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）
企业
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）基于蛋白的治疗药物在治疗人类疾病中的重要性正日益提升。它们之所以受到青睐，主要是因为它们具有高度的目标专一性和广泛的应用范围[1]。这些产品中的活性药物成分可能包括单克隆抗体（mAb）、抗体-药物结合物（ADC）、Fc融合蛋白、酶类、激素、干扰素或白细胞介素等[2]。Protein-based therapeutics are becoming increasingly important in the treatment
of human diseases. The primary reason for their popularity is the high target
specificity and broad applicability [1]. The active pharmaceutical
ingredient in these products may include a monoclonal antibody (mAb),
antibody-drug conjugate, Fc-fusion protein, enzyme, hormone, interferon or
interleukin [2].生产治疗性蛋白质及其转化为药物制剂可能是一个复杂且成本高昂的过程。为了最大化生产效率，生产活动中通常会制备大量的蛋白质溶液。这些蛋白质药物原料（DS）在最终配制成药物制剂（DP）之前，往往需要长时间储存。普遍认为，在低温条件下储存可以维持蛋白质的稳定性，进而延长其有效期。在这种情况下，DS通常在添加了适宜的稳定剂后进行冷冻，然后在配制成DP之前进行解冻。冷冻状态带来了额外的好处，比如降低了微生物生长的风险，以及在运输过程中减少了泡沫或摇晃的问题[3]。商业化的蛋白质产品，包括Jetrea®、以及细胞和基因治疗产品如Glybera®、IMLYGIC®和Luxturna®，也采用了冷冻储存的方式[4]。The production of therapeutic proteins and their formulation into drug products
can be complex and expensive. In order to optimize capacity use, bulk protein
solutions are often produced in manufacturing campaigns. This protein drug
substance (DS) may be then stored for long time periods before being formulated
into a drug product (DP). The bulk DS are often stored in the frozen state with
the assumption that the protein stability will be preserved at the low
temperature of storage and will increase the shelf-life. In such situations,
the DS, usually frozen in the presence of suitable stabilizers, is thawed
before it is formulated into the DP. The frozen state offers additional
advantages, such as the reduced possibility of microbial growth and alleviation
of foaming or shaking issues during transport [3]. Frozen storage
has also been used for commercial protein products such as Jetrea®, cell and
gene therapy products including Glybera®, IMLYGIC®, and Luxturna®[4].市场上的大多数蛋白质制剂通常通过非肠道途径给药。考虑到经济性和使用的便捷性，人们更倾向于使用现成的液体制剂。然而，许多蛋白质在液体制剂中容易遭受化学（如去酰胺化或氧化）和物理（如聚集和沉淀）降解的影响[5]。如果无法开发出具有预期货架寿命的液体剂型，冻干技术则提供了一种制备稳定制剂的替代方法。在冻干过程中，含有药物原料（DS）和辅料的溶液会经历三个连续的阶段——冷冻、一次干燥（主要通过升华去除冰晶），以及二次干燥（去除结合水），具体细节见图1。在使用前，将冻干粉末重新溶解成溶液以供给药。值得注意的是，目前市场上约有30%的生物制剂以冻干产品的形式销售[6]。A large fraction of the marketed protein formulations are administered
parenterally. From both economic and ease-of-use considerations, a ready-to-use
liquid formulation is desired. However, many proteins are susceptible to
chemical (e.g. deamidation or oxidation) and physical degradation (e.g.
aggregation and precipitation) in a liquid formulation [5]. If a
solution dosage form with the desired shelf-life cannot be developed,
freeze-drying provides an avenue to prepare stable formulations. In the
freeze-drying process, a solution containing the DS and the excipients is
subjected to three sequential steps - freezing, primary drying predominantly to
remove ice by sublimation, and secondary drying to remove the sorbed water
(more details in Fig. 1). Right before use, the lyophilized powder is
reconstituted into a solution for administration. Interestingly, ~30% of
currently marketed biologics are available as lyophilized products [6].尽管冷冻保存和冻干技术的主要目的是维持生物制剂的稳定性和保护其生物活性，但在冷冻和干燥过程中，蛋白质仍然可能面临多种压力。这些压力可能引起蛋白质结构的可逆或不可逆变化，甚至导致生物活性的损失。为了“保护”蛋白质不受这些压力的损害，通常的做法是在制剂中添加一些稳定剂。这些稳定剂包括糖类、多元醇、聚合物、表面活性剂和氨基酸等多种化合物[7]。深入理解这些压力的来源以及稳定剂的作用机制，对于开发出稳定的生物制剂至关重要。While frozen storage and lyophilization are intended to preserve and stabilize
a biologic formulation, a protein may be subjected to several stresses both
during freezing and drying. These stresses can lead to reversible or
irreversible changes and may also result in a loss in its biological activity.
In order to ‘protect’ the protein from these stresses, it is a common practice
to add stabilizing excipients. These encompass a wide variety of compounds
including sugars, polyols, polymers, surfactants, and amino acids [7]. An
understanding of these stresses and functional mechanisms of stabilizers is
critical to the design of stable biologic formulations.本文综述专注于为冷冻和冻干制剂合理选择辅料。文中将探讨各类稳定剂及其稳定机制。我们力图归纳总结近十年来的重要科研成果。同时，我们将不涉及一些相关且重要的议题，比如冻干工艺的周期优化和包装设计，这些议题已在近期的一些杰出文献中得到了深入讨论[7-9]。The present review focusses on the rational selection of excipients for use in
frozen and freeze-dried formulations. The various types of stabilizers and the
stabilization mechanism will be discussed. We have attempted to summarize the
important research findings of the past decade. We will refrain from discussion
of several related and important topics, such as lyophilization cycle optimization
and packaging design. Some of these topics have been covered in excellent
recent publications [7-9].Processing-induced stresses and stabilization mechanisms
在冷冻干燥的冷冻和干燥阶段中，蛋白质所面临的压力通常被视为一个整体来考虑。这两个阶段都包括水分的去除，如冷冻时通过冰晶形成以及干燥时通过升华和解吸过程。然而，每一步中与溶质相关的未冻水存在细微差别。在冷冻过程中，未冻水保留在冷冻浓缩液中，而在干燥过程中则被移除。因此，蛋白质在这些不同步骤中所承受的压力本质上是不同的[10]。基于此，在本综述中，我们将辅料在冷冻阶段和冷冻储存期间所遇到的稳定化机制及其压力归为一类，而将干燥和储存期间的压力分别进行讨论。The stresses to which a protein is exposed during the freezing and drying steps
of freeze-drying are generally considered together. Both processes involve
removal of water, such as by ice crystallization during freezing and by
sublimation/desorption during drying. However, there is a subtle difference
with respect to the unfrozen water associated with the solutes during each
step. While the unfrozen water is retained in the freeze-concentrate during
freezing, the process of drying leads to its removal. As a result, the stresses
experienced by a protein during these steps are fundamentally different[10]. Hence in this review, the stresses and mechanisms of stabilization
by an excipient during the freezing step and frozen storage are grouped
together while those during drying and storage are discussed separately.2.1 冷冻和冷冻储存Freezing and frozen storage
蛋白质的天然（折叠）形态是其发挥治疗作用的基础，并且在热力学上，相比于其展开状态，它是最稳定的形态，具有更低的自由能。这种天然蛋白质的稳定性可以通过其展开自由能（ΔGunf）来量化，它代表了蛋白质展开所需克服的能量障碍。在生理条件下，生物活性的天然构象与非活性的展开构象之间的自由能差异通常很小（大约5到20千卡/摩尔）。因此，即使是温度、pH值或离子强度等外部条件的微小变化，也可能触发蛋白质结构的展开[5]。The native (folded) structure of the protein is responsible for its therapeutic
activity and is thermodynamically the most stable form with a lower free energy
when compared to the unfolded state. The energy barrier for a native protein to
unfold can be characterized by its free energy of unfolding (ΔGunf). Under
physiologic conditions, the free energy difference between the native
(biologically active) and folded (inactive) protein conformation can be very
low (~5 to 20 kcal/mol). Hence a minor change in the external conditions such
as temperature, pH, ionic strength among others, can result in unfolding of the
protein [5].在冷冻阶段，概念上包括两个不相互排斥的物理事件：过冷和冰晶形成，这导致形成最大程度的冷冻浓缩溶液（见图1）。相应地，蛋白质在每个事件中所经历的压力以及辅料通过可能的冷冻保护机制来稳定天然蛋白质状态的作用可以被区分开来。
The freezing stage conceptually involves two mutually nonexclusive physical
events, namely supercooling and ice crystallization resulting in the formation
of maximally freeze-concentrated solution (Fig. 1). Accordingly,
the stresses experienced by protein and the possible cryoprotection mechanisms
by which the excipients stabilize the native protein state during each event
can be distinguished.
