冻融（Freezing/Thawing）对蛋白分子的影响

生物药因存在分阶段生产的情况，所以原液经常会冷冻（如-40℃）后储存，再转移至制剂生产场地进行后续的灌装。在这种情况下，原液就存在冻融的工艺步骤，在设计和验证原液的冻融用设备＆工艺参数时应考虑哪些方面？本文引述一篇关于某种特殊装置用于原液冻融的文献中的部分内容，尝试给出一些思考的角度。

For the majority of these biological molecule formats, the drug substance (DS) is typically frozen in bulk before shipment to the fill-finish site to increase storage stability and prolong shelf-life by reducing reaction kinetics, limiting microbial growth of potential bio-burden, and to reduce potential interfacial stresses during transportation. 生物药原液因为各种原因，通常经过冷冻后储存（提高储存稳定性以延长效期，抑制微生物生长，减少运输过程的界面应力作用），待进一步用于制剂生产。

Decoupling of DS from DP manufacturing adds flexibility to the manufacturing process and adds significant opportunities with regards to decoupled shelf lives.将原液和制剂的生产工艺分开定义有更多的优势。 However, freezing/thawing (F/T) processes are critical unit operations during fill/finish processing, that can impact product quality significantly. During freezing and thawing, physical instabilities manifesting e.g., as protein aggregates and particles, and are of major concern as they may impact product quality and safety but may also lead so significant issues in downstream unit operations (e.g., filter clogging). 原液冻融过程主要的风险是蛋白可能聚集并产生颗粒，从而影响产品质量，或者导致后续除菌过滤时堵塞滤膜。

In brief, solutes in a formulation are only partially entrapped in the growing ice during freezing, and thus, solutes in the liquid are transported along with the moving ice front and are eventually heterogeneously distributed in the frozen bulk. This is described as the so-called macroscopic cryo-concentration.冷冻过程中，药液中只有部分溶质包裹在形成的冰中。所以，在溶液冷冻时，液体中的溶质随着冰晶界面移动（冰晶不断增大）而移动，最终导致冷冻后溶质的不均匀分布，这就是常说的低温浓缩现象。

Crystallizationof excipients during freezing may lead to a loss of stabilizing effect as it has been reported for example for trehalose and sorbitol.有研究显示，一些辅料（如海藻糖、山梨醇）在冷冻过程中的结晶导致其对蛋白的稳定性作用降低。Likewise, selective crystallization of buffer system components such as dibasic sodium phosphate or a temperature-dependent shift in pKa during freezing can lead to a shift in pH, which may result in an increase in protein self-interactiondue to differences in the charge heterogeneity profile followed by aggregation. 冷冻过程中缓冲体系的辅料（如磷酸盐缓冲体系）结晶或温度导致的pKa变化都会使得药液产生pH梯度，从而增加蛋白之间因电荷异质性而相互作用，最终导致蛋白聚集。Also, surface-induced denaturation at the ice-liquid interface has been reported, whereas the air–liquid interface from air bubbles formed during freezing has been only recently proposed as a potential destabilizing factor.冷冻过程中，冰-液界面应力、因气泡产生的气-液界面应力对于蛋白来说都是不稳定的因素。A higher risk for protein oxidation has been also discussed in the context of air bubbles.药液中的气泡也产生了氧化蛋白的风险。Most importantly, cryo-concentration can impact protein stability by locally changing protein and excipient concentrations, which may for example lead to an increase in ionic strength. Colddenaturation of proteins may increase susceptibilities against other freezing induced degradation mechanisms. Also, the mechanical stress due to volume expansion of the liquid during freezing, that was observed as significant pressure rise in DS bottles, was proposed as a potential stress during freezing.蛋白在冷冻过程中存在降解的风险。使用瓶子储存原液时，冷冻过程产生的机械应力作用更强。In fact, slow cooling rates typically lead to a higher degree of cryo-concentration while the resulting specific ice surface area is expected to be smaller, andvice versa反之亦然for fast cooling rates.慢速冷冻导致更强的低温浓缩效应，形成的冰晶更小。快速冷冻则相反。

During the thawing process, freeze-concentrated fractions start to melt andsediment沉淀, which leads to a vertical concentration gradient of solutes in the thawed solution. These inhomogeneous conditions in solution were shown to destabilize proteins due to a salting out effect at high concentration at the bottom or due to self-association effects.解冻过程中，底部融化的药液浓度高，容易因盐析作用而沉淀，导致蛋白不稳定。Further, slow thawing rates may lead to (re-) crystallization of the liquid.慢速解冻可能还会导致融化后的药液出现重结晶。Overall, previous studies indicate that optimal freezing rates are specific to the protein molecule and formulation, due to molecule specific liabilities, while for thawing, faster thawing rates are generally considered less critical, while care should be taken to not exceed maximum allowable temperatures and holding times.快速解冻过程相对不那么关键，只要确保解冻的温度、时间不超范围即可。

In contrast to bags, freezing in DS bottles or carboys is typically conducted by placing the containers into freezer with forced convection (air-blast freezers) or in conventional freezers with stagnant air (without air convection), which is referred to as passive freezing. Even though conventional freezers are not designed to freeze large amounts of liquids, in practice they are often used for the same. Passive freezing is dependent on several process parameters, such as the type of freezer, the freezer load, the container size, and the container position in the freezer, etc., which can lead to considerable variability in the process. 假如使用传统（被动式）冰箱冷冻原液，那么冰箱型号、冰箱装载、容器尺寸和摆放位置就比较重要。As freezing and thawing of DS is typically performed at different facilities/sites, a controlled and transferrable F/T process is envisaged resulting in constant and reproducible F/T rates independent of equipment properties and process parameters (e.g., container size, the freezer type, freezing temperature, or freezer load). While air-blast freezers, to our knowledge, currently offer the best attempt towards controlled freezing of DS bottles, they are typically not readily available (like ULT freezers) resulting in investment costs. 由于冷冻原液、解冻原液通常在不同的生产车间，所以开发原液冻融工艺参数时，需要考虑二边的条件。主动式冰箱对于这方面有优势，但是比较贵。

