred to as an immunoglobulin) in its monomeric or polymeric form, and any fragment, chain, domain, or any modification thereof that is capable of specific binding to an antigen that is derived from a full-length antibody. An antigen-binding polypeptide or protein may belong to any of the IgG, IgA, IgD, IgE, or IgM immunoglobulin isotypes or classes. Fusion proteins and antibody-drug conjugates comprising antibody fragments that are capable of specific binding to an antigen are also within the definition of an antigen-binding polypeptide or protein as used herein.
全长抗体是Y型糖蛋白，其包含具有Fc(可结晶片段)结构域和Fab(抗原结合片段)结构域的通式。这些在结构上由经由二硫键互连以形成Y型结构的两个重(H)链和两个轻(L)链多肽结构组成。每种类型的链包含可变区(V)和恒定区(C)；重链包含可变链区(VH)和各种恒定区(例如，CH1、CH2等)并且轻链包含可变链区(VL)和恒定区(CL)。V区还可表征为另外的亚结构域/区，即框架(FR)区，其包含更保守的氨基酸残基，和高变(HV)或互补决定区(CDR)，从氨基酸氨基的角度来看其包含可变性增加的区域。链的可变区决定抗体的结合特异性并且形成抗体的抗原结合Fab结构域。Full-length antibodies are Y-shaped glycoproteins that contain a general formula with an Fc (crystallizable fragment) domain and a Fab (antigen-binding fragment) domain. Structurally, these are composed of two heavy (H) chains and two light (L) chain polypeptide structures interconnected via disulfide bonds to form a Y-shaped structure. Each type of chain contains a variable region (V) and a constant region (C); the heavy chain contains a variable chain region (VH ) and various constant regions (e.g.,CH1 ,CH2 , etc.), and the light chain contains a variable chain region (VL ) and a constant region (CL ). The V region can also be characterized as additional subdomains/regions, namely the framework (FR) region, which contains more conserved amino acid residues, and the hypervariable (HV) or complementarity determining region (CDR), which contains regions of increased variability from the perspective of amino acid amino groups. The variable regions of the chains determine the binding specificity of the antibody and form the antigen-binding Fab domain of the antibody.
在本发明的优选实施方式中，组合物包含抗原结合的多肽或蛋白质，其中抗原结合多肽或蛋白质选自单克隆抗体、多克隆抗体、抗体片段、包含抗体片段的融合蛋白、抗体-药物偶联物、或它们的任何组合。In a preferred embodiment of the present invention, the composition comprises an antigen-binding polypeptide or protein, wherein the antigen-binding polypeptide or protein is selected from a monoclonal antibody, a polyclonal antibody, an antibody fragment, a fusion protein comprising an antibody fragment, an antibody-drug conjugate, or any combination thereof.
在本发明的特别优选的实施方式中，组合物包含选自单克隆抗体(mAb)的抗原结合的多肽或蛋白质。单克隆抗体是指得自特定朝向抗原上的单表位或结合位点的同质抗体群的抗体。单克隆抗体可使用本领域已知的抗体工程技术，诸如经由杂交瘤或重组DNA法来制备。In a particularly preferred embodiment of the present invention, the composition comprises an antigen-binding polypeptide or protein selected from a monoclonal antibody (mAb). A monoclonal antibody is an antibody derived from a homogeneous population of antibodies that specifically targets a single epitope or binding site on an antigen. Monoclonal antibodies can be prepared using antibody engineering techniques known in the art, such as via hybridoma or recombinant DNA methods.
另外，在抗原结合多肽和蛋白质的范围内，单克隆抗体是抗体片段。如本文所定义的，抗体片段包括其可与抗原相互作用或特异性结合的抗体的任何区域、链、结构域或其任何结构或偶联物，并且相对于结合能力可以是一价、二价或甚至多价的。此类抗体片段可由本领域已知的方法制成，这些方法例如，全长天然抗体的分解(例如，通过蛋白水解)、蛋白质合成、基因工程/DNA重组过程、化学交联或它们的任何组合。抗体片段通常衍生自在全长抗体的可变V区中起主要作用的各种结构域或区域的组合。In addition, within the scope of antigen-binding polypeptides and proteins, monoclonal antibodies are antibody fragments. As defined herein, antibody fragments include any region, chain, domain or any structure or conjugate of the antibody that can interact with antigen or specifically bind, and can be monovalent, divalent or even multivalent relative to binding capacity. Such antibody fragments can be made by methods known in the art, such as, the decomposition (for example, by proteolysis), protein synthesis, genetic engineering/DNA recombination process, chemical crosslinking or any combination thereof of full-length natural antibodies. Antibody fragments are usually derived from the various domains or regional combinations that play a major role in the variable V regions of full-length antibodies.
在本发明的一个实施方式中，组合物包含选自抗体片段的抗原结合的多肽或蛋白质，其中所述抗体片段为抗原结合片段(Fab)、单链可变片段(scFv)、单域抗体、微抗体、双价抗体。In one embodiment of the present invention, the composition comprises an antigen-binding polypeptide or protein selected from an antibody fragment, wherein the antibody fragment is an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a single-domain antibody, a minibody, or a diabody.
特别优选的抗体片段为抗原结合结构域片段(Fab，也称为Fab’)或包含由二硫键连接的两个Fab片段的Fab二聚体。Fab的实例为阿昔单抗、赛妥珠单抗、digifab、和雷珠单抗。优选的Fab为赛妥珠单抗(也称为聚乙二醇化赛妥珠单抗)，其是与聚乙二醇偶联的重组人源化抗体Fab’片段。赛妥珠单抗具有91kDa的分子量并且针对肿瘤坏死因子α(TNFα)。Particularly preferred antibody fragments are antigen-binding domain fragments (Fab, also known as Fab') or Fab dimers comprising two Fab fragments linked by a disulfide bond. Examples of Fab are abciximab, certolizumab pegol, digifab, and ranibizumab. A preferred Fab is certolizumab pegol (also known as certolizumab pegol), which is a recombinant humanized antibody Fab' fragment coupled to polyethylene glycol. Certolizumab pegol has a molecular weight of 91 kDa and is directed against tumor necrosis factor alpha (TNFα).
在本发明的另一个实施方式中，组合物可包含单链可变片段(scFv)，诸如包含由其接头或复杂的多聚/多价结构接合的重(VH)和轻(VL)链可变结构域的那些，例如双价抗体(二价二聚体)、三价抗体(三价三聚体)、或四价抗体(四价四聚体)。多聚抗体片段还可以为多特异性的，例如双特异性双价抗体可包含两个片段，其各自具有对不同抗原的特异性。另外优选的抗体片段包括单域抗体(daBs)，诸如包含能够特异性结合到抗原的单个VH或VL结构域的那些。也在本发明范围内的抗体片段包括scFv-CH二聚体结构，即微抗体。In another embodiment of the invention, the composition may comprise a single-chain variable fragment (scFv), such as those comprising heavy (VH ) and light (VL ) chain variable domains joined by a linker or a complex multimeric/multivalent structure, such as a diabody (bivalent dimer), a trivalent antibody (trivalent trimer), or a tetravalent antibody (tetravalent tetramer). Multimeric antibody fragments may also be multispecific, for example, a bispecific bivalent antibody may comprise two fragments, each of which has specificity for a different antigen. Additionally preferred antibody fragments include single-domain antibodies (daBs), such as those comprising a singleVH orVL domain capable of specific binding to an antigen. Antibody fragments also within the scope of the invention include scFv-CH dimer structures, i.e., minibodies.
