稳定剂及其与冷冻和冻干蛋白质制剂中制剂成分的相互作用（下）
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科学
2024-11-11 07:35
上海
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文献：Advanced Drug Delivery Reviews (IF 15.2) Pub Date: 2021-03-17, DOI: 10.1016/j.addr.2021.03.003作者：Seema Thakral, Jayesh Sonje, Bhushan Munjal, Raj Suryanarayanan
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本综述旨在概述冷冻和冷冻干燥过程中蛋白质稳定性与这些过程中常见的压力条件相关的当前知识。介绍了赋形剂稳定蛋白质的传统和精细机制。这些稳定剂包括多种化合物，包括糖、糖醇、氨基酸、表面活性剂、缓冲剂和聚合物。提出了用于冷冻和冷冻干燥蛋白质制剂的赋形剂的合理选择。冻干蛋白质制剂通常是多组分系统，提供了多种赋形剂-赋形剂和蛋白质-赋形剂相互作用的可能性。This review aims to provide an overview of the current knowledge on protein
stabilization during freezing and freeze-drying in relation to stress
conditions commonly encountered during these processes. The traditional as well
as refined mechanisms by which excipients may stabilize proteins are presented.
These stabilizers encompass a wide variety of compounds including sugars, sugar
alcohols, amino acids, surfactants, buffers and polymers. The rational
selection of excipients for use in frozen and freeze-dried protein formulations
is presented. Lyophilized protein formulations are generally multicomponent
systems, providing numerous possibilities of excipient-excipient and
protein-excipient interactions. The interplay of different formulation
components on the protein stability and excipient functionality in the frozen
and freeze-dried systems are reviewed, with discussion of representative
examples of such interactions.
蛋白处方（Protein formulation）、聚集（Aggregation）、冷冻保存（Frozen storage）、冻干（Freeze-drying）、稳定剂（Stabilizers）、冷冻保护剂（Cryoprotectants）、冻干保护剂（Lyoprotecants）前文见：稳定剂及其与冷冻和冻干蛋白质制剂中制剂成分的相互作用（上）
3.6 其他辅料Additional excipients
功能化的环糊精（CD），如羟丙基β环糊精（HPβCD）和磺丁基醚β环糊精，已获批用于注射，并且研究了其在冻干蛋白质制剂中的潜在应用。当以约0.1% w/v的浓度使用时，HPβCD能够在冷冻-融化和重新配制过程中提供类似于非离子表面活性剂的抗界面应力稳定作用[95]。在更高浓度下，HPβCD可能通过水分替代和玻璃化来稳定蛋白质。通过比较亲本环糊精和功能化环糊精的冻干保护活性，提供了水分替代的证据。亲本环糊精形成分子内氢键，这限制了它们与其他分子形成氢键的能力。这些环糊精可能在水分替代方面的效果有限。有趣的是，使用羟丙基化环糊精（HPαCD、HPβCD或HPγCD）冻干的乳酸脱氢酶（LDH）的相对活性高于使用亲本环糊精（αCD、βCD或γCD）冻干的LDH。功能化环糊精观察到的活性增加归因于它们额外的羟基，可能有助于增强氢键作用[96]。环糊精通常产生无定形玻璃态；例如，与IL-2一起冻干的β环糊精的Tg约为108℃（残留水分2-3% w/w），这可能在储存期间提高蛋白质的稳定性[95]。当单独使用时，用于在冷冻干燥及随后40℃下储存3个月期间稳定免疫球蛋白所需的海藻糖或HPβCD的量大约是4:1（稳定剂:蛋白质）[97]。另一方面，当以优化的重量比3.3:1（海藻糖:CD）使用，在稳定剂:蛋白质比例约为1:1时，即使在45℃下储存2个月后，也未观察到冻干制剂中的蛋白质聚集。因此，有证据表明海藻糖和HPβCD之间存在协同效应[98]。Functionalized cyclodextrin (CD) such as hydroxypropyl b cyclodextrin (HPβCD)
and sulfobutylether βCD are approved for parenteral use and their potential use
in freeze-dried protein formulations has been investigated. When used at a
concentration of ~0.1% w/v, HPβCD provided stabilization against interfacial
stress during freezing-thawing and reconstitution, in a manner similar to
non-ionic surfactants [95]. At higher concentrations, HPβCD
stabilizes protein possibly by water replacement and vitrification. Evidence
for water-replacement was provided by comparison of lyoprotectant activity
between parent and functionalized CDs. Parent CDs form intramolecular hydrogen
bonds which hinder them to hydrogen bond with other molecules. These are likely
to exhibit little degree of water replacement. Interestingly, the relative
activity of LDH lyophilized with hydroxypropylated CDs (HPαCD, HPβCD or HPγCD)
was higher than that of LDH lyophilized with parent CDs (αCD, βCD or γCD). The
increased activity observed for functionalized CDs was attributed to their
additional hydroxyl moieties, possibly contributing to hydrogen-bonding [96].
CDs usually yield amorphous glasses; for example, freeze-dried βCD with IL-2
had a Tg ~ 108℃ (residual moisture 2–3% w/w), which might enhance protein
stability during storage [95]. When used alone, the amount of
trehalose or HPβCD, required for the stabilization of immunoglobulin during
freeze-drying and subsequent storage at 40℃ for 3 months, was ~4:1 (stabilizer:
protein) [97]. On the other hand, when used in combination, in an
optimized weight ratio of 3.3:1 (trehalose: CD), with stabilizer: protein ratio
of ~1:1, no protein aggregation was observed in the lyophile even after storage
for 2 months at 45℃. Thus, there was evidence of synergistic effect between trehalose
and HPβCD [98].氯化钠（NaCl），通常用于调节注射溶液的渗透压，但由于其低共熔点（-21℃），在冻干制剂中并不理想。在冷冻溶液中，NaCl的结晶被无定形溶质如蔗糖[99]和结晶溶质如甘露醇和甘氨酸[100]所抑制。NaCl不能完全结晶降低了体系的塌陷温度。此外，NaCl还抑制了冷冻溶液中甘露醇的结晶[100]。金属离子，特别是二价阳离子如锌、铜或钙，可以结合到特定蛋白质结合位点，可能稳定（但在某些情况下也可能破坏）天然蛋白质构象[24]。例如，氯化钙常用作含有凝血因子的冻干产品中的络合剂，其活性和稳定性在钙离子存在下得到提升[6]。在一项有趣的研究中，向基于蔗糖的牛血清白蛋白（BSA）或重组人血清白蛋白（rHSA）制剂中添加氯化钠，改善了它们的储存稳定性。这些结果与传统的制剂理念相悖[101]。Sodium chloride (NaCl), commonly used to adjust the osmotic pressure of
parenteral solutions, is not desired in lyophilized formulations, due to the
low eutectic temperature (-21℃). In frozen solutions, NaCl crystallization is
suppressed by amorphous solutes such as sucrose [99] and by
crystallizing solutes, such as mannitol and glycine [100]. This
inability of NaCl to crystallize completely lowers the collapse temperature of
the system. In addition, NaCl also inhibits crystallization of mannitol in
frozen solutions [100]. Metal ions, specifically divalent cations
like zinc, copper, or calcium, can bind to specific protein binding sites and
may stabilize (but in some cases also destabilize) the native protein
conformation [24]. As an example, calcium chloride is commonly used
as a complexing agent in lyophilized products containing coagulation factors,
whose activity and stability is promoted in presence of calcium ions [6].