过冷现象。当含有药物原料和辅料的溶液被冷却时，可能不会立即观察到自发的冰晶形成，系统可能会进入过冷状态。在非受控冷冻情况下，这种情况通常会持续到-10至-12摄氏度[12]。影响超冷程度的因素包括冷却速率以及溶质的类型和浓度。冷变性是指蛋白质药物原料在冰晶形成前，由于低温引起的结构展开而发生的变性过程。然而，在冷冻干燥过程中，冷变性并不是一个主要问题，因为与冷冻干燥的时间尺度相比，蛋白质的展开速度相对较慢[13,14]。值得注意的是，某些添加剂（如蔗糖和海藻糖）以及较高浓度的蛋白质（例如β-乳球蛋白）可以降低冷变性的温度[4]。Supercooling.When a solution containing the DS and excipients is cooled, spontaneous
ice crystallization may not be observed and the system tends to supercool. This
situation generally persists up to -10 to -12℃ in the case of uncontrolled
freezing [12]. The factors determining the degree of supercooling
include cooling rate and the type and concentration of solutes. Cold
denaturation is the process by which protein DS can denature due to unfolding
at low temperatures prior to ice crystallization. However, cold denaturation is
not a major concern during freeze-drying. Protein unfolding is relatively slow
when compared to the timescales of freeze-drying [13,14].
Interestingly, the presence of additives (such as sucrose and trehalose) and
high protein (β-lactoglobulin) concentration, lower the cold denaturation
temperature [4].![]()
Fig. 1. Typical steps involved in
lyophilization of biologics. The y-axis represents temperature (color-coded).
The temperature range, during different stages of freeze-drying, broadly
follows the color scheme. Rectangles with sharp corners represent intermediate
stages while rectangles with rounded edges represent the final product. The
alphabets adjacent to the arrows represent the possible mechanisms of protein
stabilization during each stage (any of the possible mechanisms may be
operative; more details in the text). All the solutes are assumed to remain
amorphous, both in the frozen DS/DP and lyophilized DP. A typical freeze-drying
process consists of three stages; freezing, primary drying, and secondary
drying. Freezing is an efficient desiccation step where most of the solvent is
separated from the solutes to form ice. As freezing progresses, the solute
phase becomes highly concentrated and is termed the ‘freeze concentrate’.
During primary drying, ice is transferred from the product to the condenser by
sublimation. The primary drying stage is generally the longest and its
optimization has a large impact on process economics. During secondary drying,
water is desorbed from the freeze concentrate, usually at elevated temperatures
and low pressures. Secondary drying normally takes only a few hours. Typically,
drying is conducted below Tg’ (glass transition temperature of the
freeze-concentrate) during primary drying and below Tg (glass transition
temperature of the lyophile) during secondary drying[11].图1展示了生物制品冻干过程中的典型步骤。图中的y轴以颜色编码表示温度。在冻干过程的不同阶段，温度变化大致遵循颜色方案。尖角矩形表示中间过程，而圆角矩形表示最终产品。箭头旁的字母代表每个阶段可能的蛋白质稳定机制（可能同时存在多种机制；具体细节见文本）。在整个冻干过程中，所有溶质均假定保持无定形态，无论是在冻结干燥的DS/DP中还是在最终的冻干DP中。典型的冻干过程包括三个阶段：冻结、一次干燥和二次干燥。冻结是一个高效的脱水步骤，大部分溶剂在此过程中与溶质分离形成冰晶。随着冻结的进行，溶质相变得高度浓缩，形成所谓的“冻结浓缩相”。在一次干燥阶段，冰晶通过升华作用从产品转移到冷凝器。一次干燥通常是耗时最长的阶段，其优化对提高过程的经济性有显著影响。在二次干燥阶段，通常在较高温度和低压力条件下，从冻结浓缩相中脱除水分。二次干燥通常只需要几小时。通常，一次干燥在Tg'（冻结浓缩相的玻璃化转变温度）以下进行，而二次干燥在Tg（冻干产品的玻璃化转变温度）以下进行。冰晶形成与冷冻浓缩。进一步降低温度最终会诱导冰核的形成和随后的晶体生长。冰晶的形成会产生冰-水和冰-空气界面，这可能会对蛋白质造成压力，导致其不稳定[15,16]。水分以冰的形式被移除，导致含有溶质的溶液在未冻水中的浓度增加，这可能会改变其离子强度、粘度和pH值。此外，在多组分系统中，这个过程可能导致不同溶质组成的相分离，这些变化可能会使蛋白质不稳定[13,14]。Ice
crystallization and freeze-concentration. Further lowering of
temperature eventually induces the formation of ice nuclei followed by crystal
growth. Ice crystallization creates ice-water and ice-air interfaces which can
induce stress leading to protein destabilization [15,16]. Water
removal (as ice) concentrates the solution (containing the solutes dissolved in
the unfrozen water) in the interstitial region, and may alter its ionic
strength, viscosity and pH. Additionally, in multicomponent systems, this
process may lead to separation of phases differing in their solute composition.
The attendant changes have the potential to destabilize the proteins[13,14].冷冻储存。如上所述，蛋白质药物原料通常进行冷冻储存。产品通常储存在大型容器中，冷却速率可能不受控制。最佳储存温度可以通过在不同温度下储存前后对蛋白质进行特性分析来确定[3]。在冷冻储存的背景下，需要仔细考虑由于：（i）冷冻和解冻过程，以及（ii）在冷冻状态下长时间储存所引起的压力。长期储存可能会促使某些辅料结晶，从而导致蛋白质不稳定（见第4.2.2节）。Frozen
storage. As mentioned above, protein DS are often stored frozen. The
product is often stored in large containers and the cooling rate can be
uncontrolled. The optimal storage temperature can be determined by characterizing
the protein before and after storage at different temperatures [3].
In the context of frozen storage, the stresses due to: (i) freezing and
thawing, and (ii) extended storage in the frozen state mandate careful
consideration. The longterm storage can induce crystallization of some
excipients leading to protein instability (Section 4.2.2).存在多种化学多样性的化合物，统称为冷冻保护剂，它们可以保护蛋白质免受冷冻引起的压力。为了解释它们在冷冻系统中的稳定效果，已经提出了多种机制。A wide variety of chemically diverse compounds, known as cryoprotectants, can
protect proteins from freezing-induced stresses. Multiple mechanisms have been
proposed to explain their stabilizing effects in frozen systems.优先排斥（也称为溶质排斥或优先水合）：在水溶液中，水分子与蛋白质表面的极性基团相互作用，使蛋白质得到优先水合。在冷冻过程的早期阶段，冷冻保护剂分子被选择性地排除在蛋白质表面之外。这种溶质分子的排除增加了蛋白质展开的自由能（ΔGunf），从而有利于并稳定了其天然状态[10]。Preferential exclusion (also known
as solute exclusion or preferential hydration): In aqueous
solutions, the water molecules interact with the polar groups on the protein
surface making the protein preferentially hydrated. In the initial stages of
the freezing process, the cryoprotectant molecules are selectively excluded
from the immediate vicinity of the protein surface. This exclusion of solute
molecules increases the free energy of unfolding (ΔGunf), thus favoring and
stabilizing the native state [10].玻璃化假说（或玻璃基质理论）：冰晶形成和冷冻浓缩最终推动系统接近玻璃态，这是一种流动性受限的状态。冷冻保护剂如糖和聚合物的存在导致冷冻浓缩液的粘度增加，这降低了分子的流动性，从而减慢了所有动态过程。蛋白质实际上变得几乎不动。由于流动性是变性和降解反应的先决条件，因此在玻璃态中限制的流动性使蛋白质在冷冻干燥的实际时间尺度上相对稳定[14,17]。然而，长期储存可能会因储存温度和随后的辅料结晶而显示出一些流动性，这可能导致蛋白质不稳定（见第4.2.2节）。Vitrification hypothesis (or
glassy matrix theory): Ice crystallization and
freeze-concentration eventually drives the system to approach a glassy state
having restricted mobility. The presence of cryoprotectants such as sugars and
polymers result in an increase in viscosity of the freeze-concentrate, which
reduces molecular mobility, thereby slowing down all dynamic processes. The
protein becomes virtually immobilized. Since mobility is a pre-requisite for
denaturation and degradation reactions, the restricted mobility in the glassy
state makes the protein relatively stable in the timescales of practical
interest in freeze-drying [14,17]. However, the long-term storage
can show some mobility based on the storage temperature and consequent
excipient crystallization leading to protein instability (Section 4.2.2).水分替代（替代）假说（或优先溶质相互作用）：由于冷冻浓缩，可能没有足够的水分子与极性蛋白质表面形成充分的氢键。这允许溶质与蛋白质发生选择性相互作用，提出了另一种可能的机制，即水分替代（替代）假说或“优先溶质相互作用”[18]。使用原位拉曼技术对细胞冷冻保存的研究显示，蔗糖的冷冻保护作用可能归因于其与细胞膜的直接相互作用[19]。这包括了糖的羟基与蛋白质形成氢键，替代了水与蛋白质之间的氢键。这种氢键的替代有助于维持蛋白质的天然构象。Water replacement (substitute)
hypothesis (or preferential solute interaction): As a result of
freeze-concentration, sufficient water molecules may not be available to
adequately hydrogen bond with the polar protein surfaces. This allows selective
interaction of solutes with the protein, suggesting another plausible mechanism
known as the water replacement (substitute) hypothesis or ‘preferential solute
interaction’ [18]. An investigation on cryopreservation of cells
using in situ Raman suggested that the cryoprotective action of sucrose could
be attributed to its direct interaction with the cell membrane [19].
