蛋白类冻干制剂工艺开发与优化要点梳理

For a stable product with acceptable quality characteristics, a short lyophilization cycle has the advantage of higher throughput with lower manufacturing cost. In order to design an optimal drug formulation and lyophilization process, the formulation and process development scientists need to understand the critical properties of the formulation and how to apply this information to the formulation and process design. 在不影响产品质量的前提下，冻干工艺时间越短，经济效益越好。The critical formulation properties include the collapse temperature (Tc) of the formulation, the stability of the protein drug, and the characteristics (e.g. cake appearance, moisture content and reconstitution time) of the lyophilized drug product impacted by the excipients used. 药液配方直接影响塌陷温度、蛋白稳定性、冻干后的粉饼外观、水分和复溶时间。The macroscopic collapse temperature of the formulation (Tc) is the temperature above which the lyophilized drug product loses macroscopic structure and collapses during lyophilization. Tc is usually a few degrees above the Tg’, which is associated with the glass transition temperature in the frozen state or close to the eutectic temperature (Teu, the temperature at which a crystalline bulking agent mixture system melts) if solutes are crystallized in the frozen solution. 塌陷温度Tc一般略微高于玻璃转化温度Tg’（非晶型辅料）或共晶点温度Teu（晶型辅料）In a mixture of crystalline and amorphous freeze concentrate where the crystalline phase is in excess, the amorphous phase collapses if the product temperature is above Tg’ (or collapse temperature of amorphous phase), but gross or “macroscopic” collapse will not occur unless the product temperature is above both Tg’ and Teu. 如果配方中同时包含晶型和非晶型的辅料，且主要为晶型辅料时，只有在制品温度同时高于璃转化温度Tg’和共晶点温度Teu时，才会发生宏观的塌陷。In order to produce an acceptable lyophilized drug product, it is always required to lyophilize a formulation at the temperature below the collapse temperature. The product quality attributes such as drug product stability, cake appearance, visible and subvisible particles, and reconstitution time are particularly important for protein formulations. 药液配方严重影响蛋白稳定性、粉饼外观、复溶时间、药液的可见异物和不溶性微粒。Proteins are sensitive to the stresses imposed by lyophilization and are prone to degradation during the process and storage. The low temperature of lyophilization process does not guarantee protein stability because proteins could experience stresses of dehydration, cold denaturation or denaturation at interfaces (protein–air and protein–ice). Fortunately, thermodynamic instability caused by lyophilization stresses does not necessarily mean protein unfoldingduring lyophilization if the rate of unfolding is sufficiently slow on the time scale of the process in a viscous frozen formulation containing a sufficient amount of stabilizer(s) so that the process is complete before significant unfolding can occur. Therefore, the protein might safely be lyophilized at a temperature far above the Tg’, allowing a much faster drying process.蛋白质解折叠是个相对很慢的过程，所以配方中充分的蛋白保护剂可以确保蛋白在相对较短的冻干过程中免受热力作用而引发解折叠。