In an interesting study, the addition of sodium chloride to sucrose-based BSA
or rHSA formulations improved their storage stability. These results are in
contradiction with conventional formulation wisdom [101].
Interaction of formulation components
从上述讨论中可以明显看出，冻干蛋白质制剂通常是由多个组分构成的系统，这为辅料之间以及辅料与蛋白质之间的相互作用提供了众多可能性。这些相互作用能够影响冻干过程中不同阶段的辅料物理形态，以及最终药品中的形态。辅料的物理形态可以影响其功能性，进而影响活性成分的稳定性。活性成分作为蛋白质，很可能保持无定形态。在本节中，我们将重点介绍辅料之间以及辅料与蛋白质相互作用的典型案例。As is evident from the discussion above, lyophilized protein formulations are
typically multi-component systems, providing numerous possibilities of
excipient-excipient and protein-excipient interactions. Such interactions can
influence the physical form of some excipients during the different stages of
the freeze-drying process as well as in the final drug product. The physical
form can influence the excipient functionality and thereby the stability of the
active. The active ingredient being a protein, is likely to be retained
amorphous. In this section, we highlight representative examples of
excipient-excipient and protein-excipient interactions.
4.1 辅料之间的相互作用Excipient- excipient interactions
4.1.1糖类与其他辅料的相互作用Sugars with other excipients
糖类作为蛋白质制剂中最广泛使用的辅料，已显示出对共溶质相行为的潜在影响。蔗糖和海藻糖作为冻干制剂中广泛使用的稳定剂，它们对其他辅料的影响通常预期在性质上是类似的。然而，如第3.1节所述，当冷冻溶液长时间退火时，海藻糖可能会以二水合物形式结晶[37,102,103]。因此，这两种糖对共溶质的影响可能存在显著差异。海藻糖的结晶可能会损害其稳定功能。表4列出了糖类与其他共溶质在冷冻溶液中以及冻干过程中相互作用的一些例子，并将在下面详细讨论几个案例。Sugars being the most extensively used excipients in protein formulations have
shown potential influence on the phase behavior of cosolutes. Both sucrose and
trehalose are widely used stabilizers in freeze-dried formulations and their
effect on other excipients is often expected to be qualitatively similar.
However as mentioned in Section 3.1, when
frozen solutions are annealed often for longer durations, trehalose can
crystallize as trehalose dihydrate [37,102,103]. As a result, there
may be remarkable differences in the way these two sugars affect the cosolutes.
Trehalose crystallization has the potential to compromise its stabilizing
functionality. Some examples of the interaction of sugars with other cosolutes,
in frozen solutions and during lyophilization, have been tabulated (Table 4). A few case studies are discussed in detail
below.糖类与填充剂的组合。糖-填充剂的组合为获得具有短冻干时长的稳健冻干产品提供了可能[11,53,111]。糖保持无定形态并作为稳定剂。另一方面，填充剂结晶并赋予蛋糕坚硬的结构，从而实现快速冻干（短冻干时长），并经常改善复融特性[112]。然而，两种辅料的物理状态以及它们在冷冻溶液中的功能已被发现取决于糖与填充剂的比例（R，重量比）。糖类已被证明对填充剂如甘露醇和甘氨酸的结晶表现出浓度依赖性的抑制作用[104-107]。不适当的糖与填充剂比例可能会保留填充剂无定形态或促进糖结晶，从而丧失其期望的功能。以下例子说明了这一点。Sugar(s) with bulking agent(s). A sugar-bulking agent combination offers an
avenue to obtain robust lyophilized products with short cycle times [11,53,111].The sugar remains amorphous and serves as a stabilizer. The bulking agent, on
the other hand, crystallizes and gives a rigid structure to the cake, thus
enabling rapid lyophilization (short cycle time) and often improving the
reconstitution properties [112]. However, the physical state of the
two excipients and hence their functionality in frozen solution has been found
to depend upon the ratio of sugar to bulking agent (R, w/w). Sugars have been shown to exhibit a concentration
dependent inhibitory effect on the crystallization of bulking agents like
mannitol and glycine [104–107]. An inappropriate sugar to bulking
agent ratio poses the risk of retaining the bulking agent amorphous or
facilitating sugar crystallization, thereby resulting in loss in their desired
functionalities. This is evident from the following examples.高蔗糖与甘氨酸比例（R > 4:1）抑制了甘氨酸的结晶，使其主要保持在无定形态（见图5）[106,113]。即使在这些条件下进行退火（-15℃下90分钟），甘氨酸也未能结晶。另一方面，在低蔗糖与甘氨酸比例（R <2:3）时，即使没有退火，甘氨酸的结晶也完全发生了。中间比例的组成在最终冻干制剂中使甘氨酸保持部分结晶状态[106]。当海藻糖与甘露醇一起使用时，这种相互作用变得更加复杂[108]。高海藻糖与甘露醇比例（R = 3:1）在冷冻状态下完全抑制了甘露醇的结晶，使其保持无定形态。在R = 1:1时，甘露醇促进了海藻糖作为二水合物的结晶。然而，在更低的比例下，R = 1:3，甘露醇结晶了，但海藻糖没有[108]。理想的场景是填充剂结晶，但稳定剂保持无定形态，即对于蔗糖-甘氨酸和海藻糖-甘露醇组合，分别R < 2:3和≤1:3。A high sucrose to glycine ratio (R > 4:1), inhibited glycine
crystallization, retaining it predominantly in the amorphous state (Fig. 5)[106,113]. Annealing (at -15℃ for 90
min) also failed to crystallize glycine under these conditions. On the other
hand, at low sucrose to glycine ratios (R < 2:3), crystallization of glycine
was complete even without annealing. Intermediate compositions retained glycine
in a partially crystalline state in the final lyophile [106]. Such
an interaction became even more complex, when trehalose was used with mannitol [108].
A high trehalose to mannitol ratio (R = 3:1) completely inhibited mannitol
crystallization in the frozen state, retaining it amorphous. At R = 1:1,
mannitol facilitated the crystallization of trehalose as trehalose dihydrate.