It encompasses the concept that the hydroxyl groups (of the sugar) hydrogen
bonds with the protein, thereby replacing hydrogen bonds between water and the
protein. This replacement of hydrogen bonds enables the protein’s native
conformation to be maintained.在冷冻过程中，蛋白质的稳定可能通过这些机制中的任何一个来实现（见图1）。特别是，优先排斥预计在冷冻过程的早期阶段占主导地位，而随着溶液的冷冻浓缩，玻璃化和水分替代假说可能变得更加重要[15]。为了使稳定剂有效，它必须是无定形的，并且是冷冻浓缩液的一部分。The protein stabilization during the freezing process may occur through any of
these mechanisms (Fig. 1). In particular,
preferential exclusion is expected to be predominant in the initial stages of
the freezing process, while vitrification and water replacement hypotheses may
take over as the solution freeze-concentrates [15]. For the
stabilizer to be effective, it must be amorphous and a part of the
freeze-concentrate.2.2 干燥和存储Drying and storage
在冷冻干燥的干燥阶段，冰首先通过升华（一次干燥）被去除，随后通过解吸（二次干燥）去除未冻结的“结合”水。鉴于水与蛋白质之间的氢键对蛋白质的热力学稳定性极为关键，水分的移除在干燥过程中构成了主要压力，有时会导致一些敏感蛋白质发生不可逆的生物活性损失[20]。许多有效的冷冻保护剂在干燥过程中无法维持其稳定作用。能够在冷冻干燥和储存过程中保护蛋白质的稳定剂通常被称为冻干保护剂[21]。此外，蛋白质在干燥和储存期间的物理状态具有相似性，主要区别在于无定形相中水分含量的不同。从这个角度来看，可以假设，干燥过程中起稳定作用的基本机制同样适用于干燥产品在储存期间的稳定[21]。储存期间的额外压力因素包括意外的温变和辅料在产品有效期内的相分离。此外，在储存期间，还观察到蛋白质通过化学过程（例如去酰胺化和氧化）发生降解[22]。
During the drying stage of lyophilization, ice is removed by sublimation
(primary drying) and unfrozen ‘bound’ water by desorption (secondary drying).
Since hydrogen bonding between water and protein is critical to the
thermodynamic stability of protein, removal of water constitutes the major
stress during drying and can cause irreversible loss of biological activity for
some labile proteins [20]. Many effective cryoprotectants fail to
retain their stabilizing effect during drying. Stabilizers that can protect the
protein during freeze-drying as well as storage are often referred as
lyoprotectants [21]. Furthermore, the physical state of the protein
during both drying and storage is similar, with the only difference being the
water content of the amorphous phase in these conditions. From this
perspective, it is postulated that the fundamental mechanisms governing
stability during drying also govern stability during storage of the dried
product [21]. The additional stress factors during storage include
unintended temperature excursions and excipient phase separation during the
product shelf-life. In addition, the protein degradation via chemical processes
such as deamidation and oxidation are observed during storage [22].
在干燥和储存期间，辅料诱导的蛋白质稳定机制与冷冻储存的机制有相似之处。在干燥和储存期间广泛接受的两种稳定机制是“玻璃化”和“水分替代”，这两种机制通过减少分子运动和防止蛋白质结构变化来保护蛋白质结构。尽管这两种假设背后的机制不同，但都需要蛋白质和稳定剂处于同一无定形相中[23,24]。The mechanisms of excipient-induced protein stabilization during drying and
storage have some parallel with that of the frozen storage. The two
widely-accepted mechanisms of stabilization during drying and storage are
‘vitrification’ and ‘water replacement’ which result in the preservation of the
protein structure, by reducing molecular mobility and preventing changes in
protein structure respectively. Although the underlying mechanisms differ, both
hypotheses require the protein and the stabilizer to be in the same amorphous
phase [23,24].玻璃化理论基于将蛋白质固定在坚硬的无定形糖基质中的概念。人们认为，限制蛋白质的平移和松弛过程可以防止其展开[20]。无定形糖的动态特性通常由全局流动性特征，即α-松弛来表征，这是最慢的动态过程，并且具有强烈的温度依赖性。在玻璃化转变温度(Tg)附近及以上，α-松弛动态随温度的快速变化，糖的动力学固定作用和相关的稳定能力在很大程度上会丧失[21]。而“水分替代”理论则假设，优秀的稳定剂能够像水一样与蛋白质相互作用，促进其天然构象，因此在干燥过程中通过替代被移除的水分来稳定蛋白质。傅里叶变换红外光谱(FTIR)对冻干蛋白质-糖基质的测量结果证实，糖通过与干燥蛋白质形成氢键，替代了失去的水，防止了脱水引起的蛋白质展开。理论上，围绕蛋白质分子的单糖层足以通过替代所有氢键位点的水来维持蛋白质的完全活性[21,22]。As mentioned above, the vitrification theory is based on the concept of
immobilizing the protein in a rigid, amorphous glassy sugar matrix. The
restriction of translational and relaxation processes is thought to inhibit
protein unfolding [20]. The dynamics of amorphous sugars are usually
characterized by global mobility, measured as α-relaxation.