根据另一个实施方式，组合物包含具有选自有下列的分子量的抗原结合的多肽或蛋白质：至少10KDa、至少15kDa、至少35kDa、至少50kDa、至少70kDa、或至少90kDa。还优选具有至少100kDa，诸如100-150kDa，或甚至高于150kDa的分子量的抗原结合的多肽或蛋白质。特别优选的是分子量在70kDa至160kDa范围内的抗原结合的多肽或蛋白质。According to another embodiment, the composition comprises an antigen-binding polypeptide or protein having a molecular weight selected from the group consisting of at least 10 kDa, at least 15 kDa, at least 35 kDa, at least 50 kDa, at least 70 kDa, or at least 90 kDa. Also preferred are antigen-binding polypeptides or proteins having a molecular weight of at least 100 kDa, such as 100-150 kDa, or even greater than 150 kDa. Particularly preferred are antigen-binding polypeptides or proteins having a molecular weight in the range of 70 kDa to 160 kDa.
在本发明的另一个实施方式中，抗原结合的多肽或蛋白质可选自包含抗体片段的融合蛋白。抗体片段可融合到另一种生物活性蛋白或多肽片段，例如，多肽毒素、酶、细胞因子、膜蛋白等。包含抗体片段的融合蛋白的实例包括依那西普和阿塞西普。依那西普是分子量为150kDa的重组人蛋白，其包含融合到IgG1的Fc部分的75kDa的肿瘤坏死因子受体(TNFR)的配位结合部分。In another embodiment of the present invention, the antigen-binding polypeptide or protein can be selected from a fusion protein comprising an antibody fragment. The antibody fragment can be fused to another biologically active protein or polypeptide fragment, such as a polypeptide toxin, enzyme, cytokine, membrane protein, etc. Examples of fusion proteins comprising antibody fragments include etanercept and atacicept. Etanercept is a recombinant human protein with a molecular weight of 150 kDa that comprises the 75 kDa ligand binding portion of the tumor necrosis factor receptor (TNFR) fused to the Fc portion of IgG1.
在本发明的另一个实施方式中，抗原结合的多肽或蛋白质可选自抗体-药物偶联物，其中所述抗原结合的多肽或蛋白质例如经由接头或化学交联而与小分子药物或放射性标记组分(如放射性核素)共价连接。抗体-药物偶联物的实例包括妥珠单抗奥唑米星(gemtuzumab ozogamicin)、brentuximab vedotin、90Y-标记的替伊莫单抗、131I-标记的托西莫单抗、99mTc-标记的阿西莫单抗。如本文所使用的，术语抗体-药物偶联物还可以是指例如通过PEG化(例如，聚乙二醇化赛妥珠单抗，人源化TNF抑制剂单克隆抗体的聚乙二醇化Fab’片段)或脂质化而实质上化学改性的抗原结合的多肽或蛋白质。In another embodiment of the present invention, the antigen-bound polypeptide or protein can be selected from antibody-drug conjugates, wherein the antigen-bound polypeptide or protein is covalently linked to a small molecule drug or a radiolabeled component (such as a radionuclide) via a joint or chemical cross-linking. Examples of antibody-drug conjugates include gemtuzumab ozogamicin, brentuximab vedotin,90 Y-labeled ibritumomab tiuxetan,131 I-labeled tositumomab,99m Tc-labeled acitumomab. As used herein, the term antibody-drug conjugate can also refer to, for example, a substantially chemically modified antigen-bound polypeptide or protein by PEGylation (e.g., certolizumab pegol, PEGylated Fab' fragments of humanized TNF inhibitor monoclonal antibodies) or lipidation.
如本文所理解的，抗原结合多肽和蛋白质可以是嵌合的、人源化或人的。嵌合单克隆抗体例如是指包含重或轻链的结构域或区域的杂交单克隆抗体，其源自来自多于一个物种的抗体序列，例如来自鼠和人抗体序列的抗体序列。人源化单克隆抗体是指结构上主要源自人抗体序列的那些，通常具有至少85-95％源自人的序列的贡献，然而术语人单克隆抗体是指仅源自人胚系抗体序列的那些。在优选的实施方式中，组合物包含选自单克隆抗体的抗原结合的多肽或蛋白质，其中所述单克隆抗体为嵌合抗体、人源化抗体或人抗体。As understood herein, antigen-binding polypeptides and proteins can be chimeric, humanized or human. A chimeric monoclonal antibody, for example, refers to a hybrid monoclonal antibody comprising a domain or region of a heavy or light chain that is derived from antibody sequences from more than one species, such as antibody sequences from murine and human antibody sequences. A humanized monoclonal antibody refers to one that is structurally derived primarily from human antibody sequences, typically with a contribution of at least 85-95% from sequences derived from humans, whereas the term human monoclonal antibody refers to one that is derived solely from human germline antibody sequences. In a preferred embodiment, the composition comprises an antigen-binding polypeptide or protein selected from a monoclonal antibody, wherein the monoclonal antibody is a chimeric antibody, a humanized antibody or a human antibody.
在另一个实施方式中，组合物可包含多克隆抗体、或能够识别抗原的多于一个表位的抗体的异质混合物。In another embodiment, the composition may comprise a polyclonal antibody, or a heterogeneous mixture of antibodies capable of recognizing more than one epitope of an antigen.
在优选的实施方式中，抗原结合的多肽或蛋白质为治疗或诊断化合物或疫苗。如本文所用，治疗化合物是可用于预防疾病或病症、缓解疾病或病症的任何症状、改善任何疾病或病症、延缓疾病或病症的发展等的化合物。诊断化合物可用于确定生物体的状态，或用于诊断疾病、病症、症状、或患者表型。治疗化合物必须给药于患者，然而诊断试剂可根据具体情况体内或体外使用。为避免疑义，将治疗或诊断化合物以治疗或诊断有效量掺入本发明的组合物中。In a preferred embodiment, the antigen-binding polypeptide or protein is a therapeutic or diagnostic compound or vaccine. As used herein, a therapeutic compound is a compound that can be used to prevent a disease or condition, alleviate any symptom of a disease or condition, improve any disease or condition, slow the development of a disease or condition, etc. Diagnostic compounds can be used to determine the state of an organism, or to diagnose a disease, condition, symptom, or patient phenotype. The therapeutic compound must be administered to the patient, but diagnostic reagents can be used in vivo or in vitro, depending on the specific circumstances. For the avoidance of doubt, the therapeutic or diagnostic compound is incorporated into the composition of the present invention in a therapeutically or diagnostically effective amount.
在特别优选的实施方式中，本发明的组合物包含单克隆抗体或抗体片段，其是治疗有效的或可给药用于治疗疾病或病症，诸如自身免疫性疾病或炎性疾病、神经障碍、或癌症。用于治疗癌症的示例性单克隆抗体或抗体片段包括阿仑单抗(alemtuzumab)、贝伐单抗(bevacizumab)、西妥昔单抗(cetuximab)、吉妥珠单抗(gemtuzumab)、易普利姆玛(ipilimumab)、替伊莫(ibritumomab)、尼妥珠单抗(nimotuzumab)、奥法木单抗(ofatumumab)、帕尼单抗(panitumumab)、利妥昔单抗(rituximab)、托西莫单(tositumomab)、和曲妥珠单抗(trastuzumab)。用于治疗自身免疫性或炎性疾病的示例性单克隆抗体或抗体片段包括阿达木单抗、阿仑单抗、贝利单抗、briakinumab、康纳单抗、依库珠单抗、依帕珠单抗、依法珠单抗、戈利木单抗、英利昔单抗、美泊利单抗、那他珠单抗、奥法木单抗、ocrelizumab、otelixizumab、奥马珠单抗、瑞利珠单抗、利妥昔单抗、teplizumab、托珠单抗、优斯它单抗和维多珠单抗。可给药用于治疗、预防或诊断疾病或病症的单克隆抗体或抗体片段的其它实例包括巴利昔单抗、达利珠单抗、地诺单抗、依库珠单抗、帕利珠单抗和莫维珠单抗。In a particularly preferred embodiment, the compositions of the present invention comprise a monoclonal antibody or antibody fragment that is therapeutically effective or can be administered for the treatment of a disease or condition, such as an autoimmune disease or inflammatory disease, a neurological disorder, or cancer. Exemplary monoclonal antibodies or antibody fragments for the treatment of cancer include alemtuzumab, bevacizumab, cetuximab, gemtuzumab, ipilimumab, ibritumomab, nimotuzumab, ofatumumab, panitumumab, rituximab, tositumomab, and trastuzumab. Exemplary monoclonal antibodies or antibody fragments for treating autoimmune or inflammatory diseases include adalimumab, alemtuzumab, belimumab, briakinumab, canakinumab, eculizumab, epratuzumab, efalizumab, golimumab, infliximab, mepolizumab, natalizumab, ofatumumab, ocrelizumab, otelixizumab, omalizumab, reslizumab, rituximab, teplizumab, tocilizumab, ustekinumab, and vedolizumab. Other examples of monoclonal antibodies or antibody fragments that can be administered for treating, preventing, or diagnosing a disease or condition include basiliximab, daclizumab, denosumab, eculizumab, palivizumab, and motavizumab.