In general, there are different considerations and preferences applicable to first in human or late phase lyophilized formulation and lyophilization process. For an early phase lyophilized formulation, the major considerations are usually speed to clinic with limited material availability and product knowledge. A platform formulation and lyophilization process based on past product characteristics and formulation experiences can usually serve the purpose. The platform formulation and process approach can be relatively conservative to ensure successful technical transfer to different fill finish manufacturing sites, and fast development timeline with limited resource requirements although it may compromise process efficiency. 药品研发的早期阶段，通常采用平台相对保守的配方和冻干工艺参数，以更快地先把项目往前推进，后续再优化。In contrast, the late phase formulation and lyophilization process are designed and optimized for product quality and process efficiency at commercial scale with a full consideration of formulation and process robustness and product cost effectiveness. A full characterization of the lyophilization process with an acceptable design space is often desired for a commercial lyophilized drug product, which can be achieved through lyophilization process quality by design (QbD) and design of experiment approaches. Considerable material, time, and resources are required to optimize and characterize the formulation and lyophilization process. 研发的后期阶段则需要基于商业化规模去优化配方和冻干工艺，并完成工艺表征的研究，以获得可靠稳定的生产工艺和良好的经济性For example, the late phase lyophilized drug product usually requires 24 months or longer shelf-life at 2°–8°C with a 30-day room temperature time out of refrigeration (TOR) for commercial distribution/storage, acceptable room temperature post reconstitution stability for administration or maybe even stable for room temperature storage and distributions. 研发的后期阶段，应开展长期稳定性研究、室温暴露的研究、复溶后药液的稳定性研究。The formulations of the late phase and commercial products are often optimized to have a relatively high collapse temperature, which makes it more efficient and easy to lyophilize. The osmolality of the reconstituted drug product should be close to isotonic with acceptable viscosity for the intended route of administration (e.g. subcutaneous injection) and the reconstitution time is relatively short for clinical convenience. 配方研究时，应尽可能实现更高的塌陷温度，以提高干燥的效率，复溶后药液的渗透压应尽可能接近等渗，粘度在合理范围内，复溶时间尽可能短。

During the lyophilization process, proteins are subjected to multiple stresses that can adversely affect their stability. These include cold denaturation, freeze-concentration, ice-water interface, phase separation, and dehydration. This section will explore the primary stresses encountered during lyophilization. 蛋白在冻干过程中受到冷变形、低温浓缩、固液界面应力、相分离、脱水的影响。

Cold denaturation refers to the spontaneous protein unfolding at the temperatures typically well below zero degrees Celsius. Cold denaturation is largely an enthalpy-driven焓驱动process, arising from the increased solubility of non-polar groups and the consequent weakening of the hydrophobic core of proteins at lower temperatures. 冷变性指的是蛋白在很低的温度下发生自发地解折叠，主要由焓变驱动（非极性基团溶解度的增加，蛋白疏水性核心弱化）。In contrast, the thermal denaturation is mainly an entropy-driven熵驱动process.相反，蛋白热变性则是由熵驱动的。Cold denaturation can occur during the freezing process of lyophilization, where the product temperature is often lowered to −20°C to −50°C. Proteins are more resistant to cold denaturation at higher protein concentrations or in the presence of stabilizers such as sugars and polyols. 蛋白在高浓度或保护剂（如糖类、醇类）的作用下，更能抵抗冷变性。The rate of cold denaturation is typically slow on the timescale of freeze-drying, allowing primary drying to be conducted above Tg’. 在冷冻干燥的时间尺度上，冷变性的速率一般很慢（干燥都结束了，冷变性还没完成），所以一次干燥可以在高于玻璃转化温度（Tg’）下进行。

Freeze-concentration refers to the phenomenon where the solute concentration dramatically increases during freezing. A dramatic increase in salt concentration can reduce protein colloidal stability and lead to protein aggregation. A significant increase in protein concentration can increase the tendency of protein aggregation. 蛋白浓度越大，则越容易聚集。Freeze-concentration can also increase the concentration of impurities in the formulation such as metals,reactive oxygen species and proteases, accelerating protein degradation. 低温浓缩过程会提高配方中金属、氧化物质、蛋白酶的浓度，从而加速蛋白降解。Differential crystallization of buffer salts resulting from freeze concentration can lead todramatic shift in pH (≥ 3 pH unit). 低温浓缩导致缓冲盐冰晶差异，从而导致较大的pH梯度。pH can also change due to temperature dependent pKa values for amine-based buffer systems, such as histidine, TRIS and etc. 组氨酸/Tris等缓冲液，因温度相关的pKa变化也会导致pH的改变。Additionally, excipients may crystallize or precipitate when frozen. 辅料也会在冷冻时发生结晶或者沉淀。