However, at a much lower ratio, R = 1:3, mannitol crystallized, but trehalose
did not [108]. An ideal scenario would be where the bulking agent
crystallizes, but the stabilizer is retained amorphous, i.e. R < 2:3 and
≤1:3, for sucrose-glycine and trehalose-mannitol combinations respectively.![]()
Fig. 5. The effect of sucrose concentration and annealing on the crystallinity
offreeze-dried glycine. Reproduced from Bai et al [106] with permission from
Elsevier.Table 4糖类与填充剂（R = 糖与填充剂的比例，重量比）和缓冲液的相互作用示例。![]()
同样，蔗糖与甘露醇比例为1:4，已成功用于在冻干制剂中稳定多种蛋白质[53,55]。然而，蔗糖可以影响结晶甘露醇的物理形态。至少一部分甘露醇以亚稳态的MHH结晶。如上所述，不希望在冻干蛋糕中出现MHH，因为其脱水可能导致水分释放。释放的水分可能与其它处方组分相互作用。例如，在储存期间，由脱水诱导的MHH在冻干制剂（由含有4% w/w甘露醇和1% w/w蔗糖的溶液制备）中释放的水分诱导了蔗糖的结晶[55]。因此，在二次干燥期间，希望实现MHH的完全脱水。Similarly, a sucrose to mannitol ratio of 1:4, has been used to successfully
stabilize numerous proteins in lyophilized formulations [53,55]. However,
sucrose can affect the physical form of the crystallizing mannitol. At least a
fraction of mannitol crystallizes as the metastable MHH. As mentioned above,
MHH is not desired in the freeze-dried cake, since its dehydration can lead to
water release. The released water may interact with the other formulation
components. For example, the water released by the dehydration of MHH in the
lyophile (prepared from solution containing 4% w/w of mannitol and 1% w/w of
sucrose) during storage induced sucrose crystallization [55]. Hence, it is
desirable to cause complete MHH dehydration during secondary drying.糖-填充剂相互作用的另一个有趣含义是填充剂的冷冻保护能力的显现。当甘氨酸保持无定形态，在蔗糖存在下，对蛋白质在冷冻过程中施加了额外的稳定效应。虽然蔗糖在固态中保持为蛋白质的主要稳定剂，但一些无定形态的甘氨酸的加入显著增加了稳定性[114]。如果填充剂在随后的干燥步骤中完全结晶，这可能是一个潜在有用的方法。否则，从物理稳定性的角度来看，不希望在冻干制剂中保留无定形态的填充剂。无定形态的填充剂可能在储存或运输过程中结晶，从而释放相关的吸附水。这种释放的水可能与其它处方组分相互作用[108]。Another interesting implication of the sugar-bulking agent interaction is the
emergence of the cryoprotective ability of the bulking agent. Glycine when
retained amorphous, in presence of sucrose, exerted additional stabilizing
effect on the protein during freezing. While sucrose remained the primary
stabilizing agent for the protein in the solid state, inclusion of some
amorphous glycine caused a significant increase in stability [114]. This could
be a potentially useful approach if the bulking agent crystallizes completely
during the subsequent drying steps. Otherwise, from the perspective of physical
stability, retention of the bulking agent in the amorphous state in the
lyophile is not desirable. The amorphous bulking agent may crystallize, during
storage or transport, thereby releasing the associated sorbed water. This
released water may interact with the other formulation components [108].总结来说，处方研究员必须审慎选择糖与填充剂的重量比例，并优化加工条件，以实现具有所需属性的稳定的冻干产品。To summarize, the formulator must judiciously select the weight ratio of sugar
to bulking agent and optimize the processing conditions so as to achieve a
stable freeze-dried product with the desired attributes.糖类与缓冲剂的相互作用。糖类可以抑制冷冻溶液中缓冲盐的结晶，从而减轻由缓冲组分选择性结晶引起的任何pH值变化。这种效应的大小取决于糖和缓冲剂的各自浓度，以及初始处方的pH值。海藻糖和蔗糖以浓度依赖性的方式，抑制了磷酸盐缓冲剂（160 mM，初始pH 7.2）在冷冻期间结晶引起的pH值变化[110]。当糖（5.5% w/v；约160 mM）和缓冲剂的摩尔浓度相同时，pH值变化被完全抑制。在另一个案例研究中，蔗糖和海藻糖有效地抑制了琥珀酸盐缓冲剂组分的结晶，无论pH值如何，当缓冲剂和糖的摩尔浓度大致相同时（50 mM缓冲剂与2% w/v糖；约58.5 mM）。然而，在更高的缓冲剂浓度（200 mM缓冲剂与2% w/v糖）时，结果取决于pH值。当初始pH值为6.0时，海藻糖和蔗糖抑制了缓冲剂结晶，但在pH 4.0时未能做到。在较低的pH值下，琥珀酸盐缓冲剂组分的结晶诱导了海藻糖的结晶。另一方面，蔗糖降解，产生了结晶分解产物，因此未能防止pH值变化[93]。这种相互作用在选择缓冲剂和糖浓度时需要谨慎。具体来说，在低缓冲剂浓度（≤50 mM）下，预计糖的抑制作用将有助于保持冷冻溶液中缓冲剂的无定形态，从而防止其功能的丧失。此外，当缓冲剂保持无定形态时，糖类可以减少冻干诱导的离子化和表观pH值的变化[86]。Sugars with buffers. Sugars can inhibit buffer salt crystallization in frozen
solutions, thereby attenuating any pH shift brought about by selective
crystallization of buffer components. The magnitude of this effect depends upon
the individual concentrations of sugar and buffer, and the initial formulation
pH.Trehalose and sucrose, in a concentration dependent manner, inhibited the pH
shift associated with crystallization of phosphate buffer (160 mM, initial pH
7.2) during freezing [110]. The pH shift was completely inhibited
when the molar concentration of sugar (5.5% w/v; ~160 mM) and buffer were
identical. In another case study, sucrose and trehalose effectively inhibited
succinate buffer component crystallization irrespective of pH, when the molar
concentrations of the buffer and sugar were about the same (50 mM buffer with
2% w/v sugar; ~58.5 mM). However, at a higher buffer concentration (200 mM
buffer with 2% w/v sugar), the outcome was pH dependent. Both trehalose and
sucrose inhibited buffer crystallization when the initial pH was 6.0 but failed
to do so at pH 4.0. At the lower pH, crystallization of succinate buffer
component induced trehalose crystallization. Sucrose, on the other hand,
degraded, yielding crystalline decomposition products thus failing to prevent
pH shift [93]. Such interplay warrants caution while selecting the
buffer and sugar concentration. Specifically, at low buffer concentrations (≤50
mM), the inhibitory effect of sugars is expected to assist in retaining the
buffer amorphous in frozen solutions, hence preventing loss of its functionality.
Additionally, sugars can reduce the freeze-drying induced changes in ionization
and apparent pH when the buffer remains amorphous [86].