This is the slowest dynamic process and exhibits a strong temperature
dependence. Near and above the glass transition temperature (Tg), where there
is a rapid change in α-relaxation dynamics with temperature, the kinetic
immobilization and the associated stabilizing power of the sugar are largely
lost [21]. On the other hand, the ‘water replacement’ theory
hypothesizes that good stabilizers interact with the protein as does water,
promote the native conformation, and therefore stabilize the protein during
drying by replacing the water that is removed. FTIR (Fourier-transform infrared
spectroscopy) measurements of lyophilized protein–sugar matrix established that
sugars prevent dehydration-induced unfolding by hydrogen bonding to the dried
protein in place of the lost water. Theoretically, a sugar monolayer around the
protein molecule should be adequate to retain complete protein activity by
replacing water at all hydrogen bonding sites [21,22].然而，在实践中这一理想状态尚未实现。已有充分证据表明，处方的Tg值经常无法准确预测蛋白质的稳定性。所有与稳定性相关的运动都与Tg强烈耦合的基本假设并非普遍有效。尽管有大量证据显示，玻璃态中接近天然构象与抗降解稳定性之间存在相关性，但这种关系尚未量化，并且并不适用于所有蛋白质-糖组合[21,25]。在过去十年中，我们对无定形糖基质中固态蛋白质稳定性的理解有了显著提高。However, this has not been realized in practice. It has been well documented
that formulation Tg often fails to predict protein stability. The basic
assumption that all the motion relevant to stability is strongly coupled with
the Tg, is not universally valid. Though there is much evidence for a
correlation between near-native conformation in the glass and stability against
degradation, this relation has not been quantifiable, and does not extend over
the full range of protein–sugar compositions [21,25]. The past
decade has witnessed a significant improvement in our understanding of
solid-state protein stability specifically in amorphous sugar matrices.局部分子运动的相关性：近期的研究表明，封存在糖玻璃中的蛋白质的稳定性直接与相对高频的β-松弛过程或局部运动相关，而非糖基质的全局运动（α-松弛）。这被推测是β-松弛与局部蛋白质运动和玻璃中小分子活性物质的扩散耦合的结果。通过使用抗增塑剂进一步确认了这一假设，抗增塑剂是加快玻璃中α-松弛而减慢β-松弛（局部运动）的添加剂。向蔗糖-抗体处方中添加少量抗增塑剂（山梨糖醇或水）导致Tg和全局运动（α-松弛）单调下降，但在添加山梨糖醇或水的中间水平时获得最佳稳定化效果[26]。有趣的是，局部运动（β-松弛）随抗增塑剂浓度的变化与稳定性的变化趋势相同，表明更快的分子动态与蛋白质降解速率的耦合。在蔗糖或海藻糖玻璃中冻干的超过100种蛋白质的聚集或化学降解速率常数与β-松弛时间成反比[25,27,28]。图2A展示了两种蛋白质处方[重组人生长激素（rhGH）或角化细胞生长因子-2（KGF-2）]在糖基质中的玻璃化转变温度。右侧y轴显示的是冻干后红外(FTIR) α-螺旋峰宽度在半高处的变化量，Δw1/2。随着蔗糖（或海藻糖）质量分数的增加，处方与天然蛋白质结构的相似性增加（Δw1/2较低），同时处方的玻璃化转变温度(Tg)降低。另一方面，冻干rhGH的聚集常数与氢原子的均方位移（<u2>），即局部“β-快速”动态的度量（由中子反向散射确定）相关。这在40℃和50℃储存后都很明显。图2B绘制了均方位移的倒数，<u2>-1。<u2>-1越小，表明局部运动性越高[29-31]。尽管大多数关于β-松弛测量的工作都是使用中子反向散射技术进行的，但这种技术不适用于常规表征[28]。Relevance of local molecular
mobility: Recent studies showed that stability of proteins
sequestered in sugar glasses is directly correlated with the relatively high
frequency β-relaxation processes or the local
mobility, rather than global mobility (α-relaxation) of the sugar matrix. This
is presumed to be derived from coupling of b-relaxations to local protein
motions and to diffusion of small molecule reactive species in the glass. The
hypothesis was further confirmed using anti-plasticizers, additives which speed
up a-relaxation in the glass while slowing down β-relaxation(local mobility). Adding small amounts of antiplasticizer (sorbitol or water)
to sucrose-antibody formulations resulted in a monotonic decrease in Tg and
global mobility (α-relaxation), but optimal stabilization was obtained at an intermediate
level of added sorbitol or water [26]. Interestingly, local mobility
(β-relaxation) changed with antiplasticizer concentration in
the same monotonic way as did stability, suggesting coupling of the faster
molecular dynamics to protein degradation rates. The aggregation or chemical
degradation rate constant for >100 proteins freezedried in sucrose or
trehalose glasses, was inversely proportional to β-relaxation
time [25,27,28]. In Fig. 2A, the glass transition temperature of two protein
formulations [recombinant human growth hormone (rhGH) or keratinocyte growth
factor-2 (KGF-2)] in sugar matrices is presented. The right y-axis is the
change upon lyophilization of the IR a-helical peak width at half height, Δw1/2.
As the sucrose (or trehalose) mass fraction increased, the formulations showed
more structural similarity to native protein (lower Δw1/2). This was
accompanied by a decrease in glass transition temperature (Tg) of the
formulation. On the other hand, the aggregation constant for lyophilized rhGH
correlated with the mean square displacement (<u2>) of
hydrogen atoms, a measure of local ‘‘β-fast” dynamics (as determined by neutron
backscattering). This was evident following storage both at 40 and 50℃. In Fig.
2B, reciprocal of mean square displacement, <u2>-1,
is plotted. Smaller the <u2>-1, higher the local
mobility. [29–31]. While most of the work on b-relaxation
measurements was performed with neutron backscattering, this technique cannot
be used for routine characterization [28].锚定假设：蛋白质的动态与糖基质的动态通过一个水桥耦合，该水桥与两者都形成氢键[32,33]。一些模拟和实验研究表明，界面水可以在将玻璃态宿主（糖）与蛋白质的动态耦合中发挥重要作用。尽管水与蛋白质和糖都形成氢键，但界面水更有效地锚定到海藻糖玻璃而不是蔗糖玻璃，这可能归因于海藻糖形成分子间而非分子内氢键的更高倾向。Anchorage hypothesis. The dynamics of the protein are coupled to dynamics of
the sugar matrix via a water bridge that is hydrogen bonded to both [32,33].
A number of simulation and experimental studies suggest that interfacial water
can play an important role in coupling the dynamics of the glassy host (sugar)
with the protein. While the water hydrogen bonds to both protein and sugar,
interfacial water anchors more effectively to trehalose glasses than to sucrose
glasses. This may be attributed to the higher propensity of trehalose to form
intermolecular rather than intramolecular hydrogen bonds.堆积密度：作为水分替代理论的进一步改进，更小且分子上更灵活的糖在冻干后的储存期间更能稳定蛋白质，比更大且分子上更刚性的糖更好。更小的糖分子在与蛋白质相互作用时受到的立体阻碍较小，能够形成更多氢键。更强的相互作用和更紧密的堆积导致这些处方的密度增加，从而降低了自由体积[27,34]。Packing density. As a further refinement of water replacement theory, smaller
and molecularly more flexible saccharides are better able to stabilize the
protein during storage after lyophilization, than larger and molecularly more
rigid counterparts. The smaller sugar molecules are less sterically hindered in
interacting with the protein enabling the formation of hydrogen bonds. The
stronger interactions and a tighter packing leads to an increased density and
thus a decreased free volume of these formulations [27,34].此外，普遍认为，为了有效的稳定化，蛋白质和糖必须处于同一相。糖可以通过形成不同的无定形区域或结晶来相分离。这个过程导致必要的相互作用丧失，同时可能对蛋白质产生剪切应力[35]。In addition, it is generally accepted that for effective stabilization, the
protein and sugar must be in the same phase. The sugar can phase separate
either by forming a distinct amorphous region or by crystallization. This
process causes a loss of necessary interactions, coupled with induction of shear
stresses on the protein [35].这些机制并不是相互排斥的，通常很难将稳定化归因于某一特定机制。然而，了解这些机制使处方研究员可以尝试不同的方法来设计稳定的冻干处方。These mechanisms are not mutually exclusive and it is often difficult to
attribute stabilization to one specific mechanism. However, an understanding of
these mechanisms enables the formulator to try different approaches for
designing stable lyophilized formulations.针对蛋白质在冷冻干燥及储存过程中所遭遇的各种压力，必须在制剂处方中添加适宜的辅料，以预防蛋白质的聚集与降解。蛋白质制剂是一个包含多种组分的系统，通常由冷冻或冻干保护剂、缓冲剂、填充剂和表面活性剂构成。这些辅料以特定的蛋白质与溶质的比例混合使用，已被证实是稳定蛋白质的有效策略In light of the various stresses experienced by protein during freeze-drying
process and storage, suitable excipients need to be added to the formulation to
prevent protein aggregation and degradation. A protein formulation is a multicomponent
system usually consisting of a cryo- or lyo-protectant, buffer, bulking agent
and surfactant. A combination of these excipients in certain protein: solute
ratios has been shown to be a useful strategy to stabilize the protein.
3.1 糖和多元醇Sugars and sugar alcohols
许多糖类和多元醇在溶液中作为蛋白质的稳定剂，在冷冻、冻融、冷冻干燥或产品储存期间发挥作用。它们所提供的稳定化水平依赖于其浓度。通常，至少需要达到糖与蛋白质1:1的重量比以获得良好的稳定性，而最佳稳定性比例约为3到5:1[20]。进一步提高糖的浓度可能会达到稳定化极限，甚至在冷冻干燥和储存过程中导致蛋白质不稳定。使用最小有效浓度的糖，是因为蛋白质与糖的比例会影响玻璃化转变温度[17,27]。Many sugars or polyols are used as protein stabilizers in solution, in the
frozen state, during freeze-thawing, freeze-drying or product storage. The
level of stabilization afforded depends on their concentration. Generally, a
weight ratio of sugar to protein of at least 1:1 is required for good stability,
with optimal stability around 3 to 5:1 [20]. Further increase in
sugar concentration may reach the limit of stabilization or even destabilize a
protein during freeze-drying and storage. The minimal effective concentration
of sugar is employed, because of the effect of protein: sugar ratio on the
glass transition temperatures [17,27].