The formulation components, such as buffer salts, stabilizers and bulking agents, can separate into distinct phases during lyophilization. 相分离后，比如出现溶液浑浊、分层或微小液滴。Liquid-liquid phaseseparationhas been reported for many systems relevant to protein formulation, such as polymer-polymer,polymer-salt and protein-polysaccharides. Liquid-liquid phase separation can lead to the depletion of stabilizers from proteins and therefore compromise protein stability. 比如二种高分子聚合物之间可能发生液-液相分离（比如浓度不合适），高分子聚合物和盐之间也可能发生液-液相分离（比如盐改变了高分子聚合物的溶解性），蛋白和多糖之间可能发生液-液相分离（比如二者电荷相反形成复合物）。Crystalline-amorphous phaseseparationcan also occur when buffer salts, bulking agents and stabilizerscrystallize due to freeze-concentration. Crystallization of bulking agents such as mannitol and glycine from amorphous proteins can lead to the loss of their stabilizing effect. 晶型-非晶型相分离也可能因缓冲盐、填充剂（如甘露醇、甘氨酸）、保护剂在冷冻阶段出现结晶而发生。

Proteins are exposed to ice-water interfacesduring the freezing process of lyophilization. The area of ice-water interfaces depends on the number, size and morphology of the ice crystals. Direct adsorptionof proteins to the ice-water interface can lead to protein denaturation and aggregation. 蛋白直接吸附在冰-水界面会导致变性、聚集。In addition to direct adsorption, the ice-water interface can promote the cold denaturation of proteins by weakening thehydrophobic core that is critical for maintaining protein structure. 冰-水界面还会弱化蛋白疏水内核，破坏蛋白结构，使得发生冷变性。

Proteins experience dehydration stressduring the drying process of lyophilization when water moleculesbound to the protein surface are removed. Water molecules bind to protein surface via hydrogen bonding, and this interaction plays an essential role in stabilizing protein structure. 水分子和蛋白之间通过氢键相互作用，起到稳定蛋白的作用。Disruption of water-protein interactions can lead to protein denaturation and aggregation.Stabilizers such as sugars and polyols that can replace water molecules to form hydrogen bonds with amino acid residues at protein surfaces, thereby protecting proteins against dehydration stress. 干燥过程中水分丢失，保护剂（糖类、醇类）可以替代水分子与蛋白的氨基酸残基结合形成氢键，起到稳定蛋白的作用。In contrast, polymeric stabilizers like PEG are not effective against dehydration stress due to their inability to replace the hydrogen bonds between protein and water. 但聚合物（如PEG）作为保护剂时，因不能和蛋白形成氢键，所以在干燥阶段起不到保护蛋白的作用。

Considerations for the development of stable lyophilized protein formulations：

Buffers: It is preferable to select buffers, such as histidine, Tris and citrate, that do not crystallize during freezing. Volatile buffers, such as acetate, are not suitable for lyophilized formulations. 推荐使用非晶型的缓冲体系（如组氨酸、Tris、柠檬酸盐），不推荐醋酸盐缓冲体系。To avoid dramatic pH change resulting from differential crystallization of buffer components, it is recommended to avoid the use of high concentration phosphate saltbuffers such as sodium phosphate. 避免使用高浓度的磷酸盐缓冲体系（如磷酸钠）以避免冻干过程中剧烈的pH变化。When the use of phosphate salt buffers is necessary, the risk of buffer crystallization can be mitigated by minimizing the buffer concentration (≤ 20 mM) or adding excipients that can inhibit the crystallizationof phosphate salts, such as trehalose and sucrose. 如果必须使用磷酸盐缓冲体系，则建议降低其浓度（≤20mM），或者添加抑制其结晶的辅料（如海藻糖、蔗糖）。