4.1.2 填充剂与其他辅料的相互作用Bulking agents with other excipients
填充剂通常用于那些自身体积不足以支撑结构的低剂量蛋白质制剂。尽管甘露醇和甘氨酸是最常用的填充剂，但葡萄糖、蔗糖、乳糖、右旋糖酐和氨基酸也是其他可用的选择。在最终的冻干制剂中，填充剂可能是结晶态或无定形态[115]。对于如甘露醇和甘氨酸这样的结晶性填充剂，它们的结晶倾向及多晶型形式会受到其他处方组分的影响。Bulking agents are generally used for low dose protein formulations that per se
do not have the necessary bulk to support their own structure. Though mannitol
and glycine are the most common bulking agents, glucose, sucrose, lactose,
dextran and amino acids are other examples. In the final lyophile, a bulking
agent may be crystalline or amorphous [115]. For crystallizing
agents, such as mannitol and glycine, their crystallization propensity as well
as the polymorphic form is influenced by other formulation components.与缓冲剂的相互作用：如前所述，冷冻溶液中缓冲剂组分的选择性结晶可能导致显著的pH变化。重要的是，填充剂和缓冲剂可能会相互影响对方的结晶倾向。了解共溶质对缓冲剂结晶的影响至关重要，因为这将影响冷冻浓缩液的pH值，进而影响蛋白质的稳定性。With buffers: As already mentioned, selective crystallization of a buffer
component in frozen solution can result in a pronounced pH shift. Importantly,
bulking agent and buffers may influence the crystalline propensity of each
other. It is important to understand the influence of cosolutes on buffer
crystallization, since this will influence the freeze-concentrate pH and hence
the protein stability.当含有磷酸钠缓冲剂（10或100 mM）和甘氨酸（≥0.8% w/v）的溶液被冷冻时，两种成分的结晶都得到了促进。尽管这并未影响甘氨酸作为填充剂的功能，但缓冲盐的显著结晶导致了冷冻过程中的pH变化[116]。有趣的是，甘氨酸的存在增加了10和100 mM磷酸钠缓冲剂在冷冻过程中的pH变化。然而，在较低浓度（≤0.4% w/v）时，甘氨酸防止了磷酸盐缓冲剂在冷冻过程中的pH变化。但在这样的低浓度下，甘氨酸可能无法提供足够的体积和形成蛋糕状结构。因此，需要谨慎选择处方组分及其配比，以确保两个组分的功能，并得到一个稳定而优雅的处方。含有甘氨酸（2% w/v）和50 mM磷酸钠缓冲剂的溶液在-20℃下进行退火处理。这个退火步骤成功地促使甘氨酸结晶，同时最小化了缓冲盐的结晶[117]。该研究表明了在药物学上相关的浓度下保留两个组分功能的成功方法，即填充剂结晶，而缓冲剂组分不结晶。When solutions containing sodium phosphate buffer (10 or 100 mM) and glycine
(≥0.8% w/v) were frozen, crystallization of both the components was
facilitated. Though this did not affect the functionality of glycine as a
bulking agent, the pronounced crystallization of the buffer salt led to pH
shifts upon freezing [116]. Interestingly, the presence of glycine
increased the pH shifts upon freezing, for both 10 and 100 mM sodium phosphate
buffers. However, at low concentrations (≤0.4% w/v), glycine prevented any pH
shifts in phosphate buffers upon freezing. However, at such a low
concentration, glycine might not provide the bulk and cake structure. Thus,
judicious selection of both the formulation components and their composition is
needed to ensure the functionality of both the components and obtain a stable
and elegant formulation. Solutions containing glycine (2% w/v) and 50 mM sodium
phosphate buffer were annealed at -20℃. This annealing step led to the desired
glycine crystallization along with minimal buffer salt crystallization [117].
The study demonstrated a successful approach to retain functionality of both
the components at pharmaceutically relevant concentrations, i.e.
crystallization of bulking agent, but not of the buffer component.同样，甘露醇的结晶也被磷酸盐缓冲剂（浓度≥100 mM）所抑制[118]。当甘露醇和甘氨酸组合使用时，磷酸盐缓冲剂盐显著抑制了这两种溶质在冷冻溶液中的结晶。甘露醇-甘氨酸组合的最终冷冻保护效果取决于在冻干制剂中保留无定形态的溶质比例。增加缓冲剂浓度降低了甘露醇和甘氨酸的结晶度，这反过来又提高了酶活性的保留[119]。通常，只有无定形的共溶质才能抑制缓冲剂结晶。然而，当共溶质结晶时，也会观察到缓冲剂结晶。与表面活性剂的相互作用：当甘露醇溶液中逐渐增加聚山梨酯80的浓度并进行冻干时，正如预期，冻干制剂的比表面积逐渐增加。表面活性剂浓度也影响了最终冻干制剂中甘露醇的物理形态。低聚山梨酯浓度导致δ-甘露醇优先结晶，而在更高浓度下，得到了β-甘露醇[120]。Similarly, mannitol crystallization was also inhibited by phosphate buffer
(concentration ≥100 mM) [118]. When mannitol and glycine were used
in combination, the phosphate buffer salts significantly inhibited the
crystallization of both of these solutes in frozen solutions. The resultant
lyoprotectant action of the mannitol-glycine combination was a function of the
fraction of the solutes that were retained amorphous in the lyophile. An
increase in buffer concentration decreased the crystallinity of mannitol and
glycine and this, in turn, translated to a higher retention of enzyme activity [119].
In general, only an amorphous cosolute inhibited buffer crystallization.
However, when the co-solute crystallized, buffer crystallization was also
observed. With Surfactant. When mannitol solutions, with progressively
increasing concentrations of polysorbate 80 were lyophilized, as expected,
there was a progressive increase in specific surface area of the lyophile. The
surfactant concentration also influenced the physical form of mannitol in the
final lyophile. While a low polysorbate concentration led to the preferential
crystallization of δ-mannitol, at higher concentrations, β-mannitol was
obtained [120].与氯化钠的相互作用：在氯化钠存在的情况下，甘露醇在冷冻过程中可能保持无定形态。即使在冷冻前的溶液中NaCl含量低至0.5% w/v，这种效应也很明显。然而，接近Tg'的退火处理导致了两种组分的结晶。将NaCl浓度增加到1% w/v显著增加了甘露醇结晶的难度。已有充分证据表明，使用如蔗糖这样的非结晶糖可以抑制NaCl的结晶。有趣的是，作为结晶溶质的甘露醇也抑制了NaCl的结晶。这些相互作用对Tg'乃至主要干燥温度都有重要影响[100]。NaCl还抑制了含有甘露醇和蔗糖的制剂在冷冻过程中甘露醇的结晶，尽管在冻干过程中甘露醇和NaCl都结晶了[104]。含有氯化钠的中性甘氨酸溶液也未观察到甘氨酸结晶[117]。With sodium chloride. In the presence of sodium chloride, mannitol may be
retained amorphous during freezing. The effect was evident even when the NaCl
content in the prelyophilization solutions were as low as 0.5% w/v. However,
annealing near Tg’ led to crystallization of both the components. Increasing
the NaCl concentration to 1% w/v made mannitol crystallization significantly
more difficult. Inhibition of NaCl crystallization with noncrystallizing sugars
such as sucrose is well documented. Interestingly, mannitol which is a
crystallizing solute, also inhibited NaCl crystallization. These interactions
can have significant implications on Tg’ and hence the primary drying
temperature [100]. NaCl also inhibited the crystallization of
mannitol during freezing in formulations containing mannitol and sucrose,
though both mannitol and NaCl crystallized during lyophilization [104].