根据本发明的混悬液或分散体组合物可具体地包含癌症治疗试剂，所述癌症治疗试剂选自抗原结合的多肽或蛋白质，诸如单克隆抗体、多克隆抗体、抗体片段、包含抗体片段的融合蛋白、抗体-药物偶联物、或它们的任何组合，以及液体载体，所述液体载体包含式RFRH的半氟化烷烃，其中RF为具有4至12个碳原子的直链全氟化烃片段，并且其中RH为具有4至8个碳原子的直链烷基基团。充当血管生成抑制剂或能够抑制肿瘤细胞增殖(抗增殖剂)的抗原结合的多肽或蛋白质是尤其相关的。抗体片段可以为抗原结合片段(Fab)、单链可变片段(scFv)、单域抗体、微抗体、或双价抗体。The suspension or dispersion composition according to the present invention may specifically comprise a cancer therapeutic agent selected from an antigen-binding polypeptide or protein, such as a monoclonal antibody, a polyclonal antibody, an antibody fragment, a fusion protein comprising an antibody fragment, an antibody-drug conjugate, or any combination thereof, and a liquid carrier comprising a semifluorinated alkane of the formula RFRH, wherein RF is a linear perfluorinated hydrocarbon fragment having 4 to 12 carbon atoms, and wherein RH is a linear alkyl group having 4 to 8 carbon atoms. Antigen-binding polypeptides or proteins that act as an angiogenesis inhibitor or are capable of inhibiting tumor cell proliferation (antiproliferative agents) are particularly relevant. The antibody fragment may be an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a single-domain antibody, a mini-antibody, or a bivalent antibody.
例如，根据本发明的组合物可包含贝伐单抗和液体载体，其中所述液体载体包含式RFRH的半氟化烷烃，其中RF为具有4至12个碳原子的直链全氟化烃片段，并且其中RH为具有4至8个碳原子的直链烷基基团；并且其中贝伐单抗以分散体或混悬液的形式掺入组合物中。还可考虑贝伐单抗的抗原结合片段。贝伐单抗(商品名)是靶向VEGF-A(血管内皮生长因子A)并充当血管生长抑制剂的人源化鼠抗体。包含贝伐单抗的本发明组合物可用于治疗或预防疾病或病症，诸如结肠直肠癌、肺癌、乳腺癌、肾或脑(胶质母细胞瘤)癌、以及眼部疾病，如老年性黄斑变性(AMD)。For example, a composition according to the present invention may comprise bevacizumab and a liquid carrier, wherein the liquid carrier comprises a semifluorinated alkane of the formula RFRH, wherein RF is a linear perfluorinated hydrocarbon fragment having 4 to 12 carbon atoms, and wherein RH is a linear alkyl group having 4 to 8 carbon atoms; and wherein bevacizumab is incorporated into the composition in the form of a dispersion or suspension. Antigen-binding fragments of bevacizumab are also contemplated. Bevacizumab (trade name) is a humanized murine antibody that targets VEGF-A (vascular endothelial growth factor A) and acts as an angiogenesis inhibitor. Compositions of the present invention comprising bevacizumab can be used to treat or prevent diseases or conditions such as colorectal cancer, lung cancer, breast cancer, kidney or brain (glioblastoma) cancer, and eye diseases such as age-related macular degeneration (AMD).
其它组合物可包含以传递途径名Fsn0503和Fsn1006已知的抗体。Fsn1006是能够结合到人EGFR(表皮生长因子受体)配体双调蛋白和HB-EGF(肝素结合表皮生长因子)的双特异性抗体，并且其可用于抑制细胞增殖。Fsn1006是人源化IgG1/κ同种型。已经展示Fsn1006独立于细胞的K-ras突变状态起作用，从而具有优于当前EGFR靶向特性如西妥昔单抗的优点。Fsn0503也是人源化IgG1/κ抗体，其靶向并抑制人组织蛋白酶S的蛋白质水解活性。Fsn0503可用于治疗癌症，和其它血管相关疾病，尤其是涉及组织蛋白酶S介导的细胞外基质重塑的疾病。Other compositions may include antibodies known by the delivery names Fsn0503 and Fsn1006. Fsn1006 is a bispecific antibody capable of binding to the human EGFR (epidermal growth factor receptor) ligand amphiregulin and HB-EGF (heparin-binding epidermal growth factor) and can be used to inhibit cell proliferation. Fsn1006 is a humanized IgG1/κ isotype. Fsn1006 has been shown to function independently of the K-ras mutational status of the cell, thus offering advantages over current EGFR targeting properties such as cetuximab. Fsn0503 is also a humanized IgG1/κ antibody that targets and inhibits the proteolytic activity of human cathepsin S. Fsn0503 can be used to treat cancer and other vascular-related diseases, particularly those involving cathepsin S-mediated extracellular matrix remodeling.
在另外优选的实施方式中，根据本发明的混悬液或分散体组合物可包含TNF抑制剂，其选自抗原结合的多肽或蛋白质，诸如单克隆抗体、多克隆抗体、抗体片段、包含抗体片段的融合蛋白、抗体-药物偶联物、或它们的任何组合，以及液体载体，所述液体载体包含式RFRH的半氟化烷烃，其中RF为具有4至12个碳原子的直链全氟化烃片段，并且其中RH为具有4至8个碳原子的直链烷基基团。示例性THF抑制剂为英利昔单抗、依那西普和赛妥珠单抗及其生物仿制药。本发明的组合物可包含这些TNF抑制剂和液体载体，其中所述液体载体由选自下列项中的半氟化烷烃组成：F4H5、F4H6、F4H8、F6H4、F6H6、F6H8和F6H10。具体地，这些组合物可用于治疗影响胃肠系统的自身免疫性疾病，诸如克罗恩病、溃疡性结肠炎，或者影响关节和皮肤的病症，诸如类风湿性关节炎、银屑病性关节炎、强直性脊柱炎和斑块状银屑病。In another preferred embodiment, the suspension or dispersion composition according to the present invention may comprise a TNF inhibitor selected from an antigen-binding polypeptide or protein, such as a monoclonal antibody, a polyclonal antibody, an antibody fragment, a fusion protein comprising an antibody fragment, an antibody-drug conjugate, or any combination thereof, and a liquid carrier comprising a semifluorinated alkane of the formula RFRH, wherein RF is a linear perfluorinated hydrocarbon fragment having 4 to 12 carbon atoms, and wherein RH is a linear alkyl group having 4 to 8 carbon atoms. Exemplary THF inhibitors are infliximab, etanercept, and certolizumab pegol and biosimilars thereof. The composition of the present invention may comprise these TNF inhibitors and a liquid carrier, wherein the liquid carrier is composed of a semifluorinated alkane selected from the group consisting of F4H5, F4H6, F4H8, F6H4, F6H6, F6H8, and F6H10. In particular, these compositions can be used to treat autoimmune diseases affecting the gastrointestinal system, such as Crohn's disease, ulcerative colitis, or conditions affecting the joints and skin, such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and plaque psoriasis.