Stabilizers: The most effective stabilizers for lyophilized protein formulations are sugars, such as sucrose and trehalose. Other stabilizers for lyophilized protein formulations include amino acids or sugar alcohols, such as mannitol and sorbitol, and cyclodextrin. 蔗糖和海藻糖是最常用的保护剂，其次是甘露醇、山梨醇、环糊精。Sugars can protect proteins against various stresses during lyophilization (e.g. cold denaturation, ice-water interface, freeze-concentration and dehydration) and long-term storage. 糖类可以保护蛋白免受各种应力。Trehalose can crystallize from an aqueous solution during freezing. The β, β-trehalose crystallizes much more rapidly from a freezing aqueous solution than does the α-α trehalose. 海藻糖在冷冻过程中能够从水中结晶出来，β, β海藻糖的结晶速度比α-α海藻糖更快The stabilizing effect of sugars in solid-state can be attributed to the formation of sugar glass and the replacement of hydrogen bonding between protein and water. The formation of sugar glass greatly reduced the mobility of molecules and the rate of physical and chemical degradation. 糖玻璃态的形成，能够降低蛋白分子的迁移和降解速率。Increased protein stability is typically observed at a higher stabilizer to protein molar ratio for lyophilized products. 对于冻干后的药品，保护剂和蛋白分子的比例越高越稳定。It is worth noting that reducing sugars, such as glucose and lactose, should be avoided in the selection of stabilizers. While reducing sugars may be effective at preventing protein folding, they tend to react with proteins via Maillard reaction, leading to protein glycation that can adversely affect product quality. 选择糖类保护剂时，应避免使用还原糖（如葡萄糖、乳糖）。虽然还原糖能够避免蛋白发生异常折叠，但是他们容易和蛋白发生美拉德反应，导致糖基化的发生，影响糖型。Polymeric stabilizers such as PEG and dextran can be used to increase the collapse temperature and therefore achieve better cake structure or shortening drying time. 聚合物（如PEG）能够提高塌陷温度，从而获得好的粉饼外观、提高干燥效率。Studies have shown that the combination of polymeric stabilizers and disaccharides can provide good stabilizing effect. 聚合物和二糖共同作为辅料时，能够起到良好地稳定蛋白的作用。

Surfactants: Surfactants are often included in lyophilized protein formulation to protect proteins against interfacial stresses from ice-water interfaces during freezing and air-liquid interfaces during reconstitution. 表面活性剂在冷冻阶段和起到保护蛋白免受冰-水界面应力的作用，在复溶后起到保护蛋白免受气-液应力的作用。It is generally recommended to maintain the surfactant concentration above its critical micelle concentration to achieve sufficient stabilizing effects. 通常表面活性剂的浓度在临界胶束浓度（CMC）之上。Meanwhile, it is advisable to limit the concentration of surfactants to mitigate the risk of protein degradation linked to surfactants or their impurities. 也不能浓度过高，导致表面活性剂或其相关杂质引起蛋白降解。Peroxides, which can be introduced into protein formulation as residual impurities from polysorbateor as by-products of polysorbate auto-degradation, can cause protein oxidation and aggregation during lyophilization. 由聚山梨酯引入的过氧化物杂质，可能在冻干过程中氧化蛋白，导致蛋白聚集。

Viscosity reducers: Viscosity reducers such as arginine hydrochloride, sodium chloride and proline may be considered when the viscosity of the formulation is a concern. 盐酸精氨酸、氯化钠、脯氨酸通常作为粘度控制剂。Arginine hydrochloride is a commonly used viscosity reducer for lyophilized formulation due to its effectiveness and its tendency to remain in a single amorphous phase with proteins during lyophilization. 其中盐酸精氨酸因为其在冻干过程中能够与蛋白处在单一的非晶型相中而最常用。In contrast, sodium chloride has a much higher tendency to crystallize, leading to its separation from the amorphous phase containing proteins. 氯化钠更容易结晶，容易和蛋白产生相分离。