Neutral glycine solutions containing sodium chloride also showed no glycine
crystallization [117].Table 5 辅料结晶对蛋白稳定性的影响。结晶的实验条件及相应的辅料结晶用粗体字表示。![]()
4.2 蛋白质与辅料相互作用Protein-excipient interaction
尽管围绕辅料的合理处方设计是一个核心议题，了解蛋白质如何影响辅料的相行为同样重要（见表5）。可以归类为以下几个类别的代表性例子突出了蛋白质与辅料之间的相互作用。While rational design of formulations with respect to excipients is a central
theme, it is equally important to be aware of the effect of protein on
excipient phase behavior (Table 5).
Representative examples which highlight the interplay of proteins and excipients
can be classified into the following categories.
4.2.1蛋白质对辅料相行为的影响Effect of protein on excipient phase behavior
多元醇结晶的抑制：在多组分系统中，非结晶性溶质（如冻干保护剂和蛋白质）可以影响填充剂或其他可结晶性溶质的结晶行为。单克隆抗体（mAb）能够以浓度依赖性的方式抑制甘露醇的结晶，特别是在mAb浓度超过20 mg/mL时效果显著[105]。类似的，当Fc融合蛋白浓度达到80 mg/mL时，也能观察到对山梨醇结晶的抑制作用[121]。这种结晶抑制可能带来的影响已在表5中讨论。Inhibition of sugar alcohol crystallization. In multicomponent systems,
non-crystallizing solutes (lyoprotectant and protein) can influence the
crystallization behavior of bulking agents or other crystallizable solutes.
Mannitol crystallization was inhibited by a mAb, the effect being concentration
dependent and pronounced at mAb concentrations >20 mg/mL [105]. A
similar effect was observed when a Fc-fusion protein at concentrations 80 mg/mL
resulted in inhibition of sorbitol crystallization [121]. The
potential implications of such crystallization inhibition are discussed in Table 5.缓冲剂结晶的抑制：如前所述，磷酸钠缓冲剂因选择性结晶而发生pH变化。在低浓度的磷酸钠缓冲剂（10 mM）条件下，BSA和β-半乳糖苷酶（10 mg/mL）能够抑制缓冲盐的结晶，减轻pH变化。当缓冲剂浓度增至100 mM时，蛋白质未能显著抑制缓冲结晶。由此产生的pH变化导致蛋白质聚集[92]。Inhibition of buffer crystallization. As discussed above, sodium phosphate
buffers are known to undergo pH shift as a consequence of selective buffer
component crystallization. At low sodium phosphate buffer concentration (10
mM), BSA and b-galactosidase (10 mg/mL) inhibited buffer salt crystallization
and attenuated the pH shift. When the buffer concentration was increased to 100
mM, the protein did not significantly inhibit buffer crystallization. The
consequent pH shift resulted in protein aggregation [92].辅料物理形态的调整：预期在冻干过程中结晶的填充剂，如甘露醇和甘氨酸，在蛋白质与辅料比例较高时，蛋白质可以抑制填充剂的结晶；在较低比例时，它们似乎会影响填充剂的多晶形态。例如，在甘露醇-溶菌酶的冻干制剂中，随着蛋白质浓度的增加，甘露醇的多晶形态优势从β-甘露醇转变为δ-甘露醇[122]。重要的是，无水甘露醇的多晶形态似乎对药品的质量属性没有任何显著影响，而亚稳态MHH的形成可能具有重大影响。MHH形成受蛋白质影响的方式并不能一概而论。使用Fc融合蛋白时，在中等蛋白质浓度下观察到最多的MHH含量，而更高蛋白质含量则抑制了甘露醇的结晶。处方中添加蔗糖也有助于MHH的形成[123]。Liao等人表明，在白蛋白融合蛋白存在下，MHH的形成受到抑制[124]，而Cao等人报告称，Fc融合蛋白以浓度依赖性的方式促进了MHH的形成（在-20℃下退火）[125]。在甘露醇-蔗糖（1:1）的处方中，增加溶菌酶或BSA含量促进了MHH的形成。另一方面，在甘露醇对蔗糖比例为4:1时，增加蛋白质含量减少了MHH的形成[126]。Modification of the physical form of excipients. Bulking agents, such as
mannitol and glycine, are expected to crystallize during lyophilization. While
at higher protein: excipient ratios, proteins can inhibit the crystallization
of bulking agent, at lower ratios they appear to influence the polymorphic form
of bulking agent. For example, in freeze-dried mannitol-lysozyme formulations,
with increasing protein concentration, the prevalence of mannitol polymorphic
form shifted from β- to δ-mannitol [122]. Importantly, the polymorphic
form of anhydrous mannitol does not seem to have any measurable impact on the
quality attributes of the drug product, while formation of the metastable MHH
can have significant implications. It is not possible to generalize the manner
in which MHH formation is influenced by proteins. With an Fc-fusion protein,
the maximum MHH content was observed at an intermediate protein concentration,
with higher protein content inhibiting mannitol crystallization. Addition of
sucrose to the formulation also facilitated MHH formation [123].
Liao et al have shown that MHH formation was inhibited in the presence of an
albumin fusion protein [124], while Cao et al reported that a
Fc-fusion protein facilitated MHH formation in a concentration dependent manner
(upon annealing at -20℃) [125]. In a mannitol-sucrose (1:1)
formulation, increasing lysozyme or BSA content promoted MHH formation. On the
other hand, with mannitol to sucrose ratio 4:1, increasing the protein content
reduced MHH formation [126].
4.2.2 辅料相行为对蛋白质稳定性的影响Effect of excipient phase behavior on protein stability
非预期结晶：冷冻是生物制剂制造中冻干和冷冻储存的常见单元操作。蛋白质原料在添加稳定剂后通常在配制成药品前冷冻储存数月。从经济和实用的角度来看，-20℃（±5℃）是一个方便的储存温度[4]。然而，大多数蛋白质制剂在这个温度下并没有完全冻结，可能以冰和冷冻浓缩液的混合物形式存在。储存温度在决定冷冻系统的长期稳定性方面至关重要。Unintended crystallization. Freezing is a common unit operation in
freeze-drying and frozen storage in the manufacturing of biologic formulations.
The protein DS in presence of stabilizers is often stored frozen for months
prior to its formulation into a drug product. From an economical and practical
standpoint, -20℃ (±5℃) is a convenient storage temperature [4].