According to the equation, the freezing process is determined by the container geometry (for a bottle the square dimension), the temperature gradient present between the liquid and temperature outside the bottle (temperature in the freezer), and the heat transfer coefficient. 冷冻参数和容器尺寸、原液的温度梯度、环境温度、传热系数相关。The heat transfer coefficient is substantially different for the following heat transfer mechanisms listed in order of increasing contribution: natural convection,forced convection and conduction.自然对流、强对流、传导这三个因素影响传热系数，且作用程度逐步增加。

Besides its implications on F/T rates, the device notably changed the F/T geometry. The channeled air stream causes the heat to be dissipated in the same direction, from underneath the bottle along the walls towards approximately half height of the bottle, where the air stream is directed away from the bottle. 研究的装置显著影响冻融过程的原液几何性状，被引导的气流使得热量从瓶底沿着瓶壁至瓶子一半高度的位置消散而直接离开瓶子。The upper part of the bottle isshielded隔离from the air stream and surrounded by a head space that effectively insulates it, preventing the liquid from starting to freeze at the surface as observed for passive freezing without the device (Fig. 3).瓶子上部位置被隔离而不会与气流接触，防止液体从表面开始冻结（普通冰箱容易出现该现象），见下图：

Uni-directional freezing from bottom to top was proposed as beneficial because the ice front is progressing in the opposite direction to the movement of solutes, which are transported downwards due to the density gradient-driven natural convection in the not fully solidified fraction.冰晶生长方向与溶质在液相中的对流迁移方向相反，因而有助于把溶质持续排出正在形成的冰相，减少其被包埋在冰中，有利于蛋白的稳定性（但冷冻速度过快也不行，可能导致溶质来不及迁移）。The time span between nucleation of ice until reaching Tg’ at the last point in the bottle (stress time) was therefore selected as read-out indicating the critical time span for cryo-concentration. In their study, directional freezing in 2 L DS bottles filled with a bovine serum albumin (BSA) solution was "passively" induced by thermally insulating the top part of the bottle, which also resulted in a bottom-up freezing geometry. While only a small reduction of the maximumCF浓缩倍数 within the bottle was observed, the distribution pattern of BSA changed, and aggregation levels, especially near the top, were lower.通过瓶子上部隔热措施而产生的自下而上的冷冻，虽然浓缩倍数只有轻微的下降，但降低了蛋白聚集水平，尤其是瓶子上部。

After the thawing process, vertical concentration gradients in the thawed solution with a highly diluted region on top and increasing solute concentrations towards the bottom have been previously reported. 解冻后，底部液体浓度相对更高。The segregation occurs due to gravity-driven sedimentation of denser freeze-concentrated matrix released from ice mainly consisting of water. The floating ice continuously dilutes the top region until melting is completed.解冻过程中，上方漂浮的冰逐步融化而持续稀释上方的浓度。After complete thawing, diffusion of solutes towards an initial equilibrium concentration has been shown to be very slow, though faster for small molecules (e.g., His). 完全解冻后，溶质扩散至平衡浓度的速度非常慢，小分子的速度相对快一些。Consequently, a pronounced concentration gradient of His persistent up until 48 h after thawing as reported in a study by Bluemel, a condition that is well known to be critical for protein stability. 据研究显示，溶液中的组氨酸在解冻后持续至24h后的浓度梯度才消失。Thus, the time during which a protein molecule is exposed to these unfavorable conditions should be minimized, and faster thawing can reduce exposure time, but might also reduce the extent of concentration gradients formed in solutions after thawing.快速解冻可以减少蛋白分子在非友好环境下的暴露时间，缩小解冻后浓度梯度的范围。

In our study, we pulled liquid samples at different heights (positions E0, E1, E2), immediately after thawing was complete, in order to investigate, whether the faster thawing times achieved by the device will reduce the concentration gradients studied by osmolality, and PS80 and His concentrations. Even though thawing times were reduced from 16.1 h without the device to 12.8 h with the device, there was no substantial reduction in vertical concentration gradients. Nevertheless, the exposure time and therefore holding time during DP manufacturing was reduced by the shorter thawing times, assuming timely homogenization/mixing of the solution upon complete thawing. 本冻融装置的研究数据显示，解冻时间相较于被动解冻方式缩短了3.3小时，但是浓度梯度没有明显降低，但至少可以提高制剂生产的效率。（缩短了解冻时间，也就减少了蛋白分子在非友好环境下的暴露时间，对质量控制也是有益的）。

In order to be able to conduct a sufficiently large number of experiments, we used a surrogate solution for a mAb formulation including representative formulation compounds and representative solution properties (e.g., Tg′ of −34°C, osmolality of 310 mOsmol/kg). Different analytical read-outs (osmolality, His and PS80 concentration) have been discussed identifying osmolality as a sensitive read-out to investigate cryo-concentration, in combination with the surrogate solution, that has arelatively low viscosity, which reflects a rather worst-case for cryo-concentration, further increasing the sensitivity of this model.渗透压指标对于研究低温浓缩最为敏感，溶液粘度低是研究低温浓缩的更差条件。To note, the regions within a bottle, where freezing-induced protein aggregation was found varied for different protein molecules and were not necessarily found in regions of high protein concentration, but for example in the top region near the ice mound due to the reasons elaborated above, or at regions with shifted mAb to His ratio.