Bulking agents: Crystalline bulking agents such as mannitol and glycineshould be considered when there is a challenge in forming mechanically strong and elegant cake. 甘露醇和甘氨酸通常作为粉饼的骨架支撑剂。These bulking agents can crystallize and provide support to cake structure. While crystalline bulking-agents typically do not protect protein against lyophilization stress, it has been demonstrated that the mixing of mannitol and glycine at certain ratios can lead to partial crystallization. The fraction of mannitol and glycine remaining in amorphous phase can provide sufficient stabilizing effect. 将甘露醇和甘氨酸按照一定比例混合后可以实现部分晶型的状态，从而这部分非晶型的甘露醇和甘氨酸可以起到保护蛋白的作用。Crystalline mannitol hydrate formed during lyophilization can release water to the amorphous protein drug during storage, potentially compromising the stability of the lyophilized protein formulation. 药品储存过程中，晶型的甘露醇水合物会释放出水被非晶型蛋白吸收，从而影响药品的稳定性。

High protein concentration formulations: The development of high protein concentration lyophilized formulations often presents unique challenges, such as high opalescence in the reconstituted drug product, long reconstitution time and the need to balance between stability and osmolality. Special considerations in formulation design are required to address these challenges effectively. 高浓度蛋白的产品，需要考虑药液的乳光高、复溶时间长、在质量稳定性和渗透压之间做好平衡的问题。

优化配方以提高冻干效率：

Primary drying is generally the most resource-intensive process, accounting for ~40% of the total resource required for a lyophilization process. A higher shelf temperature can accelerate the rate of sublimation and shorten the time required for primary drying. For example, a 1°C increasein product temperature during primary drying can lead to a 13% reductionin primary drying time. To produce lyophilized protein products with satisfactory appearance and long-term stability, the product temperature typically needs to be maintained at 2–3°C below the collapse temperature. Therefore, the formulation should be optimized to achieve the highest collapse temperature to shorten primary drying. 一次干燥在整体冻干工艺中所占的时间最长，提高1℃大概能够降低13%的一次干燥时间。所以，配方设计时应考虑获得较高的塌陷温度，以能够使用较高的一次干燥温度。

One strategy to increase collapse temperature is to increase protein concentration in the formulation. Depaz et al. reported that the collapse temperature of two monoclonal antibodies increased from approximately –30°C to approximately –20°C when the protein concentration was increased from 0 to 100 mg/ml. 提高蛋白浓度可以提高塌陷温度。However, an increase in protein concentration may require higher stabilizer concentration, which can result in a significant increase in total solid content. This can lead to difficulty in reconstitution and primary drying. 提高蛋白浓度的同时，保护剂的浓度也会跟着提高，导致粉饼重量增加，从而提高复溶和一次干燥的难度。

Another strategy to increase the collapse temperature is to add crystalline bulking agents, such as mannitol and glycine. 配方中增加骨架支撑剂（甘露醇和甘氨酸）时另一种提高塌陷温度的方法。A drawback of this strategy is that a higher bulking agent to stabilizer ratio is required to ensure their crystallization prior to the primary drying. Since the crystalline bulking agents are typically used as fillers to achieve desirable cake structure and do not provide significant stabilizing effect, a low bulking agent concentration is preferred. 这种方法的缺点是为了使得在一次干燥之前完成结晶，需要更高的骨架剂浓度。It has been reported that crystallizing amino acids such as phenylalanine, leucine and isoleucine can serve as alternative bulking agents. These amino acids can crystallize at a lower bulking agent to stabilizer ratio, enabling fast drying at low bulking agent concentrations.

容器密封系统（CCS）的考虑：

Glass vials are commonly used as the primary container for lyophilized biopharmaceutical drug products. Amber glass vials are available for light-sensitive products. Glass vials specifically designed for lyophilized products may contain a hydrophobic inner surface coating, comprised of non-covalent silicone oil or a covalent hydrophobic silicone to improve cake appearance,reduce fogging, and maximize withdrawable volume. 玻璃材质的瓶子最常用，如果药品对光敏感，则可以考虑使用棕色瓶子。使用内表面经过疏水性处理（硅油涂层或共价硅涂层）的瓶子，可以降低爬壁等外观问题，降低复溶后抽取时的残留体积。