However, most of the protein formulations are not completely frozen at this
temperature and can exist as a mixture of ice and a freeze-concentrate. The
storage temperature is critical in determining the long-term stability of
frozen systems.含有山梨醇（5% w/v）的Fc融合蛋白（2 mg/mL）在-20、-30和-70℃下冷冻储存了超过1年。在-30℃的中间温度储存的稳定性最差[127]。在-30℃，比基质的Tg'（-45℃）高15℃，非预期的山梨醇结晶导致其冷冻保护活性的丧失。在后续研究中，两种单克隆抗体（IgG1和IgG2）在含有274 mM山梨醇和10 mM醋酸钠缓冲剂的溶液中冷冻，浓度范围从0.1到120 mg/mL。当IgG2在-30℃储存时，观察到山梨醇结晶的蛋白质浓度依赖性抑制。在浓度小于60 mg/mL时观察到山梨醇结晶和蛋白质聚集。然而，在更高浓度80到120 mg/mL时，山梨醇结晶被抑制，防止了蛋白质聚集（见图6）。An Fc-fusion protein (2 mg/mL) containing sorbitol (5% w/v) was stored frozen
at -20, -30 and -70℃ for >1 year. Storage at the intermediate temperature of
-30℃ resulted in least stability [127]. At -30℃, which is 15℃ higher
than the Tg’ (-45℃) of the matrix, unintended sorbitol crystallization resulted
in loss of its cryoprotectant activity. In a follow-up study, two mAbs (IgG1
and IgG2) were kept frozen in solutions containing 274 mM sorbitol and 10 mM
sodium acetate buffer at concentrations ranging from 0.1 to 120 mg/mL. When
IgG2 was stored at -30℃, a protein concentration dependent inhibition of
sorbitol crystallization was observed. Sorbitol crystallization and hence
protein aggregation was observed at concentrations <60 mg/mL. However, at
higher concentrations 80 to 120 mg/mL, sorbitol crystallization was inhibited
preventing protein aggregation (Fig. 6).含有海藻糖（8.4% w/v）和其他处方组分的IgG2单克隆抗体（20 mg/mL）溶液在-10、-20和-40℃下储存了12个月。在-20℃下的聚集最多。假设基质的Tg'（-29℃）以上的温度海藻糖结晶导致在-20℃时冷冻保护活性的丧失。尽管在-10℃时海藻糖结晶了，但由于系统中的流动性较高，导致蛋白质重新折叠，从而减少了聚集。在-40℃，远低于系统的Tg'，海藻糖保持无定形态，没有观察到聚集。值得注意的是，在-10℃时海藻糖的结晶并没有导致冷冻保护的丧失。这项研究强调，仅仅冷冻保护剂的结晶并不是导致蛋白质聚集的原因。应彻底调查储存温度、冰界面变性、冷冻系统中的流动性以及冷变性效应等因素[128]。An IgG2 mAb (20 mg/mL) solution containing trehalose (8.4% w/v) and other
formulation components was stored for 12 months at -10, -20 and -40℃.
Aggregation of mAb was highest at -20℃. It was hypothesized that, trehalose
crystallization at a temperature above the Tg’ (-29℃) of the matrix resulted in
loss in the cryoprotectant activity at -20℃. Although trehalose crystallized at
-10℃, the mobility in the system was high leading to refolding of the protein
resulting in lesser aggregation. At -40℃ which is well below the Tg’ of the
system, trehalose was retained amorphous and no aggregation was observed.
Notably, crystallization of trehalose at -10℃did not result in loss in
cryoprotection. This study highlighted that crystallization of cryoprotectant alone
is not responsible for protein aggregation. Factors such as storage
temperature, ice-interface denaturation, mobility in the frozen system along
with cold denaturation effects should be thoroughly investigated [128].![]()
图 6. 在10mM醋酸钠缓冲液中加入274 mM山梨醇和0.004% w/v聚山梨醇酯20的IgG2（40 至 120 mg/mL）水溶液在-30℃下储存24个月。图中显示了各时间点的聚集体形成（%）与IgG2浓度的函数关系。填充符号表示结晶山梨醇，空符号表示无定形山梨醇。当浓度≤60 mg/mL时，山梨醇结晶导致较高的聚集百分比，而当浓度>80 mg/mL时，山梨醇结晶受到抑制。在IgG2浓度较高时观察到的聚集体百分比略高，这可能是由于其他因素造成的，包括蛋白质与蛋白质之间的相互作用。Fig. 6. IgG2 (40 to 120 mg/mL) aqueous solutions with 274 mM sorbitol in 10 mM
sodium acetate buffer and 0.004% w/v polysorbate 20 were stored at -30℃ for24
months. Aggregate formation (%) at respective time points as a function of
IgG2concentration are shown. The filled symbols denote crystallized sorbitol
whereasempty symbols denote amorphous sorbitol. At concentrations ≤60 mg/mL,
sorbitolcrystallized resulting in higher % aggregates whereas, at
concentrations >80 mg/mLsorbitol crystallization was inhibited. Slightly
higher % aggregates observed athigher IgG2 concentration maybe due to other
factors including protein–proteininteractions. Reproduced from Piedmonte et al [121]with permission of Elsevier.比较了温度对甘露醇、海藻糖和蔗糖水溶性的影响，无论温度如何，蔗糖的溶解度都比甘露醇和海藻糖高得多（见图7a）。甘露醇和海藻糖的较低溶解度转化为它们更高的结晶倾向以及mAb聚集。这些处方在-20℃下储存了28天，并用各自的辅料进行诱导结晶以加速结晶。在蔗糖处方中未检测到聚集，而在海藻糖和甘露醇处方中聚集分别增加了1%和3%（见图7）[103]。甘露醇结晶与蛋白质聚集之间的关系是众所周知的。甘露醇在冷冻、退火和长期储存期间有很强的结晶倾向，这可能会引起蛋白质聚集[49,118,129,130]。The effect of temperature on the aqueous solubility of mannitol, trehalose and
sucrose were compared and irrespective of temperature, sucrose had a much
higher solubility than mannitol and trehalose(Fig. 7a).The lower solubility of mannitol and trehalose translated to their much higher
crystallization propensity as well as mAb aggregation. These formulations were
stored at -20℃ for 28 days and seeded with respective excipients to
acceleratecrystallization. No aggregation was detected in sucrose formulations,
while aggregates increased by 1 and 3% in trehalose andmannitol formulations
respectively (Fig. 7) [103]. The
relationshipbetween mannitol crystallization and protein aggregation is well-known.
