胶塞硅油与蛋白颗粒形成的相关性研究：高浓度单抗及PX188制剂中可见颗粒的影响因素

In this second study, I focused on visible particle formation in vial formulations with high concentration mAb and PX188 as actually employed in commercial products, and examined the impact on particle formation by changing several factors that might affect it. The factors in question specifically included whether or not there was drug solution adherence to the stopper, the orientation of storage (upright or inverted), the presence or absence of a silicone coating on the stopper, the material of the vial (glass or COP), and the hydrophobicityof PX188 (low or high).使用高浓度单抗和PX188作为辅料，研究药液黏附/不黏附胶塞上、灌装了药液的瓶子放置的朝向、含硅油/不含硅油的胶塞、容器的材质（玻璃/COP）、PX188疏水性强弱这些因素对于可见颗粒形成机制的影响。

Findings suggest that the formation of protein–PDMS VPs in mAb formulations is enhanced when the stopper directly contacts the drug solution and is stored upright, retaining some of the solution on the stopper.研究表明，正置存放的药液中，胶塞和药液接触过的样品，其可见颗粒多于胶塞和药液未接触的样品。

胶塞和瓶子之间残留药液与异物形成的相关性示意图

To assess the effect of the vials’ storage orientation, I compared A01 with A03, which had the same vial and stopper configuration (glass vial, Si-coated stopper) but was stored upside down at 25°C for up to 12 months. Visual inspection and subsequent chemical identification of observed VPs showed that only one vial in A03 (inverted) formed protein–PDMS VP, while there was marked protein–PDMS VP formation in A01 (upright). MFI measurement revealed a lower number of protein-like SVPs in A03 than in A01. BCA assay indicated that the amounts of protein adsorbed on stoppers in A03 were lower than in A01. To sum up the results for invertedstorage, the amount of protein adsorbed on the stopper, which is considered a primary site for VP formation, was reduced. This may be a factor in the suppression of subsequent particle formation.研究的数据表明，倒置样品的可见颗粒、不溶性微粒、胶塞上吸附的蛋白都明显比正置样品的要少。倒置样品吸附在胶塞上的蛋白减少了，这可能是抑制颗粒形成的原因。One distinction between the upright and inverted positions is the presence or absence of an air phase near the stopper. Consequently, it is plausible that the air phase has affected VP formation in some manner. For instance, it has been reported that protein aggregation is increased at the air–liquid–solid triple interface, so the formation of a similar triple interface on the stopper could be one explanation. 正置样品颗粒更多的原因可能是气-液-固三重界面应力的作用促使颗粒更容易形成。Alternatively, it is possible that the presence of the surrounding air phase itself, not just the triple interface, may have had an impact. 也可能是黏附在胶塞上的药液受到附近气相作用的单独影响。Further research centering on this point is warranted to clarify how the air phase near the drug solution adhered to stopper promotes the VP formation. To assess the effect of PDMS on the stopper, A01(glass vial, silicone stopper) was compared with A04 (glass vial, Si-free stopper) that was stored upright at 25°C for up to 12 months. Visual inspection and subsequent chemical identification of detected VPs showed no proteinaceous VP formation in A04, while there was substantial formation of protein–PDMS VPs in A01. MFI measurement revealed a lower number of protein-like SVPs in A04 than in A01. BCA assay indicated that the amounts of protein adsorbed on stoppers in A04 were lower than in A01. These results implies that the presence of PDMS on the stopper somehow contributes to the formation of proteinaceous VPs.进一步对比研究表明，胶塞上的硅油和蛋白异物的形成具有相关性。There are two potential explanations for why the presence of PDMS on the stopper encouraged VP formation: PDMS either promoted protein adsorptionto the stopper, and/or it facilitated the desorption of aggregatesformed on the stopper. 对于为何胶塞上的二甲基硅油会促进颗粒的形成，可能存在二种解释：二甲基硅油促进蛋白吸附在胶塞上，二甲基硅油促进聚集体的解析。To investigate the first hypothesis, I compared the amount of protein adsorbed on the stoppers of A03(silicone stopper) and A05(Si-free stopper), both of which were stored upside down, allowing the entire stopper to continuously contact the drug solution. 进一步研究二甲基硅油是否会促进蛋白吸附在胶塞上，设置对比实验，均倒置存放。Both A03 and A05 displayed similar adsorption value, implying that PDMS itself does not inherently encourage antibody adsorption, at least when the impact of the air phase is negligible. 都是倒置时，胶塞是否含有硅油对于胶塞吸附蛋白的情况无相关性。A08 (COP vial, Si-free stopper), which was stored upright, exhibited the same level of adsorption as A01(glass vial, silicon stopper). This suggests again that a Si-free stopper does not always result in decreased adsorption, even when the air phase is in close proximity.同样都是正置，不同材质的瓶子、含硅油和不含硅油的胶塞，对于蛋白吸附水平没有差异。To explore the latter theory, we then assessed the quantities of PDMS and protein adsorbed on the stoppers prior to and following mechanical stress, as well as the quantities of SVPs in the solution. 进一步研究胶塞的二甲基硅油量和蛋白吸附的相关性。Data implies that the PDMS on the stopper facilitated the desorption of protein–PDMS aggregates which had been formed on the stopper into the solution, resulting in an increased occurrence of VPs.研究数据显示胶塞上的二甲基硅油会促进胶塞表面形成的蛋白-二甲基硅油聚合物解吸附后进入药液中，从而增加可见颗粒的形成To avoid such PDMS-triggered desorption of aggregates, one straightforward solution could be to eliminate PDMS. However, it is speculated that using a container where PDMS is covalently bonded to the primary container, making it difficult to desorb, could also be promising strategy.采用二甲基硅油与容器表面材质通过共价键结合的方式，可以抑制上述解吸附的过程。