Polymer vials, commonly made of COP (cyclic olefin polymer), COC (cyclic olefin copolymer), and polypropylene have also been used as the primary containers. Polymer vials offer the advantage of resistance to vial breakage with minimal leaching. However, polymers are generally less efficient in conducting heat (with lower Kᵥ value than glass vials), which potentially requires a longer primary drying time. 聚合物材质的瓶子拥有材质相容性的优势，缺点是传热差点，导致一次干燥耗时长。Oxygen and water vapor could permeate through conventional polymer vials. Novel technologies, such as adding polyamide oxygen barrier layer (COP/polyamide/COP) improved barrier properties. Other inner and outer silica-based hydrophobic coatings could also act as a gas barrier to minimize oxygen or water vapor exposure. 传统的聚合物材质的瓶子，其隔绝氧气和水汽的能力差点，新型材质（添加了COP/聚酰胺/COP）或者增加表面涂层后有所改善。

There are two types of glass vials based on manufactured process, tubular vials and molded vials. Tubular glass vials are converted from glass tubing where the wall thickness is controlled and the vial base shape can be optimized during conversion of the tubing to base. Molded glass vials are formed by pouring liquid glass into a mold. Traditionally the molded glass vials often have a thicker wall in contrast to tubular vial and a concave bottom. 模制瓶的壁厚一般比管制瓶更薄。With wide temperature ranges during lyophilization, a uniform thickness of the glass vial bottom and side with a low coefficient of expansion is desirable. The concave bottom of the vial prevents the close contact and leaves an air-filled gap between the vial and the freeze-dryer shelf. The air gap may limit the heat transfer during lyophilization, lowering K_g (Equation 1) and resulting in inefficient heat transfer. However, in the last decade, there have been improvements in molded glass vial manufacturing, such that the bottom geometry of the molded vials could be comparable to tubing glass vials, resulting in improved heat transfer performance and similar K_v. 早期的模制瓶因为瓶底存在凹面，导致不能和板层直接接触，影响传热，近些年模制瓶因生产工艺的改善而有所好转。

There are three types of neck design for glass vials, American blowback (ABB), European (blowback (EBB), and no blowback (NBB). The ABB and EBB vials reduce the risk of stopper pop-off, which are important for lyophilization application. 瓶子颈部有二种防跳塞的设计（ABB和EBB），选择的时候需要结合胶塞的设计。

For lyophilized drug product, which is often sensitive to moistures, selecting stoppers containing low moisture and with low moisture vapor transmission (MVT) is important to ensure product quality and stability. Stoppers may absorb trace amount of moisture from stopper manufacturing, sterilization, storage, and even during drug product lyophilization. The trace amount of moistures contained in the rubber stoppers may be transferred to the lyophilized product. 胶塞在制造、灭菌、储存、产品冻干过程中均可能吸收水分，随后这些水分会进一步被粉饼吸收。所以，尽可能选择含水分低、隔绝水汽能力强的胶塞。

Stoppers made of butyl or halogenated butyl (bromobutyl or chlorobutyl) elastomer are commonly used for vialled lyophilized drug products. These elastomers are low in gas permeation, low in moisture absorption and MVT, and provide low coring, good sealing and resealing properties. When used for lyophilized biopharmaceutical drug products, the stoppers are usually coated with fluoropolymer (such as tetrafluoroethylene and ethylene coating, or polyethylene and tetrafluoroethylene coating) to improve drug compatibility. The coating minimizes the extractable and leachable from the rubber components, reduces moisture transmission, and also modifies the surface properties of the stoppers. 冻干产品通常选用覆膜（比如聚四氟乙烯覆膜）胶塞，可以降低E＆L风险、提高水汽隔绝能力。The coating on the side and top of stoppers, if presents, prevents stickiness and avoids the need for siliconization. 胶塞顶部和侧面覆膜后，也可以减少胶塞之间、胶塞和部件之间的粘连问题。

The configuration of the stoppers is especially important for lyophilization. Typically, a stopper for lyophilized product has a vent opening for sublimated ice, and it has 3 types of design, single vented (igloo), dual vented, or three-legged configurations. The single-vented stoppers are commonly used attributing to ease of handling and good machinability. 单叉胶塞最为常用。Vial stopper plugs also come with many different designs, such as different height, diameter, vent position, rib design, blowback, and rings. The stopper design should match the vial neck to ensure proper sealing.

