单抗制剂表面活性剂与蛋白可见颗粒形成相关性研究要点汇总

大分子生物制剂因蛋白分子的特殊性质，容易产生可见颗粒，尤其是因蛋白聚集而形成的白点颗粒。影响因素有很多方面，比如：药物研发阶段蛋白浓度的选择、缓冲体系的选择、表面活性剂类别/浓度的选择、包装容器材质的选择、原液冻融工艺的开发、制剂工艺参数的开发等。本文引述日本一篇研究论文的部分内容，引发关于表面活性剂与蛋白颗粒相关性的一些思考。

Amongst common excipients in mAb formulations, surfactants play an indispensable role in preventing particle formation, mainly through the protection against interfacial stresses. At present, three nonionic surfactants are commonly employed in the formulations of biotherapeutic proteins: namely Polysorbate 20 (PS20), Polysorbate 80 (PS80), and Poloxamer 188 (PX188).单抗药物常用聚山梨酯20/80、泊洛沙姆188这三种辅料作为表面活性剂，三种物料的应用对比见下表：

PS20 and PS80 are commonly utilized due to their proven effectiveness in safeguarding proteins against interfacial stressand their recognized safety record. Nevertheless, a significant shortcoming of using polysorbates is its degradation through oxidative or hydrolytic routes, as well as enzymatic routes that stem from the residual host cell proteins in drug substance.聚山梨酯20/80因其良好的安全数据和保护蛋白免受界面应力的优势而被广泛应用。但是其本身存在易于因氧化/水解而降解，包括因残留的宿主细胞蛋白所产生的酶促水解。This PS20/80 degradation not only directly impacts its content in drug product and protective activity but also sometimes result in particle formation by precipitation of the insoluble free fatty acids produced by its degradation.降解产生的游离脂肪酸因沉淀产生了可见颗粒。In such situations, additional measures against PS20/80 degradation and subsequent particle formation might need to be evaluated during drug substance purification and/or further refinement of the formulation.

下图展示了聚山梨酯的降解路径：氧化后产生脂肪酸酯、醛类、酮类、羧酸类、过氧化物、烷类物质。水解后产生脂肪酸。

聚山梨酯的降解路径示意图

PX188 has also shown its effectiveness as a surfactant in safeguarding bio-therapeutics at the interface. It is generally considered to be a more stable excipient than polysorbates. However, in some instances, the formation of proteinaceous visible particle has been observed in the formulations using PX188 derived from the interaction between the protein and silicone oil (polydimethylsiloxane – PDMS二甲基硅油). Such protein-PDMS complexed particles were not observed in the corresponding polysorbate formulations, likely due to PX188’s lower surface activity compared to polysorbates.泊洛沙姆188相较于聚山梨酯更稳定，但是其更容易造成蛋白和二甲基硅油相互作用所形成的可见颗粒（其表面活性作用比聚山梨酯低）。

Primary pharmaceutical packaging components, such as vial and syringe stoppers and syringe barrels, are basically coated with PDMS to enhance handling during production and to facilitate the sliding of syringe stopper. Such PDMS applied to primary packaging material is believed to be the primary source of the PDMS that forms particles in combination with the proteins in the drug product. It is plausible as there have been reports indicating that antibodies can engage with and adsorb onto silicone-coated surfaces or silicone-oil droplet（微滴） in a solution. This is a motivation to the development of many silicone-free primary containers in recent years. In fact, it has been demonstrated that protein-PDMS VPs are hardly ever produced in vials that used rubber stoppers without PDMS coating. However, there are still unresolved issues, such as the formation of VPs made solely of aggregated protein and VPs that originate from the rubber stopper itself.已知二甲基硅油和可见颗粒（蛋白和二甲基硅油结合物）的形成相关，但仍然存在一些未解的问题，如成分仅为蛋白的可见颗粒、由胶塞本身引发的可见颗粒。

However, since PDMS coat on the primary container can migrate into the liquid phase, it is not clarified whether the protein–PDMS VPs occurs at the interface with the PDMS-coated solid phase or within the liquid phase. In fact, the prior study has demonstrated protein-PDMS VP suppression by a stopper without PDMS coating but has not mentioned where and how protein-PDMS VPs were formed. 内包材上的二甲基硅油会迁移到药液中，从而引起蛋白-二甲基硅油可见颗粒的形成。有研究表明使用不含二甲基硅油的胶塞可以抑制颗粒的形成，但是没有提到颗粒到底是在什么位置（内包材表面or药液中）、什么阶段形成的。

颗粒形成机制示意图

Besides the presence or absence of silicone, the material of the primary packaging itself can influence the formation of aggregates. For instance, while glass has been traditionally used as a standard primary container for biopharmaceuticals, it also carries risks associated with VP formation. This includes the creation of glass particles due to delamination and proteinaceous VPs caused by poor compatibility between the container's contents and the glass surface.使用传统西林瓶作为容器时，存在因容器表面脱落产生的玻璃异物风险，以及容器材质和药液兼容性问题所形成的蛋白颗粒风险。In recent years, primary containers made from materials other than glass have been developed. Among these, the cyclo olefin polymer (COP，环烯烃聚合物) offers the benefit of being conducive to protein stability in certain cases due to reduced adsorption onto the container surface. COP材质的容器可以通过避免蛋白吸附在容器表面而提高蛋白分子的稳定性。Given these impacts, the type of primary container housing the biopharmaceutical can be a crucial factor in its long-term stability. Additionally, the orientation（朝向） during storage after these containers are filled can also affect the formation of VPs. For instance, one study reported that VP formation was lessened with inverted storage as compared to upright storage.有研究表明，产品倒置时产生的可见颗粒比正置更少。