![]()
Fig. 2. (A) Influence of sugar content on the glass
transition temperature (vertical bars; left y-axis) and native secondary
structure retention (change of IR a-helical peak width at half height (Dw1/2,
cm-1) as lines; right y-axis) in the lyophilized protein
formulations. Protein was either recombinant human growth hormone (rhGH) (solid
lines/filled markers) or keratinocyte growth factor-2 (KGF-2) (dashed
lines/hollow markers). The smaller the change in the width of IR peak, the
higher the retention of native protein secondary structure after freeze-drying.
(B) Influence of sugar content on local mobility (vertical bars; left y-axis)
and the aggregation rate constant (lines; right yaxis) for rhGH formulations
stored either at 40℃ (solid bars/solid lines/filled marker) or at 50℃ (dashed
bars/dashed line/hollow markers). The <u2> is a measure of the mean
square displacement of hydrogen atoms. Smaller the <u2>-1,
higher the local mobility. Each formulation contained 1 mg/mL protein and 5%
w/v excipients, which was hydroxyethyl starch with either trehalose (blue bars
and lines) or sucrose (red bars and lines). Data adapted from Xu et al [29],
Devineni et al [30] and Chieng et al [31]. (For interpretation of the
references to color in this figure legend, the reader is referred to the web
version of this article.)图2展示了冻干蛋白质处方中糖含量对玻璃化转变温度（以垂直条形表示；左侧y轴）及蛋白质二级结构保持情况（以红外光谱中α-螺旋峰半高宽变化表示；右侧y轴）的影响。测试的蛋白质包括重组人生长激素（rhGH，以实线和实心标记表示）和角质细胞生长因子-2（KGF-2，以虚线和空心标记表示）。红外峰半高宽的变化越小，意味着冻干后蛋白质的二级结构保持得越好。图B描述了糖含量对rhGH处方在40℃（以实心条和实线表示）或50℃（以空心条和虚线表示）储存时的局部流动性（左侧y轴的垂直条形）和聚集速率常数（右侧y轴的线条）的影响。<u2>值代表氢原子的平均平方位移，该值越小，表示局部流动性越高。每个处方均含有1 mg/mL的蛋白质和5% w/v的辅料，辅料为羟乙基淀粉，可以是海藻糖（以蓝色表示）或蔗糖（以红色表示）。数据来源包括Xu等人[29]，Devineni等人[30]和Chieng等人[31]的研究。（关于图中颜色标识的详细解释，请参见文章的在线版本。）蔗糖和海藻糖是最受欢迎的稳定剂。它们的理化特性及其可能与稳定化活性的相关性在表1中展示。Sucrose and trehalose are the most popular stabilizers. Their physico-chemical
properties, likely of relevance in their stabilizing activity, are presented inTable 1.对比表1可以明显看出，蔗糖和海藻糖具有多项独特的物理化学特性，为每种糖作为稳定剂提供了明显的优势（见表2）。然而，使用这些糖也可能存在严重的局限性。对于生物制药应用特别关注的是海藻糖在冷冻期间的结晶倾向。在冷冻溶液退火时，海藻糖可能以二水合物形式结晶。在约-20℃下长期储存的冷冻蛋白质溶液中的聚集现象，已被归因于海藻糖的结晶[37]。尽管海藻糖在冷冻状态下以二水合物形式结晶，但在干燥过程中，二水合物会转化为无定形的无水物。重要的是，二水合物的结晶、脱水和无定形化可能在冷冻干燥过程中发生，因此在对最终产品进行表征时可能不明显。As is evident from the comparison in Table 1, sucrose and
trehalose are characterized by several unique physical and chemical properties,
conferring each with distinct advantages as stabilizers (Table 2).
Likewise, there can also be serious limitations with the use of these sugars.
Of particular interest for biopharmaceutical applications is the propensity of
trehalose to crystallize during freezing. Upon annealing frozen solutions,
trehalose can crystallize as a dihydrate. The aggregation in frozen protein
solutions, following long term storage at ~-20℃, has been attributed to
trehalose crystallization [37]. While trehalose crystallizes as a
dihydrate in the frozen state, upon drying, the dihydrate converts into the
amorphous anhydrate. Importantly, the dihydrate crystallization, dehydration
and amorphization can occur in the course of the freeze-drying process and thus
may not be obvious when characterizing the final product.比较蔗糖和海藻糖在治疗性蛋白质制剂中的有效性是有意义的。如图2B（第2.2节）所示，在40℃和50℃储存条件下，与蔗糖或海藻糖共冻干的KGF-2的聚集速率常数相似（分别比较红线和蓝线）[29]。然而，在某些情况下，蔗糖可能比海藻糖提供更好的保护，如在冻干溶菌酶中观察到的[38]。两种糖的保护作用差异归因于海藻糖相比蔗糖更大的相分离倾向[39,40]。相反，分子模拟研究显示，海藻糖在防止固体状态下的hGH展开方面应该比蔗糖更有效。海藻糖分子通过在它们自身之间以及与蛋白质之间形成密集、紧凑的氢键网络来保护蛋白质[41,42]。实际上，与含有海藻糖（2:1辅料/蛋白质重量比）的冻干制剂相比，含有蔗糖的制剂中β-半乳糖苷酶的酶活性下降得更慢[43]。总结来说，每种糖为特定蛋白质提供冷冻干燥和储存诱导的稳定化能力应在个案基础上确定[20]。It is worthwhile to compare the effectiveness of sucrose and trehalose in a
therapeutic protein formulation. Fig. 2B (Section 2.2)
shows that the aggregation rate constant of KGF-2 upon storage at 40 and 50℃
was similar for the protein co-lyophilized with either sucrose or trehalose
(compare redand blue lines respectively) [29].
In some cases, however, sucrose might provide better protection than trehalose
as observed in lyophilized lysozyme [38]. The difference in the
protective action between the two sugars was attributed to the greater tendency
of trehalose to phase separate when compared to sucrose [39,40]. In
contrast, molecular simulation studies suggest that trehalose should be more
efficient than sucrose in preventing hGH unfolding in the solid state.
Trehalose molecules protect proteins by forming a dense, compact hydrogen
bonding network between themselves, as well as with the protein [41,42].
In fact, the enzymatic activity of ß-galactosidase decreased more slowly in
lyophile containing trehalose (2:1 excipient/protein weight ratio) when
compared to those containing sucrose [43]. To summarize, the ability
of each of these sugars to provide lyophilization and storage-induced
stabilization for a given protein should be determined on a case-by-case basis [20].通常，二糖（如蔗糖、海藻糖、乳糖、麦芽糖；其Tg分别约为60、110、114、100℃）被报道比单糖（如葡萄糖、半乳糖、果糖、甘露糖；其Tg分别约为32、38、13、33℃）更有效的冻干保护剂[44]。这可能是因为它们具有更高的玻璃化转变温度，尽管增加的空间位阻可能干扰与干燥蛋白质的密切氢键形成。因此，辅料的选择需要在形成高Tg玻璃体与氢键强度之间取得平衡[44]。同样，棉子糖，一种三糖，尽管其Tg更高（109℃），但在储存期间作为酶稳定剂的效果不如蔗糖[45]。在-10℃退火冷冻溶液时，棉子糖可以作为五水合物结晶，这在干燥过程中脱水产生无定形的冻干制剂。棉子糖在冷冻干燥过程中的结晶伴随着蛋白质活性的显著丧失[46]。In general, disaccharides (such as sucrose, trehalose, lactose, maltose; ~Tg
60, 110, 114, 100℃ respectively) are reported to be more effective
lyoprotectants than monosaccharides (glucose, galactose, fructose, mannose; ~
Tg 32, 38, 13, 33℃ respectively) [44]. This may be due to their
higher Tg, though the increased steric hindrance may interfere with the
intimate hydrogen bonding with a dried protein. Therefore, the excipient
selection needs balancing the formation of a high Tg glass with the strength of
hydrogen bonding [44]. Similarly, raffinose, a tri-saccharide,was a less effective enzyme stabilizer than sucrose during storage, in spite of its higher Tg (109℃) [45].
Upon annealing the frozen solution at -10℃, raffinose can crystallize as
pentahydrate, which dehydrates during drying to yield an amorphous lyophile.