Dual-chamber vial, also called act-o-vial, is a vial with two compartments, lyophilized drug product compartment and diluent compartmentseparated by a stopper. Dual chamber syringes and cartridges, consisting of a barrel which is divided into two chambers via a plunger, with one chamber containing the lyophilized product and the other chamber containing the diluent, are becoming more common (Fig.1). The dual chamber cartridge is similar to syringe, except that the cartridge is sealed with a septum and the syringe is closed with a Luer cone and needle. The diluent can bypass the stopper upon activation via an external channel or internal groove for reconstitution. Dual chamber syringes and cartridges offer accurate dosing with minimized steps for reconstitution. 西林瓶、预充针、卡氏瓶都有双腔室的设计，药物成分和稀释剂通过胶塞隔离开来，可以方便临床使用，满足用药安全的需求。

双腔室设计示意图

The plungers are usually coated with fluoropolymer coating to minimize leaching. 1 ml dual chamber syringe or cartridge is the most common size for commercial products on the market. 双腔室制剂用的胶塞通常都是覆膜的，目前市场上最常见的是1mL的预充针和卡氏瓶。The dual chamber devices are also available in 1–5 ml and potentially > 5 ml. The selection of the dual chamber devices is dependent on the dose volume. The lyophilization of dual chamber syringes and cartridges are different from vials. The syringes and cartridges are usually not in direct contact with the freeze-dryer shelf and the heat transfer is primarily by convection rather than conduction. 预充针/卡氏瓶的冻干工艺原理和西林瓶不同，他俩不和板层直接接触，所以主要依靠的是气体对流传热。

蛋白类药物的冻干工艺的开发：

冷冻：Controllednucleationis a freezing technique that can manage supercooling to control ice crystal size and distribution. By inducing ice formation at a specific temperature, it ensures uniform ice crystals across all vials, resulting in a consistent and homogeneous product. There are several methods to achieve controlled nucleation, including a) ice fog method that involves introducing a fog of small ice particles into the product, acting as nucleation sites and inducing the formation of ice crystals; b) pressure drop methodthat involves pressurizing and reducing the pressure suddenly in the lyophilizer chamber, causing ice formation at the controlled ice nucleation temperature; and c) electromagnetic field method that involves using an electromagnetic field to induce nucleation. Among all the above controlled nucleation methods, the pressure drop method has been successfully adopted for GMP productions of lyophilized drug products. 有三种受控成核的技术：冰雾法、压力骤降法、电磁场法，压力骤降法（药液降低至目标成核温度但未自然冻结，然后对腔体快速抽真空，水分蒸发带走热量而使药液成核，之后进入常规冷冻、干燥步骤）已经有了用于商业化生产的成功案例。Annealing is an optional step that holds the protein formulation at a specific temperature above the Tg’for a defined period after the freezing step. This process allows for the growth and reorganization of ice crystals and leads to larger and more uniform ice crystals, which can enhance the sublimation rate and reduce the drying time. 退火是个可选的步骤，药液冷冻后，在Tg’温度之上维持一段时间，使得冰晶生长和重排，获得更大更均一的冰晶，从而有利于一次干燥效率。This step is particularly beneficial for formulations that require the crystallization of solutes, such as mannitol or glycine, to avoid amorphous phases and enhance product stability. 退火步骤通常用于需要溶质结晶（如甘露醇或甘氨酸）的配方中，以避免无定形状态。