I conducted two studies aiming at uncovering the mechanism of VP formation and crucial factors on that to provide potential VP mitigation measures. This study is the first to elucidate the primary pathway of protein-PDMS VP formation in a vial and also provide various countermeasures against protein-PDMS VP formation including the type of primary packing materials, storage orientation, the presence and absence of PDMS, and especially improvement of PX188’s potential critical material attribute, i.e., hydrophobicity. 研究了PX188关键质量属性是否与颗粒形成有关，如物料的疏水性。An increase in PX188’s hydrophobicity showed a positive correlation with a decrease in surface tension at the air-water interface, suggesting that hydrophobicity impacts adsorption behavior of PX188s at the air-water interface. PX188的疏水性越强，气-液界面的表面张力就越弱，表明物料的疏水性影响气-液界面的吸附作用。As such, the hydrophobicity of PX188, which influences surface tension and consequently the capacity to guard against interfacial stress, is anticipated to impact particle formation in antibody solutions, and should be closely monitored.应关注与PX188辅料疏水性相关的质量属性（如共聚物杂质偏高、聚氧丙烯PPO比例升高、环氧乙烷EO比例降低，意味着疏水性增加）。

Seven formulations were prepared for each of the two mAbs utilizing seven different PX188 lots to assess how PX188’s hydrophobicity influences on the formation of aggregates and proteinaceous particles. The analysis revealed that there was no significant effect on the percentage of HMWS both under the 5°C storage condition with/without mechanical stress condition. No substantial difference was observed depending on the PX188 used in the formulations, implying no substantial impact to soluble aggregate levels based on the difference in material attributes of PX188.没有发现PX188物料的疏水性强弱对HMWS水平（可溶解的蛋白聚集水平）造成显著影响。None of the formulations stored at 5°C for 6 months under static conditions formed VPs. On the contrary, visual inspection revealed the existence of VPs under all other stress condition. Most importantly, the result showed that the VP occurrence varied significantly based on the lot of PX188 used. 受到剪切力作用后的药液在稳定性研究期间发现了可见颗粒，并且颗粒水平与PX188批次具有相关性It was partially observed that PX188s with lower hydrophobicity exhibited more SVPs under thermal stress conditions. In the end, the influence of PX188 properties on SVP formation was not conclusive, as not all conditions followed the same pattern. 研究高温的影响因素时，发现疏水性弱的PX188批次，存在不溶性微粒偏多的现象，但是从研究数据上并不能得出PX188性质差异和药液中不溶性微粒之间有什么很直接的关系。To pinpoint the molecular characteristics of PX188 that affect VP formation under heat stress conditions, the correlations between VP formation, PX188 hydrophobicity and the degree of unsaturation were examined. The VP occurrence under heat stress conditions could be adequately explained by the hydrophobicity of PX188 alone.进一步研究在高温条件下，PX188物料的疏水性、不饱和程度与可见颗粒形成的关系。研究表明高温下可见颗粒的差异主要归因于PX188物料的疏水性。Under mechanical stress conditions (5°C with shaking and dropping), the observed VPs were primarily identified as protein–only VPs. The VP occurrence under mechanical stress conditions could be associated with both the degree of unsaturation and hydrophobicity.机械应力试验的研究表明，产生的可见颗粒的鉴定成分主要是蛋白，可见颗粒出现的程度与PX188物料的疏水性、不饱和程度都有相关性。PX237 has an average of approximately 30% PPO blocks and is more hydrophobic than PX188. In addition to the main data from PX188 formulations, supplemental data with PX237 was also obtained to corroborate the explanation that proteinaceous VP was reduced by higher hydrophobic PX subspecies. The compositions of VPs were identified as all protein-PDMS VP by RIM. The VP suppression in the PX237 formulation compared to PX188 formulation confirmed the effectiveness on VP reduction of higher hydrophobic.为了进一步确认辅料的高疏水性是否真的会降低可见颗粒的形成，使用更高疏水性的PX237辅料作进一步的对比研究，试验数据证明了此结论。

The VP occurrence was found to have negative correlation with the indicator of PX188’s hydrophobicity. This connection between PX188's hydrophobicity and the occurrence of VP under heat stress, and the partial correlation under mechanical stress, can be attributed to the stronger tendency of more hydrophobic PX188 to adsorb onto hydrophobic interfaces, such as the water-PDMS or air-water interfaces thereby shielding mAbs from interfacial stress. 研究表明可见颗粒的形成与PX188辅料的疏水性强弱具有负的相关性，当辅料具有更强疏水性时，辅料更容易吸附在疏水性界面（水-二甲基硅油界面、气-液界面），从而保护蛋白分子免受界面应力。Meanwhile, unlike PS80 and PS20, PX188 has been reported to prevent interfacial adsorption by directly interacting with proteins. Therefore, the difference in affinity of PX188 with the hydrophobic regions of mAbs, which is dependent on molecule’s hydrophobicity, may influence VP formation.与PS80、PS20不同，PX188还可以通过直接与蛋白质相互作用，从而抑制蛋白在界面上的吸附。因此，不同PX188分子因其疏水性不同，对单抗表面疏水区域的亲和力也会不同，从而影响可见颗粒的形成。When PX188 interact with the hydrophobic region of proteins, the hydrophilic PEO blocks may also work as a steric hindrance（空间位阻） and enhance the colloidal stability of mAbs.当PX188与蛋白的疏水区域相互作用时，其亲水性的PEO链可能伸展到水相中，形成空间位阻（阻碍蛋白分子之间聚集），从而提高蛋白分子稳定性。

Quote from “Mechanism Investigation and Mitigation Measures for Visible Particles Formation in Liquid Monoclonal Antibody Formulation”, Doctor Kohei SOEDA, 2024