在另一个实施方式中，本发明的组合物包含浓度优选为至少0.5mg/mL，诸如0.5-10mg/ml的抗原结合的多肽或蛋白质。在另外优选的实施方式中，浓度为至少1mg/mL、至少5mg/mL、至少10mg/mL、至少15mg/mL、至少25mg/mL、或至少35mg/mL。In another embodiment, the compositions of the present invention comprise an antigen-binding polypeptide or protein at a concentration of preferably at least 0.5 mg/mL, such as 0.5-10 mg/ml. In another preferred embodiment, the concentration is at least 1 mg/mL, at least 5 mg/mL, at least 10 mg/mL, at least 15 mg/mL, at least 25 mg/mL, or at least 35 mg/mL.
在另一个方面，本发明提出稳定抗原结合的多肽或蛋白质的方法，所述方法包括将抗原结合的多肽或蛋白质与包含半氟化烷烃的液体载体混合的步骤。所述半氟化烷烃具有式RFRH，其中RF为具有4至12个碳原子的直链全氟化烃片段，并且其中RH为具有4至8个碳原子的直链烷基基团。根据所述方法，进行将抗原结合的多肽或蛋白质与液体载体混合从而形成混悬液或分散体的步骤。这种稳定抗原结合的多肽或蛋白质的方法可用于制备组合物，所述组合物用作诸如治疗、预防或诊断有需要的患者的疾病或病症时的药物。任选地，这种稳定的方法还可用于制备、制造或合成抗原结合的多肽或蛋白质。In another aspect, the present invention provides a method for stabilizing an antigen-binding polypeptide or protein, comprising the step of mixing the antigen-binding polypeptide or protein with a liquid carrier comprising a semifluorinated alkane. The semifluorinated alkane has the formula RFRH, wherein RF is a linear perfluorinated hydrocarbon fragment having 4 to 12 carbon atoms, and wherein RH is a linear alkyl group having 4 to 8 carbon atoms. According to the method, the step of mixing the antigen-binding polypeptide or protein with the liquid carrier to form a suspension or dispersion is performed. This method for stabilizing an antigen-binding polypeptide or protein can be used to prepare a composition for use as a medicament, such as for treating, preventing, or diagnosing a disease or condition in a patient in need thereof. Optionally, this stabilization method can also be used to prepare, manufacture, or synthesize an antigen-binding polypeptide or protein.
如本文所用，术语稳定性被定义为维持一段时间内的抗原结合的多肽或蛋白质的化学或物理完整性和/或生物活性。稳定抗原结合的多肽或蛋白质包括防止或延缓抗原结合的多肽或蛋白质从其生物和/或治疗活性形式降解或变质成非活性形式。不稳定性可源于诸如以下情况：聚集、变性、裂解、或化学改性诸如氧化、交联、脱酰胺和与在包含抗原结合的多肽或蛋白质的组合物中起主要作用的其它组分反应。As used herein, the term stability is defined as maintaining the chemical or physical integrity and/or biological activity of an antigen-binding polypeptide or protein over a period of time. Stabilizing an antigen-binding polypeptide or protein includes preventing or delaying degradation or deterioration of the antigen-binding polypeptide or protein from its biologically and/or therapeutically active form to an inactive form. Instability can result from, for example, aggregation, denaturation, cleavage, or chemical modifications such as oxidation, cross-linking, deamidation, and reaction with other components that play a major role in the composition comprising the antigen-binding polypeptide or protein.
组合物中抗原结合蛋白质或多肽的稳定性可使用本领域已知的方法表征，已知的方法包括但不限于，利用免疫检测技术如ELISA，或者利用测定抗原结合蛋白质或多肽的纯度或物理/化学变化的其它技术(诸如尺寸排阻色谱法、毛细管凝胶电泳、圆二色性、或质谱)测量生物活性诸如抗原结合活性。通过比较经由这些类型的表征方法在初始时间点，诸如在配制或制备组合物(即，如可以为混悬液或分散体的情况)时获得的测量值，和在稍后的时间点即在给定环境或条件下储存之后获得的那些测量值来测定稳定性。The stability of the antigen-binding protein or polypeptide in the composition can be characterized using methods known in the art, including but not limited to, measuring biological activity such as antigen-binding activity using immunoassay techniques such as ELISA, or other techniques for determining the purity or physical/chemical changes of the antigen-binding protein or polypeptide (such as size exclusion chromatography, capillary gel electrophoresis, circular dichroism, or mass spectrometry. Stability is determined by comparing the measurements obtained at an initial time point, such as when the composition is formulated or prepared (i.e., as in the case of a suspension or dispersion), and at a later time point, i.e., after storage under a given environment or conditions.
本发明人已经发现在包含半氟化烷烃的液体载体中的抗原结合蛋白质在25℃下保持稳定至少6个月。更显著地，在40℃的温度下储存相同时间段时，抗原结合蛋白质是同等稳定的。即，包含抗原结合蛋白质的组合物有效保持与其初始抗原结合活性相同或相似的抗原结合活性。The present inventors have discovered that antigen-binding proteins in a liquid carrier comprising a semifluorinated alkane remain stable for at least 6 months at 25° C. More significantly, the antigen-binding proteins are equally stable when stored for the same period of time at a temperature of 40° C. That is, the composition comprising the antigen-binding protein effectively retains an antigen-binding activity that is the same as or similar to its initial antigen-binding activity.
在优选的实施方式中，本发明的组合物在25℃下，或在室温下，或在介于室温和40℃之间的温度下储存3个月期间，保持其初始抗原结合活性的至少85％或至少90％，诸如90-95％，或甚至大于95％。在另一个优选的实施方式中，本发明的组合物在25℃下，或在室温(RT)下，或在介于RT和40℃之间的温度下储存6个月期间，保持其初始抗原结合活性的至少85％或至少90％，诸如90-95％，或甚至大于95％。在另一个实施方式中，本发明的组合物在介于RT和40℃之间和50-75％之间的湿度下储存6个月期间，保持其初始抗原结合活性的至少85％。在其它实施方式中，储存时间段可以为4-6周、6-12周、或3-6个月或6-12个月。在另外的实施方式中，储存期间的湿度(RH)可以为至少40％或至少50％，或至少65％或至少75％。In a preferred embodiment, the compositions of the present invention are stored at 25 ℃, or at room temperature, or during storage for 3 months at a temperature between room temperature and 40 ℃, and keep at least 85% or at least 90% of their initial antigen-binding activity, such as 90-95%, or even greater than 95%. In another preferred embodiment, the compositions of the present invention are stored at 25 ℃, or at room temperature (RT), or during storage for 6 months at a temperature between RT and 40 ℃, and keep at least 85% or at least 90% of their initial antigen-binding activity, such as 90-95%, or even greater than 95%. In another embodiment, the compositions of the present invention are stored at a humidity between RT and 40 ℃ and 50-75% for 6 months, and keep at least 85% of their initial antigen-binding activity. In other embodiments, the storage period can be 4-6 weeks, 6-12 weeks, or 3-6 months or 6-12 months. In other embodiments, the humidity (RH) during storage can be at least 40% or at least 50%, or at least 65% or at least 75%.
如所提及的，将抗原结合的多肽或蛋白质掺入组合物中从而形成分散体或混悬液。换句话讲，抗原结合的多肽或蛋白质分散或悬浮于包含半氟化烷烃的液体载体中。混悬液是否在抗原结合蛋白质分散于液体载体中时形成取决于例如抗原结合蛋白质的性质，其在载体中的浓度，以及选择的SFA。As mentioned, the antigen-binding polypeptide or protein is incorporated into the composition to form a dispersion or suspension. In other words, the antigen-binding polypeptide or protein is dispersed or suspended in a liquid carrier comprising a semifluorinated alkane. Whether a suspension is formed when the antigen-binding protein is dispersed in the liquid carrier depends, for example, on the properties of the antigen-binding protein, its concentration in the carrier, and the SFA selected.