Raffinose crystallization during freeze-drying is accompanied by a significant
loss of protein activity [46].![]()
Table 1蔗糖和海藻糖的物理化学性质比较。表中包括了作为冻干处方稳定剂的相关性质。根据参考文献[36]中的讨论编制。赋予相对优势的性质用粗体表示，而可能限制使用（即潜在不利）的性质用斜体表示。![]()
Table 2在选择蔗糖或海藻糖作为冻干和冷冻蛋白制剂中的稳定剂时，以下是一些选择标准：多元醇如山梨糖醇、木糖醇和乳糖醇也通过优先排斥在水溶液中保护蛋白质免受热诱导变性。在冷冻状态下，山梨糖醇传统上被认为是一种无定形、不结晶的溶质，适合于冻干。然而，其低Tg'（约-44℃）和相应的低塌陷温度使其成为冻干过程的较差选择[47]。山梨糖醇（或甘油）与蔗糖一起展示了协同作用对蛋白质稳定化[26,48]。Sugar alcohols such as sorbitol, xylitol and lactitol also protect proteins
from heat-induced denaturation in aqueous solutions through preferred
exclusion. In the frozen state, sorbitol is traditionally known to be an
amorphous, non-crystallizing solute, making it amenable to lyophilization.
However, its low Tg’ (~-44℃) and consequently low collapse temperature, make it
a poor choice for efficient freeze-drying process [47]. Sorbitol (or glycerol)
along with sucrose demonstrated synergistic effect on protein stabilization[26,48].甘露醇，另一种多元醇，是冻干制剂中最受欢迎的辅料之一。当保持无定形时，甘露醇可以作为冷冻保护剂[49,50]。甘露醇传统上用作填充剂，因为它在冷冻溶液中结晶的高倾向。甘露醇通常以无水的α、β或δ形式结晶，尽管在最终冻干制剂中的物理形式受到其他处方成分的影响[51,52]。根据处方和加工参数，甘露醇也可以以亚稳态的甘露醇半水合物（MHH）结晶。在冷冻期间形成甘露醇半水合物（MHH）并在最终冻干制剂中保留需要仔细考虑，因为（i）在冷冻期间其通常不一致的形成，（ii）需要更积极的干燥才能去除它，以及（iii）如果在最终冻干制剂中保留，由于脱水在储存期间释放的水，可能对产品的物理和化学稳定性产生显著影响[53-56]。Mannitol, another sugar alcohol, is one of the most popular excipients in
lyophilized formulations. When retained amorphous, mannitol can serve as a
cryoprotectant [49,50]. Mannitol is traditionally used as a bulking
agent due to its high propensity to crystallize in frozen solutions. Mannitol
generally crystallizes as the anhydrous a-, b- or d- form, though the physical
form in the final lyophile is influenced by the other formulation components [51,52].
Depending upon the formulation and processing parameters, mannitol can also
crystallize as the metastable mannitol
hemihydrate (MHH). The formation of MHH during freezing and its retention
in the final lyophile warrants careful consideration due to (i) it’s usually
inconsistent formation during freezing, (ii) requirement of a more aggressive
drying for its removal, and (iii) if retained in the final lyophile, water
released due to dehydration during storage, can have a significant impact on
the physical and chemical stability of the product [53–56].
3.2 氨基酸Amino
acids
氨基酸（AAs）在液体制剂中稳定蛋白质天然结构方面有着长期的应用历史[57]。尽管如此，仅有少数研究探讨了氨基酸在冻干蛋白质制剂中的稳定作用[58]。表3汇总了一些代表性的实例。氨基酸通常与蔗糖或海藻糖联合使用，在冻干制剂中很少单独使用，这可能是因为它们的稳定效果不及传统的糖类冷冻保护剂[64]。然而，在几乎所有这些研究中，添加AAs都能增强糖的稳定效果，使其成为处方中的合适辅助稳定剂。Amino acids (AAs) have a long history of stabilizing native protein structure
in liquid formulations. [57]. However, only a few studies have
investigated the stabilizing role of AAs in freezedried protein formulations[58]. Some representative examples are compiled in Table 3.AAs are typically used with sucrose or trehalose and have been rarely used
alone in lyophilized formulations. This is possibly because the stabilizing
effect of AAs are lower than that of the conventional sugar cryoprotectants [64].
However, in almost all these studies, the addition of AAs enhanced the
stabilizing effect of sugars, thus making them a suitable secondary stabilizer
in the formulation.![]()
精氨酸（通常以盐酸盐形式使用）是冻干制剂中研究最为广泛的氨基酸[71,72]。它不仅对提高蛋白质的溶解性有积极作用，还能降低溶液的粘度，这是其额外的优点。但是，添加精氨酸会降低塌陷温度，因此需要谨慎选择干燥周期[72]。为解决这一问题，已尝试了多种策略：i) 使用柠檬酸、磷酸、琥珀酸和乳酸等替代反离子，虽然提高了Tg'，但在蛋白质稳定方面不如盐酸盐[59,60,69,71,73]。ii) 提高蛋白质浓度以增加Tg'，防止了冻干蛋糕的塌陷[59,73]。iii) 添加蔗糖虽能改善冻干蛋糕的外观，但未能阻止塌陷[59,73]。iv) 甘露醇或苯丙氨酸作为结晶性填充剂使用，有助于形成外观更佳的冻干蛋糕；从蛋白质稳定性角度来看，苯丙氨酸的表现优于蔗糖或甘露醇[70]。在更复杂的处方体系中（海藻糖 + 精氨酸HCl + 甘露醇），精氨酸能显著降低蛋白质聚集的程度，相比单独使用海藻糖或海藻糖与甘露醇的组合，降低幅度达到2-2.5倍[63]。Arginine (generally used as the hydrochloride salt) has been the most studied
AA in lyophilized formulations [71,72]. Its positive effect on the
solubility of proteins, and tendency to lower the viscosity of the bulk and
reconstituted solutions are additional advantages. However, the addition of
arginine lowers the collapse temperature, thereby necessitating conservative
drying cycles [72]. Several strategies have been attempted to
resolve this issue. i) Use of alternative counterions such as citric,
phosphoric, succinic and lactobionic acids, increased the Tg’ but were found
inferior to the HCl salt in terms of protein stabilization [59,60,69,71,73].
ii) High protein concentration prevented cake collapse by increasing the Tg’ [59,73].
iii) The addition of sucrose led to improved cake appearance but failed to
prevent collapse [59,73]. iv) Mannitol or phenylalanine was used as
a crystallizing bulking agent to yield an elegant cake. From the perspective of
protein stability, phenylalanine was superior to sucrose or mannitol [70].
In a more complex matrix (trehalose + arginine HCl + mannitol), arginine
lowered the degree of protein aggregation by 2–2.5 times (than trehalose alone
or trehalose + mannitol) [63].此外，已有报告称组氨酸[65,67]、丙氨酸、赖氨酸、脯氨酸[68]、丝氨酸[64]和苯丙氨酸[64]也能增强冻干产品中蛋白质的稳定性。在一项有趣的研究中，发现使用酸性氨基酸作为精氨酸和组氨酸的反离子，可以增强它们的稳定效果[67]。尽管组氨酸作为缓冲剂已经很受认可，但其作为稳定剂的潜力可能还需要更系统的探究。In addition, histidine [65,67], alanine, lysine, proline [68],
serine [64] and phenylalanine [64] have also been
reported to enhance protein stability in lyophilized products. In an
interesting study, the use of acidic AAs as counterions for arginine and
histidine were found to enhance their stabilizing effect [67]. While
histidine has already gained popularity as a buffer, its utility as a
stabilizer may warrant more systematic investigations.目前，氨基酸在固态中稳定蛋白质的机制尚未完全明了。可能的机制包括但不限于与蛋白质分子的优先相互作用[72,74]、改变自由体积[57]和分子运动性[59,75]。尽管如此，氨基酸作为冻干制剂中蛋白质稳定剂的应用，能够扩展辅料的选择范围。The mechanisms by which AAs stabilize proteins in the solidstate are not well
understood. These may include, but are not limited to, preferential interaction
with protein molecules [72,74], alteration in free volume [57]and molecular mobility [59,75]. Nevertheless, the utility of AAs as
stabilizers of proteins in lyophilized formulations can expand the excipient
options.