Mannitol has a high propensity to crystallize during freezing, on annealing and
on long-term storage which can induce protein aggregation [49,118,129,130].图7. a) 蔗糖（蓝色）、海藻糖（红色）和甘露醇（绿色）的溶解度与温度的函数关系。 b) 含有 6% w/v 蔗糖、海藻糖或甘露醇的制剂中mAb2在冷冻前（红色）和在-20℃下储存28天后（蓝色）的高分子量聚集体 (HMWS) 百分比柱状图。Fig. 7. a) Solubility of sucrose (blue), trehalose (red) and mannitol (green)
as a function of temperature. b) Bar plot of percentage high molecular weight
aggregates (HMWS) of mAb2 in formulation containing 6%
w/v sucrose, trehalose or mannitol before freezing (red) and after 28 days of storage
at -20℃ (blue). Reproduced from Connolly et al [103]with permission from Elsevier. (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this
article.)此外，研究了不同浓度的海藻糖和mAb，以确定可以导致在-20℃下储存12个月的稳定处方的最优海藻糖与mAb比例，最小化聚集。在0至100 mg/mL的所有mAb浓度下都观察到海藻糖的结晶。然而，随着mAb浓度从25 mg/mL增加到100 mg/mL，结晶的海藻糖比例从53%降低到9%。得出的结论是，海藻糖与mAb的比例在0.2:1到2.4:1的范围内是最优的稳定性。在比例小于0.2:1时，海藻糖浓度不足以作为冷冻保护剂，在比例大于2.4:1时，高浓度的海藻糖导致其结晶[103]。In addition, the effect of varying trehalose and mAb concentrations were
investigated to determine the optimum trehalose tomAb ratio that can result in
a stable formulation with minimumaggregation on 12-month storage at -20℃.
Trehalose crystallization was observed at all concentrations of mAb from 0 to
100 mg/mL. However, the fraction of trehalose that crystallized reducedfrom 53%
at 25 mg/mL mAb concentration to 9% at 100 mg/mLmAb concentration. It was
concluded that trehalose to mAb ratioin the range of 0.2:1 to 2.4:1 is required
for optimum stability. Atratios <0.2:1, trehalose concentration was not
sufficient to act asa cryoprotectant and at ratios >2.4:1, the high
trehalose concentration resulted in its crystallization [103].其他处方辅料如缓冲剂的结晶可能导致冷冻期间的pH变化，从而导致蛋白质聚集，特别是如果蛋白质稳定性是pH依赖的。当从室温冷却到-25℃时，10 mM和100 mM磷酸钠缓冲剂（初始pH 7.0）中磷酸二钠的结晶被证明分别导致pH变化1.5和3.2个单位。单体和四聚体β-半乳糖苷酶（2 mg/mL至1000 mg/mL）对磷酸二钠结晶没有抑制作用。在缓冲剂浓度较高的溶液中恢复的蛋白质活性较低。另一方面，尽管在10 mM和100 mM磷酸钾缓冲剂中磷酸钾一钠结晶，冷冻状态下的pH变化仅为0.1和0.3个单位。在这种情况下，单体和四聚体β-半乳糖苷酶的聚集较低，与磷酸钠缓冲剂相比。然而，较低的聚集归因于除缓冲结晶以外的因素，如冰界面诱导的变性或冷变性[131,132]。Crystallization of other formulation excipients such as bufferscan result in pH
shift during freezing and consequent proteinaggregation, especially if the
protein stability is pH dependent.When cooled from RT to -25℃, crystallization
of disodium phosphate in 10 mM and 100 mM sodium phosphate buffers (inital
pH7.0) was shown to cause pH shift of 1.5 and 3.2 units respectively.Monomeric
and tetrameric b- galactosidase (2 mg/mL to 1000 mg/mL) did not have an
inhibitory effect on disodium phosphate crystallization. The recovered protein
activity was lower in solutionswith higher buffer concentration. On the other
hand, in spite ofcrystallization of monopotassium phosphate in 10 and 100
mMpotassium phosphate buffer, the pH shift in the frozen state wasonly 0.1 and
0.3 units respectively. Aggregation of monomericand tetrameric b- galactosidase
in this case was lower when compared to sodium phosphate buffer. The lower
aggregation howeverwas attributed to factors other than buffer crystallization
such asice-interface induced denaturation or cold denaturation [131,132].预期结晶：在高浓度蛋白质制剂（>50 mg/mL）中，通常不需要填充剂。然而，甘露醇在含有 100 mg/mL 重组蛋白的制剂中退火结晶的能力使复溶时间大大缩短。尽管增加蛋白质浓度抑制了甘露醇的结晶，即使是甘露醇的一小部分结晶也促进了快速复溶[112]。当含有精氨酸和甘氨酸的mAb溶液（100 mg/mL）进行冻干时，蛋糕部分结晶。与无定形蛋糕相比，它的复溶时间更短[133]。Intended crystallization. In high concentration protein formulations (>50
mg/mL), a bulking agent is not often required. However,the ability of mannitol
to crystallize on annealing in formulationswith 100 mg/mL recombinant protein,
resulted in a dramaticreduction in reconstitution times. Although increasing
the proteinconcentration inhibited mannitol crystallization, even the
crystallization of a small fraction of mannitol facilitated rapid
reconstitution [112]. When a mAb solution (100 mg/mL) containing
arginineand glycine was freeze-dried, the cake was partially crystalline.
Itshowed shorter constitution time when compared to cakes thatwere amorphous [133].在含有甘露醇和蔗糖的蛋白质制剂中，甘露醇最好结晶以充当填充剂。然而，退火引起的甘露醇结晶导致LDH活性恢复下降，而蔗糖没有起到溶解保护剂的作用。加入吐温80后，LDH活性恢复增加，这表明界面现象导致的不稳定性可能是冰或甘露醇结晶造成的[134]。在海藻糖-甘露醇制剂中，当BSA浓度较低时（4% w/w），冻干物中的结晶甘露醇为半水合物和无水物形式，而曲哈糖则为二水合物形式。在较高的 BSA 浓度（10% w/w）下，冻干物只含有δ-甘露醇，而海藻糖则呈无定形状态[135]。在这种情况下，当BSA浓度较高时，海藻糖将成为有效的稳定剂，而甘露醇将成为有效的填充剂。In protein formulations containing mannitol and sucrose, mannitol
crystallization is desirable for it to act as a bulking agent.However,
annealing induced crystallization of mannitol led todecrease in LDH activity
recovery and sucrose did not act as alyoprotectant. LDH activity recovery
increased on addition ofTween 80, suggesting instability due to interfacial
phenomenonwhich can be due to ice or mannitol crystallization [134].