如本文所使用的，混悬液可定义为一类分散体，即，具有至少一个连续(或连贯)相和分散于连续相中的至少一个不连续(或内)相的系统。在混悬液中，分散相处于固态。可用于实施本发明的混悬液至少在生理温度下为液体，这是指连续相为液体。通常，混悬液在室温下也为液体。除了混悬液之外，术语分散体应理解为包括胶态体系，其中抗原结合蛋白质和多肽精细分散于液相中。在一些实施方式中，抗原结合的多肽或蛋白质还至少部分溶剂化。As used herein, a suspension can be defined as a type of dispersion, i.e., a system having at least one continuous (or coherent) phase and at least one discontinuous (or internal) phase dispersed in the continuous phase. In a suspension, the dispersed phase is in a solid state. Suspensions useful in practicing the present invention are liquid at least at physiological temperature, meaning that the continuous phase is liquid. Typically, suspensions are also liquid at room temperature. In addition to suspensions, the term dispersion should be understood to include colloidal systems in which antigen-binding proteins and polypeptides are finely dispersed in a liquid phase. In some embodiments, the antigen-binding polypeptide or protein is also at least partially solvated.
抗原结合的多肽或蛋白质的稳定化混悬液或分散体经由以下方法制备，所述方法包括将抗原结合的多肽或蛋白质与包含半氟化烷烃的液体载体混合的步骤。所得混悬液或分散体的稳定性可通过使用诸如本领域所已知的那些方法测量各种物理属性来表征，所述物理属性包括但不限于，例如，悬浮颗粒的再分散性、粒度分布和随时间推移的粒度增长。A stabilized suspension or dispersion of an antigen-binding polypeptide or protein is prepared by a method comprising mixing the antigen-binding polypeptide or protein with a liquid carrier comprising a semifluorinated alkane. The stability of the resulting suspension or dispersion can be characterized by measuring various physical properties such as those known in the art, including, but not limited to, redispersibility of the suspended particles, particle size distribution, and particle size growth over time.
在一个特定实施方式中，组合物仅包含抗原结合的多肽或蛋白质以及一种或多种SFA，即组合物由抗原结合的多肽或蛋白质以及如上定义的一种或多种SFA组成。在另一个优选的实施方式中，包含抗原结合的多肽或蛋白质以及一种或多种SFA的组合物有效地或基本上不含水，即组合物不包含水，除了可能经由其它固体或液体组分或抗原结合的多肽或蛋白质本身引入的残余水量之外。在其它情况下，包含抗原结合的多肽或蛋白质以及包含半氟化烷烃的液体载体的混悬液或分散体组合物可以不含水。In a specific embodiment, the composition comprises only the antigen-binding polypeptide or protein and one or more SFAs, i.e., the composition consists of the antigen-binding polypeptide or protein and one or more SFAs as defined above. In another preferred embodiment, the composition comprising the antigen-binding polypeptide or protein and one or more SFAs is effectively or substantially free of water, i.e., the composition does not contain water, except for residual water that may be introduced via other solid or liquid components or the antigen-binding polypeptide or protein itself. In other cases, the suspension or dispersion composition comprising the antigen-binding polypeptide or protein and a liquid carrier comprising a semifluorinated alkane may be free of water.
与现有技术中已知的一些其它混悬液或分散体相反，本发明的制剂对于其物理稳定性而言，不需要表面活性剂或仅需要少量表面活性剂。这是显著的优点，因为表面活性剂具有刺激性和局部毒性的可能性相当大，尤其是当通过皮下或肌内注射或通过滴注到眼中来给药时。根据一个优选的实施方式，本发明的组合物基本上不含表面活性剂。在另一个实施方式中，如果掺入多于一种表面活性剂，则一种或多种表面活性剂的总量分别不大于约10重量％，具体地不大于约5重量％，或优选不大于约2重量％。在另外优选的实施方式中，所述量分别不大于约1重量％，或不大于约0.5重量％。在该情况下，如本文所述的SFA，虽然由于其化学结构而具有一些两亲性，但不应被理解为是在表面活性剂的范围内，所述化学结构包括氟化或非氟化烷基(或亚烷基)基团，所述基团的特征在于不同程度的亲脂性。In contrast to some other suspensions or dispersions known in the prior art, the formulations of the present invention do not require a surfactant or only require a small amount of a surfactant for their physical stability. This is a significant advantage because surfactants are very likely to be irritating and locally toxic, especially when administered by subcutaneous or intramuscular injection or by instillation into the eye. According to a preferred embodiment, the composition of the present invention is substantially free of surfactants. In another embodiment, if more than one surfactant is incorporated, the total amount of one or more surfactants is no more than about 10% by weight, specifically no more than about 5% by weight, or preferably no more than about 2% by weight. In another preferred embodiment, the amount is no more than about 1% by weight, or no more than about 0.5% by weight. In this case, the SFAs described herein, although having some amphiphilicity due to their chemical structure, should not be understood to be within the scope of surfactants, which chemical structure includes fluorinated or non-fluorinated alkyl (or alkylene) groups characterized by varying degrees of lipophilicity.
不存在或仅少量存在的表面活性剂包括非离子、阳离子、阴离子和两性离子表面活性剂，如通常用作各种类型的药物组合物中的赋形剂，例如作为润湿剂、乳化剂、分散剂、增溶剂等。被认为是潜在可用的表面活性剂的实例包括泰洛沙泊，泊洛沙姆诸如PluronicF68LF或Lutrol F68、Pluronic L-G2LF和Pluronic L62D，聚山梨醇酯例如聚山梨醇酯20和聚山梨醇酯80，聚氧乙烯蓖麻油衍生物，脱水山梨醇酯，聚氧乙烯硬脂酸酯，卵磷脂，纯化或合成的磷脂，以及它们中的两种或更多种的混合物。Surfactants that are absent or present only in small amounts include nonionic, cationic, anionic and zwitterionic surfactants, such as those commonly used as excipients in various types of pharmaceutical compositions, for example, as wetting agents, emulsifiers, dispersants, solubilizers, etc. Examples of surfactants considered to be potentially useful include tyloxapol, poloxamers such as Pluronic F68LF or Lutrol F68, Pluronic L-G2LF and Pluronic L62D, polysorbates such as polysorbate 20 and polysorbate 80, polyoxyethylene castor oil derivatives, sorbitan esters, polyoxyethylene stearates, lecithin, purified or synthetic phospholipids, and mixtures of two or more thereof.
本发明的组合物可任选包含非氟化有机液体，例如以便改变液体载体的特性，诸如粘度。此类其它液体可以为选自甘油酯油、液体蜡和液体石蜡中的油，或表现出高度生物相容性的有机溶剂，或多于一种液体赋形剂的混合物。The compositions of the present invention may optionally contain a non-fluorinated organic liquid, for example to modify the properties of the liquid carrier, such as viscosity. Such other liquids may be oils selected from glyceride oils, liquid waxes and liquid paraffins, or organic solvents exhibiting a high degree of biocompatibility, or mixtures of more than one liquid excipient.
可与一种或多种SFA组合使用的潜在可用的油性赋形剂的实例包括甘油三酯油(即大豆油、橄榄油、芝麻油、棉子油、蓖麻油、甜杏仁油)、矿物油(即凡士林和液体石蜡)、中链甘油三酯(MCT)、油性脂肪酸、肉豆蔻酸异丙酯、油性脂肪醇、山梨醇和脂肪酸的酯、油性蔗糖酯、或在生理上由眼部耐受的任何其它油性物质。Examples of potentially useful oily excipients that can be used in combination with one or more SFAs include triglyceride oils (i.e., soybean oil, olive oil, sesame oil, cottonseed oil, castor oil, sweet almond oil), mineral oils (i.e., petrolatum and liquid paraffin), medium chain triglycerides (MCTs), oily fatty acids, isopropyl myristate, oily fatty alcohols, esters of sorbitol and fatty acids, oily sucrose esters, or any other oily substance that is physiologically tolerated by the eye.