3.3 表面活性剂Surfactants
蛋白质通常具有表面活性，易于因表面诱导的变性作用而发生聚集[76]。这种吸附或结合可能在多种界面发生，例如液体处方组分混合时形成的空气-液体界面，以及在冷冻、融化或干燥过程中形成的冰-液体或冰-空气界面。在这种情况下，向蛋白质制剂中添加表面活性剂可能通过两种可能的机制来稳定蛋白质：(1) 表面活性剂分子优先吸附在界面上。较小的表面活性剂分子比蛋白质分子更易与疏水表面结合。这些吸附的表面活性剂分子在界面上形成一层覆盖膜，防止蛋白质吸附。(2) 表面活性剂与蛋白质分子相互作用，形成表面活性剂-蛋白质复合物，阻止蛋白质进一步的相互作用[76]。Proteins are usually surface active and vulnerable to aggregation due to
surface-induced denaturation [76]. Adsorption or binding can occur at
various interfaces, for example, at the air–liquid interface due to mixing of
liquid formulation components and at ice-liquid or ice-air interfaces during
freezing and thawing or drying. In this context, addition of surfactants to a
protein formulation may stabilize proteins via two possible mechanisms: (1)
Preferential binding of surfactant molecules at interfaces. Thus, smaller
surfactant molecules outcompete larger protein molecules and bind to
hydrophobic surfaces. The adsorbed surfactant molecules form a coating at the
interface and prevent protein adsorption. (2) Surfactant interacts with protein
molecules to form a surfactant–protein complex preventing the protein from
further interactions [76].非离子表面活性剂的使用一直是防止或抑制液体和冻干制剂中由表面引起的聚集的有效策略。聚山梨酯（Polysorbates，如20和80）和Poloxamer
P188是蛋白质制剂中广泛使用的表面活性剂[23,24]。尽管聚山梨酯在蛋白质制剂中具有多方面的优势和广泛的应用，但它们已知会发生自氧化[77]。自氧化产生的过氧化物已被证明会导致蛋白质的氧化[78]。此外，根据溶液环境，聚山梨酯也可能发生长链脂肪酸的水解。使用精炼的聚山梨酯，并将溶液储存在低温、氮气环境下并避免光照，可以至少部分防止其降解[77]。The use of non-ionic surfactants has been a successful strategy to prevent or
inhibit surface-induced aggregation in both liquid and freeze-dried
formulations. Polysorbates (20 and 80)and poloxamer P188 are surfactants widely used in protein formulations[23,24]. In spite of the advantages and wide use of polysorbates in
protein formulations,they are known to undergo auto-oxidation [77].
The peroxides formed as a result have been shown to cause oxidation of proteins[78]. In addition, based on solution environment, polysorbates are also
known to undergo hydrolysis of the long chain fattyacids. Use of refined
polysorbates and storing solutions at low temperatures in a nitrogen atmosphere
and protected from light, can at least partially prevent degradation [77].聚山梨酯20和80在水中的临界胶束浓度（CMC）在25℃
± 0.5℃时分别约为0.007%和0.0017% w/v[79]。当表面活性剂溶液冷却时，冰晶的形成会导致表面活性剂显著浓缩，并在冷冻浓缩液中形成胶束。随着冷冻浓缩，CMC的增加尚未经过深入研究。在这种情况下，基于蛋白质制剂中初始表面活性剂浓度的表面活性剂-蛋白质相互作用可以作为一个很好的参考（见图3）。通常，聚山梨酯的使用浓度不超过0.1% (w/v)。在冷冻浓缩过程中，表面活性剂的浓度会指数级增加。因此，图3中的区域4和5可能反映了表面活性剂防止蛋白质变性的机制。为了最大化稳定性，建议从最低的聚山梨酯浓度开始。其他表面活性剂，如Triton-XTM和Brij®（聚氧乙烯月桂醚35和78）也作为替代的非离子表面活性剂进行了探索[80]。The critical micelle concentration (CMC) of polysorbate 20 and 80 in water (25
C ± 0.5℃) is ~0.007% and ~0.0017% w/v respectively [79]. When a
surfactant solution is cooled, the ice crystallization will cause pronounced
concentration and result in micelleformation in the freeze concentrate. The
increase in CMC as a function of freeze-concentration has not been thoroughly
investigated. In this context, the surfactant–protein interaction based on the
initial surfactant concentration in protein formulation can be a good reference(Fig. 3). Generally, polysorbates are used at
concentrations <0.1% (w/v). On freeze-concentration, the surfactant
concentration increases exponentially. Thus, regions 4 and 5 in Fig. 3 would possibly
reflect the mechanism by which surfactants prevent protein denaturation. In
order to maximize stability, a good idea is to start with minimum polysorbate
concentrations. Other surfactants such as Triton-XTM and Brij® (polyoxyethylene
lauryl ether 35 and 78) have also been explored as alternative non-ionic
surfactants [80].
3.4 缓冲液Buffers
溶液的pH值是决定蛋白质稳定性的关键因素之一。因此，冷冻干燥前的蛋白质处方普遍含有缓冲液，用以控制(i) 冷冻干燥前溶液、(ii) 最大冷冻浓缩体系，以及(iii) 重新配制后的溶液的pH值。同样，长期储存在冷冻状态下的蛋白质溶液也常采用缓冲体系，以维持“冷冻状态”及随后融化溶液的pH值。蛋白质处方中常用的缓冲体系包括组氨酸、磷酸钠、磷酸钾、柠檬酸盐、三羟甲基氨基甲烷（Tris）和琥珀酸盐缓冲液[6]。Solution pH can be one of the determinants of protein stability. As a result,
prelyophilization protein formulation compositions invariably contain a buffer,
to control the pH of (i) the pre-lyo solution, (ii) the maximally
freeze-concentrated system, and (iii) the reconstituted solution. Similarly,
protein solutions which are stored in the frozen state for prolonged time
periods are often buffered, both for maintaining pH in the ‘‘frozen state” and
in the subsequently thawed solution. The most commonly used buffer systems in
protein formulations include - histidine, sodium phosphate, potassium
phosphate, citrate, tris and succinate buffers [6].制药用蛋白质处方中缓冲液的选择通常基于所需的pH范围、缓冲容量以及缓冲液特异性催化的可能性[81]。理想情况下，缓冲液在pH值偏离酸解离常数（pKa）±1个单位时使用，且在pH = pKa时具有最大的缓冲容量。了解pKa有助于选择合适的缓冲液。例如，当所需pH值约为7.0时，广泛采用的是磷酸盐缓冲液（pKa约为7.2），而在pH值约为6.0时，组氨酸（pKa约为6.1）是首选缓冲液[6]。对于具有多个pKa值的缓冲液，如磷酸钠缓冲液（pKa值分别约为2.1、7.2和12.3）[81]，其有效范围更广。此外，对于冻干和冷冻处方，缓冲成分在冷冻浓缩过程中的结晶倾向也是一个重要考量[82]。缓冲成分在冷冻过程中的选择性结晶可能导致pH值变化，进而影响蛋白质的稳定性。磷酸钠缓冲液是一个广泛研究的例子，其在冷冻过程中的磷酸二钠十二水合物（Na2HPO4·12H2O；磷酸盐缓冲液的基本成分）结晶会降低冷冻浓缩液的pH值，可能改变蛋白质的稳定性[83-85]。因此，希望缓冲成分在冷冻过程中保持无定形态。然而，如柠檬酸盐缓冲液所示，即使缓冲成分保持无定形态，也可能观察到明显的离子化和表观pH值变化[86]。The selection of buffer for a pharmaceutical protein formulation is usually
based upon the desired pH range, buffering capacity and the possibility of
buffer-specific catalysis [81]. Ideally, the use of a buffer is desired at pH
values ± 1 unit of the acid dissocaition constant (pKa), with the highest
buffering capacity at pH = pKa. The knowledge of pKa aids in buffer selection.
For example, when the desired pH is ~7.0, phosphate buffer (pKa ~ 7.2) is
widely employed, while histidine (pKa ~ 6.1) is the buffer of choice at pH ~
6.0 [6]. The effective range is broadened for buffers with multiple
pKas, such assodium phosphate buffer (pKas ~ 2.1, 7.2 and 12.3) [81].