To assess the effect of vial material, A01 (glass vial, Si-coated stopper) was compared with A07 (COP vial, Si-coated stopper) that was stored upright at 25°C for up to 12 months. These findings further corroborate the explanation that the higher hydrophobicity of the COP vials resulted in the decrease in the solution adhesion area on stoppers and consequently the quantity of protein adsorbed, which might have also influenced the formation of protein–PDMS VPs. COP瓶子比玻璃瓶具有更高的疏水性，导致胶塞表面的药液更少（瓶子和胶塞间隙位置竞争性吸附蛋白），从而减少蛋白-二甲基硅油颗粒的形成。It might be possible that in the case of the COP vials, the drug solution was indeed concentrated and thicker on one side, making it less prone to the air-phase effects mentioned in the previous section because the droplets were thicker than in the case of glass vials and the air-liquid interface is distanced from the PDMS on stopper. COP瓶子表面的液滴比玻璃瓶更厚，气-液界面与胶塞存在更大的距离，所以蛋白受到的气-液界面应力的影响就更小。

I identified the primary location for protein–PDMS VP formation to be on the vial stopper where the drug solution adheres and where an air phase exists is nearby. Moreover, the main pathwayfor protein-PDMS VP formation was found to be the migration of protein-PDMS aggregates already formed on the stopper into the drug solution, which were then detected as VPs. In this study, I used only one type of antibody (IgG4) as the model antibody, but the characteristics of antibodies themselves also influence VP formation. Additionally, even though the antibody concentration was fixed at 150 mg/mL, but the concentration can have impact on VP formation. Therefore, additional research is warranted to determine whether the findings of this study apply to other antibodies with different properties and different protein concentrations. Nevertheless,不同蛋白分子的性质差异、浓度差异等均可能影响本研究的结论。The finding that inverted storage, often chosen in vial stability studies due to the continuous contact between the contents and both the vial and the rubber stopper, is not the worst-case scenario for protein–PDMS VP formation is also noteworthy. Given this fact, if we need to select the worst storage conditions when testing quality relevant items of drug products, then storage orientation might be one of the conditions to be worth considering. 对于稳定性研究，只把倒置当作和可见颗粒研究相关的最差条件可能是不合理的，正置和倒置研究都要做。

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