潜在可用的有机溶剂的实例包括甘油、丙二醇、聚乙二醇和乙醇。助溶剂的浓度应当优选相对于SFA或SFA混合物的浓度是低的。如果使用有机溶剂诸如乙醇，则可推荐使其保持在低于约5重量％的水平。更优选地，乙醇的含量为约0.1至约2重量％，并且最优选地不大于约1重量％。The example of potential available organic solvent comprises glycerine, propylene glycol, polyethylene glycol and ethanol.The concentration of cosolvent should preferably be low with respect to the concentration of SFA or SFA mixture.If using organic solvent such as ethanol, then can recommend making it remain on the level below about 5 % by weight.More preferably, the content of ethanol is about 0.1 to about 2 % by weight, and most preferably is not more than about 1 % by weight.
当然，所述组合物包含其它根据需要或有用的药物赋形剂。潜在有用的赋形剂包括酸、碱、抗氧化剂、稳定剂、增效剂、着色剂、增稠剂、以及(如果在特定的情况下需要的)防腐剂。然而，本发明通常提供配制微生物稳定的非水性组合物的方法。这是由于SFA通常不易于发生微生物污染的事实。因此，其可配制待填充到多用容器中的不含防腐剂的组合物。不含防腐剂的组合物由许多患者更好地耐受并能够降低最终产品的成本。Certainly, described composition comprises other as needed or useful pharmaceutical excipient.Potential useful excipient comprises acid, alkali, antioxidant, stabilizer, synergist, coloring agent, thickener and (if needed in specific case) preservative.Yet the present invention generally provides the method for preparing microbiologically stable non-aqueous composition.This is because SFA is not easy to occur the fact of microbial contamination usually.Therefore, it can prepare the composition that does not contain preservative to be filled in the multipurpose container.The composition that does not contain preservative is better tolerated by many patients and can reduce the cost of final product.
本发明的液体混悬液可通过常规方法制备。原则上，包含活性成分的固体颗粒可分散到包含SFA的液体载体中。另选地，颗粒可通过在可控条件下将活性成分(和任选地，一种或多种固体赋形剂)的(通常为有机的)溶液加入基于SFA的载体中来原位沉淀。Liquid suspensions of the present invention can be prepared by conventional methods. In principle, solid particles comprising the active ingredient can be dispersed in a liquid carrier comprising SFA. Alternatively, the particles can be precipitated in situ by adding a (usually organic) solution of the active ingredient (and optionally, one or more solid excipients) to an SFA-based carrier under controlled conditions.
固体颗粒可通过冻干或喷雾干燥抗原结合蛋白质或颗粒的溶液来制备。所述溶液可以为水性或非水性的并且还可包含可能有用或需要的药物赋形剂。Solid particles can be prepared by lyophilizing or spray drying a solution of the antigen binding protein or particles. The solution can be aqueous or non-aqueous and may also contain pharmaceutical excipients that may be useful or desired.
还可在颗粒与液体载体混合之前或之后调节分散相的粒度。在一个优选的实施方式中，提供已具有经适当选择的粒度的活性成分的颗粒。具有此类选择粒度的粉末可通过结晶工程由相应化合物的合成直接获得，或使用标准设备诸如球磨机、锤磨机、辊磨机、胶体磨、喷磨机等通过传统的研磨或铣削方法在合成之后获得。如果在制备混悬液之后减小颗粒直径，则可使用超声破碎法以及各种类型的匀浆器，诸如胶体磨或高压匀浆器。The particle size of the dispersed phase can also be adjusted before or after the particles are mixed with the liquid carrier. In a preferred embodiment, particles of the active ingredient having a properly selected particle size are provided. Powders with such selected particle sizes can be obtained directly from the synthesis of the corresponding compound by crystallization engineering, or obtained after synthesis by conventional grinding or milling methods using standard equipment such as ball mills, hammer mills, roller mills, colloid mills, jet mills, etc. If the particle diameter is reduced after preparing the suspension, ultrasonic crushing and various types of homogenizers, such as colloid mills or high-pressure homogenizers, can be used.
本发明还提供用于治疗、预防或诊断有需要的患者的疾病或病症的方法，所述方法包括对患者给药优选呈混悬液或分散体形式的组合物的步骤，所述组合物包含抗原结合的多肽或蛋白质和液体载体，其中所述液体载体包含半氟化烷烃。根据本发明的混悬液的优异的物理特性使得这些组合物尤其可用于局部给药于患者的眼部、耳部、鼻部或肺部，或通过注射肠胃外给药。例如，本发明的组合物可通过局部施用或注射而给药于患者的眼部。注射的优选模式包括真皮注射、皮下注射、肌内注射、和局部区域注射。最优选的是皮下和肌肉注射给药途径。The present invention also provides a method for treating, preventing or diagnosing a disease or condition in a patient in need thereof, the method comprising administering to the patient a composition, preferably in the form of a suspension or dispersion, comprising an antigen-binding polypeptide or protein and a liquid carrier, wherein the liquid carrier comprises a semifluorinated alkane. The excellent physical properties of the suspensions according to the present invention make these compositions particularly useful for topical administration to the patient's eyes, ears, nose or lungs, or parenteral administration by injection. For example, the compositions of the present invention can be administered to the patient's eyes by topical application or injection. Preferred modes of injection include dermal injection, subcutaneous injection, intramuscular injection, and localized regional injection. Subcutaneous and intramuscular routes of administration are most preferred.
实施例Example
实施例1Example 1
研究经过6个月的时间段，抗ECSCR(内皮细胞特异性趋化性调节器)单克隆抗体(克隆ID 13G11 1A31 A7，由结合到内皮标记ECSCR的杂交瘤细胞表达的鼠IgG/k抗体)在F6H8中在25℃和40℃下的稳定性。The stability of anti-ECSCR (endothelial cell-specific chemotaxis regulator) monoclonal antibody (clone ID 13G11 1A31 A7, a murine IgG/kappa antibody expressed by hybridoma cells that binds to the endothelial marker ECSCR) was studied in F6H8 at 25°C and 40°C over a period of 6 months.
将抗ECSCR单克隆抗体的冻干的0.25mg样品(最初储存在-80℃下PBS缓冲液中)重组成在F6H8中浓度为1mg/mL。将重组的样品储存在卷曲玻璃瓶中在25℃/60％RH和40℃/75％RH下。在储存3个月和6个月之后，测定这些样品的结合活性。Lyophilized 0.25 mg samples of anti-ECSCR monoclonal antibody (originally stored in PBS buffer at -80°C) were reconstituted in F6H8 at a concentration of 1 mg/mL. The reconstituted samples were stored in crimped glass bottles at 25°C/60% RH and 40°C/75% RH. The binding activity of these samples was determined after 3 and 6 months of storage.
为用作第一对照，将冻干抗体的其它样品以冻干形式储存在-80℃下。仅在分析之前将这些样品重组于PBS中。第二对照由抗体的PBS溶液的样品组成，其从未冻干，但保持冷藏在4℃下。To serve as a first control, additional samples of lyophilized antibody were stored in lyophilized form at -80°C. These samples were reconstituted in PBS just before analysis. A second control consisted of a sample of antibody in PBS solution that was never lyophilized but kept refrigerated at 4°C.