Additionally, for lyophilized and frozen formulations, the crystallization
propensity of buffer components during freeze-concentration, becomes an
important consideration [82]. The selective crystallization of a
buffer component during freezing may lead to pH shifts, thereby affecting
protein stability. Sodium phosphate buffer is a widely-studied example wherein
crystallization of disodium phosphate dodecahydrate (Na2HPO4.12H2O; the basic
component of phosphate buffer) during freezing lowers the pH of the freeze
concentrate and may alter the stability of proteins [83–85]. Hence,
it is desirable that the buffer components are retained amorphous during
freezing. However, as exemplified by citrate buffer, significant changes in
ionization and apparent pH may be observed even when buffer components remain
amorphous [86].![]()
图 3. 增加表面活性剂浓度和防止蛋白质变性的示意图。经 Elsevier 许可，转载自 Lee 等人 [76]。Fig. 3. Graphical representation of increasing surfactant concentration and
prevention of protein denaturation. Reproduced from Lee et al [76] with
permission of Elsevier.缓冲液的结晶行为及其在冷冻过程中相关的pH值变化取决于多种因素，包括缓冲液的类型和浓度、初始溶液pH值以及其它处方成分的浓度（见图4）。例如，柠檬酸盐缓冲液适用于初始pH值为5.0或6.0的溶液，但当初始pH值为4.0时，可能会产生约+2个单位的pH值偏移。同样，组氨酸缓冲液在pH 5.5时会出现+2个单位的pH值偏移，但在pH 6.0和7.0时则不会。琥珀酸盐缓冲液是一个例子，其pH值变化的方向受初始pH值的影响（在pH 4.0和5.0时约为+1，而在pH 6.0时约为-1.0）。在pH 6.0时，碱性缓冲成分（单钠琥珀酸盐）会结晶，而酸性成分（β-琥珀酸）在更低的pH值结晶[87]。The crystallization behavior of buffers and the associated pH shifts during
freezing depends upon several factors including the type and concentration of
buffer, the initial solution pH, and the concentration of other formulation
components (Fig. 4). For example, citrate buffer works
well for solutions with initial pH of 5.0 or 6.0 but exhibits a pH shift of ~+2
units when the initial pH is 4.0. Similarly, histidine buffer shows a pH shift
of +2 units when used at pH 5.5, but not at pH 6.0 and 7.0. Succinate buffer is
an example, where the direction of pH shift is affected by the initial pH (~+1
at pH 4.0 and 5.0 vs ~-1.0 at pH 6.0). At pH 6.0, the basic buffer component
(monosodium succinate) crystallizes, while the acidic component (β-succinic
acid) crystallizes at lower pH values [87].![]()
图 4. 不同缓冲溶液在冷冻时的 pH 值变化，按初始 pH 值从左到右的递增顺序绘制。左图：pH 值偏移大于 1 个单位的缓冲液，右图：pH 值偏移小于 1 个单位的缓冲液。填充圆圈表示每种缓冲溶液在 ~25℃时的初始 pH 值（也在相邻方框中提及）。箭头指向冷冻过程中 pH 值的移动方向，箭头尖端表示将溶液冷冻至 ~-25℃后的最终 pH 值。琥珀酸缓冲液 200 mM 显示了 pH 值的摆动，本图只显示了第一次 pH 值移动。Fig. 4. pH shift shown by different buffer solutions upon freezing, plotted in
increasing order (from left to right) of the initial pH. Left panel: buffers
showing shifts >1 pH unit and right panel: buffers showing shifts ≤1 unit.
Filled circles denote the initial pH value of the solution at ~25℃ (also
mentioned in the adjacent box) for each buffer solution. Arrows point the
direction of pH shift during freezing and the tip of the arrow marks the final
pH after freezing the solution to ~-25℃. Succinate buffer 200 mM displayed a pH
swing and only the first pH shift has been shown in this figure. Data adapted
from Bhatnagar et al [88], Kolhe et al [89], Sundaramurthi and Suryanarayanan[90,91] and Thorat et al [92].缓冲液浓度对pH值变化的幅度和性质有显著影响。例如，pH 7.4的磷酸钠缓冲液的pH值变化幅度从100 mM时的4.1降低到10 mM时的2.8。琥珀酸盐缓冲液表现出更复杂的行为，200 mM的高浓度下出现“pH摆动”，pH值先是从4.0上升到8.0，然后下降到2.2，这归因于缓冲成分的顺序结晶。在较低浓度（50 mM和100 mM）时，只观察到幅度较小的单向变化（约1个pH单位）[87]。因此，应谨慎使用最低有效浓度的缓冲液。另一种成功的方法是通过处方成分抑制缓冲结晶，从而减轻任何pH值变化。已知非结晶性溶质如蔗糖可以抑制缓冲结晶[93]。有时，蛋白质本身也可能抑制缓冲结晶，防止pH值变化[92]。这已在第4节中详细讨论。The buffer concentration can have a pronounced influence on both the magnitude
and the nature of pH shift. For example, the magnitude of pH shift for sodium
phosphate buffer pH 7.4, was reduced from 4.1 (at 100 mM) to 2.8 (at 10 mM).
Succinate buffer displayed a more complex behavior with higher concentration
(200 mM) showing a ‘pH swing’ wherein the pH first increased from 4.0 to 8.0
and then decreased to 2.2 attributed to sequential crystallization of buffer
components. At lower concentrations (50 and 100 mM), only unidirectional shifts
of a smaller magnitude (~1 pH unit) were observed [87]. It is
therefore judicious to use the buffers at their lowest effective concentration.
Another successful approach involves inhibition of buffer crystallization by
formulation components, thereby attenuating any pH shift. Non-crystallizing
solutes such as sucrose are known to inhibit buffer crystallization [93].
Sometimes, the protein itself may inhibit buffer crystallization, thereby
preventing pH shift [92]. This has been discussed in detail in Section 4.
3.5 聚合物Polymers
高分子在溶液中稳定蛋白质的应用已久，并在冷冻（或解冻）及冷冻干燥过程中发挥作用。聚乙二醇（PEG）、右旋糖酐和聚乙烯吡咯烷酮（PVP）是常用的低温保护蛋白质的高分子[10]。然而，PEG作为结晶性高分子，在干燥过程中无法为蛋白质提供保护。右旋糖酐和PVP作为无定形高分子，可以通过提升处方的玻璃化转变温度（Tg）来在干燥状态下稳定蛋白质[24]。但这些高分子通常不会与蛋白质表面的极性基团发生作用。实际上，右旋糖酐即使具有高Tg值，也无法在脱水过程中保护酶类如过氧化氢酶，这表明仅形成无定形玻璃态虽是必要条件，但不足以确保蛋白质的稳定[10,21]。使用高Tg值的高分子，如右旋糖酐，并与糖类结合使用，具有优势。最终冻干制剂的高Tg值可能允许处方在室温下长期储存。右旋糖酐（40和70 kDa）已被批准作为血浆增量剂用于注射。在稳定单克隆抗体（mAb）制剂方面，右旋糖酐40与蔗糖以1:1的重量比混合，提供了最佳的制剂效果，无论是在有效的冷冻干燥周期还是40℃下储存14天后的产品稳定性方面。使用高分子量的右旋糖酐（150和500 kDa）会导致过长的重新溶解时间，而低分子量的右旋糖酐（1 kDa）则可能导致冻干制剂中形成酸性物质[94]。当右旋糖酐单独使用时，其末端的葡萄糖单元可能在高温下引起抗体糖基化[94]。此外，高分子的柔韧性也会影响其稳定蛋白质的能力，例如，与刚性的右旋糖酐相比，柔韧性更好的菊粉展现出更好的稳定效果[34]。Polymers have been used to stabilize proteins in solution, and also both during
freezing (or thawing) and freeze-drying. Polyethylene glycol (PEG), dextran and
polyvinylpyrrolidone (PVP) are commonly used to cryopreserve proteins [10].
PEG, a crystalline polymer, however provides no protection to protein during
drying. Dextran and PVP, amorphous polymers, can stabilize proteins in the
dried state, by increasing the formulation Tg[24]. However, the
polymers are usually unable to interact with the surface polar groups of
protein. In fact, the inability of dextran to protect enzyme catalase during
dehydration, in spite of its high Tg, established that the formation of
amorphous glassy state, though a necessary condition, is not sufficient for
protein stabilization [10,21]. There is advantage in using the high
Tg polymers, for example dextran, in combination with sugars. The high Tg of
the final lyophile may permit long-term room temperature storage of the
formulation. Dextran (40 and 70 kDa) is approved for parenteral use as a plasma
expander. For stabilizing an mAb formulation, a mixture of dextran 40 with
sucrose (weight ratio 1:1) provided the best formulation, both in terms of
efficient freeze-drying cycle and product stability after storage at 40℃ (14
days). Use of high molecular weight dextrans (150 and 500 kDa) resulted in
unacceptably long reconstitution times, while low molecular weight dextran (1
kDa) led to formation of acidic species in the lyophile [94]. When
dextran is used alone, the terminal glucose moiety can induce antibody
glycation at elevated temperatures [94]. In addition, the polymer
flexibility will also influence its ability to stabilize proteins as
demonstrated by better stabilization with the molecularly-flexible inulin as
compared to rigid dextran [34].后文见：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（下）