Intrehalose-mannitol formulations, at lower BSA concentrations(4% w/w),
lyophiles contained crystalline mannitol as both hemihydrate and anhydrous
forms whereas trehalose crystallized asthe dihydrate form. At a higher BSA
concentration (10% w/w), thelyophiles contained only the d-mannitol and
trehalose wasretained amorphous [135]. In this case, at higher BSA
concentrations, trehalose will act as an effective stabilizer while
mannitolwill be an effective bulking agent.从处方角度来看，上述研究表明如海藻糖或山梨醇等稳定剂的结晶可以显著破坏蛋白质制剂的稳定性。From a formulation point of view, the above studies show that crystallization
of a stabilizer, such as trehalose or sorbitol, can significantly destabilize protein
formulations.Future directions
随着我们对多组分药物制剂中蛋白质稳定性行为认识的不断深入，蛋白质处方开发已经从依赖试错的传统方法，转变为更加系统化、科学驱动的进程。然而，处方开发过程中仍面临一些挑战。正如前文讨论的，蛋白质在冷冻/冻干过程中所承受的应力以及辅料对蛋白质稳定性的影响机制尚未完全明了。尽管已提出多种机制，但它们的适用性似乎取决于生物分子的类型和工艺条件。因此，不能简单地进行广泛推论。不过，分子动力学和模拟领域的最新研究为确定辅料比例以及评估冷冻过程中的应力对蛋白质稳定性的影响提供了希望。With an improved understanding of the stability behavior of proteins in a
multi-component DP, protein formulation development has evolved, from a
predominantly trial-and-error process, to a more systematic science-driven
process. Nonetheless, a number of challenges still exist in the formulation
development process. As is evident from the discussion above, the stresses
experienced by proteins during freezing/freeze-drying and mechanisms of
excipient-induced protein stabilization are not completely understood. Multiple
mechanisms have been proposed, but their validity seems to depend upon the type
of biomolecule (s) and the process conditions. A broad generalization may not
be appropriate. However, recent efforts in the field of molecular dynamics and
simulations look promising in identifying excipient ratios as well as
determining effect of stresses during freezing on protein stability.对蛋白质在冷冻过程中可能遇到的、以前未被充分认识的应力的兴趣与日俱增，例如冰晶形成前沿产生的气泡、冰形成过程中体积膨胀引起的局部压力和机械应力[4]。有假设认为，准液态层——即在冰晶上形成的液态水薄膜——对蛋白质稳定性起着重要作用。蛋白质在这一层次受限的物理微环境中的行为可能与在散装状态下有显著差异。冷冻引起的不稳定可能归因于蛋白质倾向于分配到这一层次，导致稳定剂无法提供有效的冷冻保护[137]。在这个领域的进一步研究可以为理解这一层的性质和功能相关性提供机理见解，并有助于识别应对冷冻相关应力的缓解策略。There is a growing interest in studying previously unidentified stresses
experienced by protein during freezing, such as air bubbles formed on the ice
crystallization front, local pressure and mechanical stresses due to volume
expansion during ice formation [4]. It is hypothesized that the
quasi-liquid layer, i.e., a thin film of liquid water formed on ice crystals,
plays an important role in protein stability. The physical microenvironment of
a protein confined within this layer may be substantially different than in the
bulk. The freeze-induced destabilization might be attributed to the preferred
partitioning of proteins into the layer and hence inability of stabilizer to
provide cryoprotection [137]. More research in this area can provide
mechanistic insights into properties and functional relevance of this layer and
to help identify mitigation strategies to overcome the freezing-associated
stresses.对表面活性剂引起的冷冻保护的了解需要更深入的研究，特别是就冷冻浓缩可能将表面活性剂浓度提高到临界胶束浓度(CMC)以上的潜力而言。与聚山梨酯相关的降解和毒性问题，以及规避这些问题的策略，是值得关注的研究方向。An understanding of surfactant-induced cryoprotection warrants further
investigations, especially with respect to the potential for
freeze-concentration to increase the surfactant concentration above CMC. The
strategies to circumvent degradation and toxicity issues associated with
polysorbates are of interest.储存温度和时间、冷冻速率、辅料和蛋白质浓度等因素对优化原料药稳定性的影响，需要进一步研究。在相对较高的储存温度(-20℃)下储存的冷冻原料药，具有足够的分子流动性来诱导辅料相行为的变化，这可能最终影响稳定性。对冷冻原料药的稳定性测试应在高于和低于玻璃化转变温度(Tg')的实际温度下进行。这将有助于确立辅料对蛋白质稳定性的影响。The effect of storage temperatures and time, freezing rates, excipient and
protein concentrations needs further investigation for optimizing DS stability.
Frozen DS, stored at relatively higher storage temperatures (-20℃), possess
adequate molecular mobility to induce changes in excipient phase behavior,
eventually compromising stability. Stability testing for frozen DS should be
carried out in real time at temperatures above and below Tg’. This will help
establish excipient effect on protein stability.预测冻干蛋白质制剂的固态稳定性依赖于识别影响蛋白质稳定性的关键属性。新型分析技术，如微拉曼光谱[42]、小角散射[138]、宽带相干反斯托克斯拉曼散射[139]、固态氢/氘交换质谱[140]，以及如能量景观模型[141]、分子动力学模拟[142]等建模工具，可以帮助进一步确立这种联系。对稳定化机制更深入的理解将有助于有效的蛋白质稳定化，并缩短开发时间。Prediction of solid-state stability of lyophilized protein formulation relies
upon identifying key properties influencing protein stability. Novel analytical
techniques, such as micro-Raman spectroscopy [42], small-angle
scattering [138], broadband coherent anti-Stokes Raman scattering[139], solid-state hydrogen/deuterium exchange mass spectrometry [140]and modelling tools such as energy landscape model [141], molecular
dynamic simulations [142] can help in further establishing the
connection. An improved understanding of stabilization mechanisms will assist
in effective protein stabilization and reduce developmental timelines.随着高浓度蛋白质制剂的日益普及，识别具有多功能属性的辅料将使处方师能够减少辅料的种类和浓度。另一方面，发现新型稳定剂可能是一种潜在的途径，尽管这种方法可能会面临毒性和监管审批的挑战[143]。With the increasing popularity of high-concentration protein formulations, the
identification of excipients with multifunctional properties will enable the
formulator to decrease the number and concentration of excipients. On the other
hand, identifyingnovel stabilizers might be a potential avenue, though the
approach would be associated with the hurdles of toxicity and regulatory
approval process [143].合理选择辅料是稳定对冷冻和/或干燥应力敏感的蛋白质的关键。尽管对稳定化机制的理解尚不完全，但很明显，稳定剂包括了糖/多元醇、氨基酸和聚合物等多种化合物。通常建议糖与蛋白质的重量比应≥1。最终的处方还可能需要包括缓冲剂、表面活性剂和填充剂。由于蛋白质可以影响辅料的物理属性，反之亦然，因此处方组成应基于充分的实验和适当的稳定性研究。The selection of the appropriate excipients provides the avenue to stabilize
proteins sensitive to freezing and/or drying stresses. While the stabilization mechanism
is incompletely understood, it is evident that the stabilizers encompass a wide
variety of compounds, including sugars/sugar alcohols, amino acids and
polymers. A sugar to protein weight ratio ≥1 is generally recommended. The
final formulation may also require incorporation of buffers, surfactants and
bulking agents. Since the protein can influence the physical properties of the
excipients and vice versa, the formulation composition should be based on sound
experimentation and appropriate stability studies.