抗体样品对ECSCR抗原的结合活性使用以下方案由ELISA测定：The binding activity of the antibody samples to the ECSCR antigen was determined by ELISA using the following protocol:
通过用ECSCR抗原和不相关的阴性抗原涂覆来制备Nunc MaxiSorpTM平底Elisa板。所述抗原以1μg/mL的浓度稀释于涂覆液中，并在4℃下用板温育过夜。然后所述板用200μL的3％封闭液封闭并在RT下在振荡器上温育1-2小时，之后用PBS-Tween 20溶液洗涤三次并吸干。将抗体样品在PBS中稀释至1μg/mL。将100ng或10ng的每个经稀释样品加入涂覆有ECSCR抗原的孔和涂覆有阴性对照抗原的孔中。所述板在振荡器上于RT下温育1h，然后用PBS-Tween 20溶液洗涤并吸干。用在PBS中以1:5000稀释的山羊抗-小鼠IgG-HRP二次抗体偶联物(Biorad，目录编号170-6516)探测抗体等分试样。所述板在振荡器上于RT下温育1h，之后用PBS-Tween 20溶液洗涤三次并吸干。所述板与100μL的TMB(3,3’,5,5’-四甲基联苯胺)在37℃下温育10min，然后加入50μL/孔的1M HCl。用分光光度计测量450nm下的吸光度。Nunc MaxiSorp™ flat-bottom Elisa plates were prepared by coating with ECSCR antigen and an irrelevant negative antigen. The antigen was diluted in the coating solution at a concentration of 1 μg/mL and incubated overnight with the plate at 4°C. The plate was then blocked with 200 μL of 3% blocking solution and incubated on a shaker for 1-2 hours at RT, followed by washing three times with PBS-Tween 20 solution and blotting. The antibody sample was diluted to 1 μg/mL in PBS. 100 ng or 10 ng of each diluted sample was added to the wells coated with ECSCR antigen and the wells coated with the negative control antigen. The plate was incubated on a shaker at RT for 1 hour, then washed with PBS-Tween 20 solution and blotted. Antibody aliquots were detected with a goat anti-mouse IgG-HRP secondary antibody conjugate (Biorad, catalog number 170-6516) diluted 1:5000 in PBS. The plate was incubated on a shaker at room temperature for 1 hour, then washed three times with PBS-Tween 20 and blotted dry. The plate was incubated with 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) at 37°C for 10 minutes, followed by the addition of 50 μL/well of 1 M HCl. The absorbance at 450 nm was measured using a spectrophotometer.
比较在初始重组时间点下和对照样品测得的结合活性示出在25℃下或者甚至在40℃的较高温度下储存的F6H8中的抗体的结合活性(由三个样品的平均值测定)在储存经过6个月时间段之后被保持(图1)。所述结合活性可比于对于在-80℃下储存的冻干抗-ECSCR单克隆抗体样品所观察的结合活性(对照1，图1)和对于在4℃下储存于PBS中的从未冻干样品所观察到的结合活性(对照2，图1)。Comparison of the binding activity measured at the initial reconstitution time point and the control sample showed that the binding activity of the antibody in F6H8 stored at 25°C or even at a higher temperature of 40°C (determined from the average of three samples) was maintained after storage for a period of 6 months (Figure 1). The binding activity was comparable to that observed for lyophilized anti-ECSCR monoclonal antibody samples stored at -80°C (Control 1, Figure 1) and for the unlyophilized sample stored in PBS at 4°C (Control 2, Figure 1).
实施例2：Example 2:
研究在25℃、50℃和70℃下经过4周时间的贝伐单抗、Fsn1006和Fsn0503的稳定性。The stability of bevacizumab, Fsn1006, and Fsn0503 was studied at 25°C, 50°C, and 70°C over a period of 4 weeks.
冻干方案：获得上文所列抗体中每一个的储备溶液；Fsn1006和Fsn0503以PBS的溶液形式，贝伐单抗(商品名)以其商业储存缓冲液形式。将80mg贝伐单抗的等分试样转移到>7000MWCO透析管中并针对3x 2L体积的PBS透析72小时。抗体溶液在PBS中稀释成0.5mg/ml，并且将500μl的每种溶液等分至单独的5ml琥珀色玻璃药用级小瓶中用于冻干。冻干经过48小时周期来完成。Lyophilization protocol: A stock solution of each of the above-listed antibodies was obtained; Fsn1006 and Fsn0503 were in PBS solution, and bevacizumab (trade name) was in its commercial storage buffer. An 80 mg aliquot of bevacizumab was transferred to a >7000 MWCO dialysis tubing and dialyzed for 72 hours against 3 x 2 L volumes of PBS. The antibody solution was diluted to 0.5 mg/ml in PBS, and 500 μl of each solution was aliquoted into separate 5 ml amber glass pharmaceutical grade vials for lyophilization. Lyophilization was completed over a 48 hour period.
冻干抗体的再悬浮：小心利用涡旋将冻干抗体以0.5mg/mL的浓度悬浮于F4H5、F6H8、50体积％F4H5的F6H8溶液、PBS(137mM NaCl、2.7mM KCl、10mM Na2HPO4、1.8mMKH2PO4，pH＝7.4)中。冻干抗体在PBS中的再悬浮形成溶液，然而利用半氟化烷烃形成混悬液。在25℃和50℃下将所得的混悬液和溶液储存在琥珀酸玻璃小瓶中4周(28天)。在23天之后，使50℃样品经受70℃条件。Resuspension of lyophilized antibodies: Carefully vortex the lyophilized antibodies at a concentration of 0.5 mg/mL in F4H5, F6H8, 50% by volume of F4H5 in F6H8, PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4 , 1.8 mM KH2 PO4 , pH = 7.4). Resuspension of the lyophilized antibodies in PBS forms a solution, whereas a suspension is formed using a semifluorinated alkane. The resulting suspensions and solutions are stored in succinic acid glass vials at 25° C. and 50° C. for 4 weeks (28 days). After 23 days, the 50° C. samples are subjected to 70° C. conditions.
抗体样品的结合活性通过在t＝0(再悬浮之后立即)、t＝2周和t＝4周时进行ELISA测试来测定。作为对照，使用从未再悬浮的抗体样品(储存在-80℃下)和从未冻干的抗体的PBS溶液(储存在4℃下)。The binding activity of the antibody samples was determined by ELISA at t = 0 (immediately after resuspension), t = 2 weeks and t = 4 weeks. As controls, unresuspended antibody samples (stored at -80°C) and unlyophilized antibody in PBS (stored at 4°C) were used.
ELISA方案：对于每种抗体的ELISA测试而言，通过添加100ng/孔的适当抗原的100μl 0.2M碳酸盐缓冲溶液(pH 9.5)用目标抗原涂覆Nunc Maxisorp96孔板，并将其在37℃下温育1小时。在用包含0.1％tween 20(PBS-T)的PBS洗涤之后，通过添加200μl 4％奶粉的PBS溶液并且在振荡器上在室温下温育2小时来封闭所述板。在进一步洗涤之后，施用抗体样品。将1ml的x0.5 PBS加入每个非水性小瓶中。通过温和摇摆5分钟将抗体萃取到溶液中。然后将1μl水层转移到包含999μl PBS的小瓶中(以获得50ng/孔的标称值)。然后以100μl的6次重复将100μl的每个样品瓶铺板。所述板在4℃下温育过夜。洗涤之后，在PBS中以1:60,000稀释施用二次抗体(山羊抗人HRP偶联物)；向每个孔中添加100μl并且将板在振摇下于室温下温育1小时。然后将所述板用3体积的PBS-T洗涤之后用2体积的PBS洗涤。然后施用100μl的TMB溶液并且所述板在37℃下温育10分钟。通过添加50μl 1M HCl停止反应。读取每个孔在λ＝450nm下的吸光度。ELISA protocol: For the ELISA test of each antibody, 100 μl of 0.2M carbonate buffer solution (pH 9.5) of the appropriate antigen added to 100 ng/well of Nunc Maxisorp 96-well plates were coated with the target antigen and incubated at 37°C for 1 hour. After washing with PBS containing 0.1% tween 20 (PBS-T), the plates were blocked by adding 200 μl of 4% milk powder in PBS and incubating on a shaker at room temperature for 2 hours. After further washing, the antibody samples were applied. 1 ml of x0.5 PBS was added to each non-aqueous vial. The antibodies were extracted into the solution by gentle shaking for 5 minutes. The 1 μl aqueous layer was then transferred to a vial containing 999 μl PBS (to obtain a nominal value of 50 ng/well). Each sample bottle of 100 μl was then plated with 6 replicates of 100 μl. The plates were incubated overnight at 4°C. After washing, a secondary antibody (goat anti-human HRP conjugate) was applied at a 1:60,000 dilution in PBS; 100 μl was added to each well and the plate was incubated at room temperature with shaking for 1 hour. The plate was then washed with 3 volumes of PBS-T followed by 2 volumes of PBS. 100 μl of TMB solution was then applied and the plate was incubated at 37°C for 10 minutes. The reaction was stopped by adding 50 μl of 1 M HCl. The absorbance of each well was read at λ = 450 nm.
表1：贝伐单抗活性(450nm下的平均OD±2SD)Table 1: Bevacizumab activity (mean OD at 450 nm ± 2SD)
表2：Fsn1006活性(450nm下的平均OD±2SD)Table 2: Fsn1006 activity (mean OD at 450 nm ± 2SD)
表3：Fsn0503活性(450nm下的平均OD±2SD)Table 3: Fsn0503 activity (mean OD at 450 nm ± 2SD)
在25℃和50℃下4周的时间段期间，包括在50℃样品增加至70℃的储存温度的该时间段的最后5天期间，以混悬液形式储存在F4H5、F6H8和50体积％F4H5的F6H8溶液中的贝伐单抗、Fsn1006和Fsn0503的活性基本上保持一致。相反，不考虑储存温度，由这些抗体的PBS缓冲溶液展示的结合活性在四周时间段末显著降低。The activities of bevacizumab, Fsn1006, and Fsn0503 stored as suspensions in F4H5, F6H8, and a 50% by volume solution of F4H5 in F6H8 remained essentially consistent over a 4-week period at both 25°C and 50°C, including during the final 5 days of this period when the 50°C sample was increased to a storage temperature of 70°C. In contrast, the binding activity exhibited by PBS-buffered solutions of these antibodies decreased significantly by the end of the four-week period, regardless of storage temperature.
实施例3Example 3
检查在50℃和80℃之间的温度下，储存在SFA和PBS缓冲液中的贝伐单抗、Fsn1006和Fsn0503的结合活性。The binding activities of bevacizumab, Fsn1006, and Fsn0503 stored in SFA and PBS buffers at temperatures between 50°C and 80°C were examined.
小心利用涡旋将冻干抗体贝伐单抗、Fsn1006和Fsn0503(如上文实例2中所制备的)以0.5mg/mL的浓度再悬浮于F4H5、F6H8和PBS中。这些冻干抗体在PBS中的再悬浮形成溶液，然而利用SFA形成混悬液。Lyophilized antibodies bevacizumab, Fsn1006, and Fsn0503 (prepared as described above in Example 2) were carefully resuspended in F4H5, F6H8, and PBS at a concentration of 0.5 mg/mL using vortexing. Resuspension of these lyophilized antibodies in PBS formed a solution, whereas SFA formed a suspension.
在PBS缓冲液萃取和使用如上文实例2中所述的ELISA方法活性分析之前，将所述混悬液/溶液样品分别在50℃、55℃、60℃、65℃、70℃、75℃和80℃下保持2h时间段，然后冷却至10℃。每个实验重复进行三次。将在40℃下加热的样品用作对照。Prior to extraction with PBS buffer and analysis of activity using the ELISA method described in Example 2 above, the suspension/solution samples were kept at 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, and 80°C for a period of 2 h, and then cooled to 10°C. Each experiment was performed in triplicate. Samples heated at 40°C were used as controls.
发现与样品的含水缓冲液溶液相比，贝伐单抗(图2)、Fsn1006(图3)和Fsn0503(图4)的F4H5和F6H8混悬液展示对热变性的显著稳定性。在这些半氟化烷烃中配制的抗体的结合活性在50℃和80℃之间保持相当恒定。相反，储存在含水PBS缓冲液中的这些抗体的活性在高于60℃的温度下急剧降低。It was found that the F4H5 and F6H8 suspensions of bevacizumab (Figure 2), Fsn1006 (Figure 3), and Fsn0503 (Figure 4) exhibited remarkable stability to thermal denaturation compared to the aqueous buffer solutions of the samples. The binding activity of the antibodies formulated in these semifluorinated alkanes remained fairly constant between 50°C and 80°C. In contrast, the activity of these antibodies stored in aqueous PBS buffer decreased dramatically at temperatures above 60°C.
实施例4Example 4
研究包含抗原结合的多肽或蛋白质以及液体载体的组合物的稳定性并与不同介质中的制剂进行比较，所述抗原结合的多肽或蛋白质选自英夫利昔单抗(具有人恒定区和鼠可变区的嵌合单克隆抗体，并且分子量为149kDa)，融合蛋白依那西普(分子量为150kDa的重组人蛋白，其包含融合到IgG1的Fc部分的75kDa TNFR(肿瘤坏死因子受体)的配体结合部分)，和赛妥珠单抗(分子量为91kDa的抗体片段，其包括偶联到约40kDa聚乙二醇的重组Fab’)或它们的生物仿制药，所述液体载体包含式RFRH的半氟化烷烃，其中RF为具有4至12个碳原子的直链全氟化烃片段，并且其中RH为具有4至8个碳原子的直链烷基基团(例如F4H5和F6H8)。The stability of a composition comprising an antigen-binding polypeptide or protein selected from infliximab (a chimeric monoclonal antibody having human constant and murine variable regions and having a molecular weight of 149 kDa), the fusion protein etanercept (a recombinant human protein having a molecular weight of 150 kDa comprising the ligand-binding portion of a 75 kDa TNFR (tumor necrosis factor receptor) fused to the Fc portion of IgG1), and certolizumab pegol (an antibody fragment having a molecular weight of 91 kDa comprising a recombinant Fab' coupled to approximately 40 kDa polyethylene glycol) or their biosimilars and a liquid carrier comprising a semifluorinated alkane of the formula RFRH, wherein RF is a linear perfluorinated hydrocarbon fragment having 4 to 12 carbon atoms, and wherein RH is a linear alkyl group having 4 to 8 carbon atoms (e.g., F4H5 and F6H8), was studied and compared with formulations in different media.
将所述抗体冻干并再悬浮于半氟化烷烃中。还在其它介质中制备比较混悬液/溶液。对于这些制剂进行以下稳定性测试：The antibodies were lyophilized and resuspended in semifluorinated alkanes. Comparative suspensions/solutions were also prepared in other media. The following stability tests were performed on these formulations:
在ICH条件(25和40℃)下的储存稳定性Storage stability under ICH conditions (25 and 40°C)
在ICH条件外的温度稳定性，即热变性Temperature stability outside ICH conditions, i.e. thermal denaturation
这些以类似于实施例1-3中所述的方法进行。进行适用于分析这些抗原结合多肽的稳定性的分析方法。在整个上文所列的稳定性实验过程中用于监测抗体活性和效力，以及监测聚集体含量的分析包括诸如类似于上文实例1-3中所述方案的ELISA在内的技术。These were performed similarly to the methods described in Examples 1-3. Analytical methods suitable for analyzing the stability of these antigen-binding polypeptides were performed. Analyses used to monitor antibody activity and potency, as well as aggregate content, throughout the stability experiments listed above included techniques such as ELISA similar to the protocols described in Examples 1-3 above.
分析方法利用由抗体混悬液直接获得的样品来进行，或根据分析技术，对为含水缓冲液萃取物的样品来进行。Analytical methods are performed using samples obtained directly from the antibody suspension or, depending on the analytical technique, on samples obtained as aqueous buffer extracts.
预计包含抗原结合的多肽或蛋白质的组合物将表现出改善的稳定性和减小的聚集体形成趋势。It is expected that compositions comprising antigen-binding polypeptides or proteins will exhibit improved stability and a reduced tendency to form aggregates.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP13177699 | 2013-07-23 | ||
| EP13177699.9 | 2013-07-23 | ||
| PCT/EP2014/065840WO2015011199A1 (en) | 2013-07-23 | 2014-07-23 | Stabilized antibody compositions |
| Publication Number | Publication Date |
|---|---|
| HK1224218A1 HK1224218A1 (en) | 2017-08-18 |
| HK1224218Btrue HK1224218B (en) | 2022-06-30 |
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