一次干燥：This process removes about 80–95% of the water in the protein product while maintaining its physical structure. 一次干燥可以去除80%~95%的水分。The fundamental concept of primary drying is to select the optimal target product temperature (Tp), a few degrees below the collapse temperature Tc, to ensure the cake structure and efficient sublimation. Shelf temperature plays a crucial role in the primary drying and it is typically set above the target Tp to provide adequate heat for ice sublimation. 一次干燥阶段时的制品温度略微低于塌陷温度，板层温度设定值高于目标制品温度。The chamber pressure (Pc) controls the rate of sublimation and influences both heat and mass transfer. Consistently maintaining a chamber pressure below 50 mTorr is challenging, and there is little benefit in exceeding 200 mTorr. It has been reported that moderate chamber pressure (100–150 mTorr) provides optimal homogeneity in heat transfer among vials. 对于西林瓶产品，冻干机腔体压力通常维持在100–150 mTorr（0.13~0.20 mbar）。Therefore, the ideal chamber pressure balances a high sublimation rate with uniform heat transfer. Equation can be used to determine the “optimal” chamber pressure for a given Tp.理想的腔体压力可以根据制品目标温度进行计算：

The recommended relationship between the target T_p, the vapor pressure of ice pressure at sublimation interference (P_ice), and the recommended chamber pressure (P_c) to achieve optimal sublimation are summarized in Table 4. 制品目标温度、升华界面冰的饱和蒸汽压力、推荐的腔体压力三者之间的关系见下表：

Ramp rate is another parameter of the primary drying stage that refers to the rate at which the temperature is increased or decreased on the shelves that hold the product. Usually, a low to moderate ramping rate (e.g. 0.1–1°C/min)is implemented during the freezing and primary drying stage considering the cooling/heating capacity of the lyophilizer and the process efficiency. 冷冻和干燥阶段，通常使用中等偏低的升/降温度速率（比如0.1–1°C/min），以确保不超出冻干机的性能。In general, the pressure within the lyophilizer chamber is monitored via a capacitance manometer and is controlled by the introduction of nitrogen during the primary drying phase. 干燥阶段的腔体压力通过电容式压力计进行监测，通过补充氮气进行压力控制。When the gas composition in the lyophilizer chamber changes from mostly water vapor to mostly nitrogen, it shows that primary drying is almost completed. 当腔体内的大部分气体组成由水汽变为氮气时，预示着一次干燥阶段完成了。A method for endpoint determination involves measuring the set-point pressure and a Pirani gauge that measures the gas's vacuum pressure-dependent thermal conductivity. Throughout the primary drying step, the Pirani gauge displays a higher value than the acute chamber pressure, indicating ice sublimation within the chamber. At the end of the primary drying, the pressure measured by the Pirani gauge decreases and aligns with the pressure recorded by the capacitance manometer. 皮拉尼压力值高于电容式压力值时，代表一次干燥正在进行中，二者趋平时预示着一次干燥完成了。

二次干燥：In this process, residual moisture remaining in the product is removed by desorption. This step uses a higher temperature, typically near or slightly above room temperature but below the glass transition temperature of the product (Tg) with a relatively slow ramping rate. 二次干燥的温度一般接近或者略高于室温，使用较慢的升温速率。Additionally, the chamber pressure is maintained at the same level as that used during the primary drying phase. Generally, temperatures and pressures in the range of 20°C to 40°C and 60 to 150 mTorr, respectively, are common, but they can be adjusted based on the needs of the drying process and product properties. Normally, drying time for secondary can vary from 3 to 12 h.二次干燥的腔体压力通常与一次干燥一致，时间一般在3-12h之间。

增加蛋白在产品储存期间的稳定性：Wolfgang's team recently focused on creating a fast-freeze-drying cycle for protein formulations containing mannitol and sucrose. The findings of this study indicate that fast-freeze-drying not only achieved protein stability throughout the process but also yielded a desirable cake appearance, significantly reducing the time required, in comparison to a conservative cycle. 研究表明，对于含有甘露醇和蔗糖的配方，采用快速冻干的参数有利于提高蛋白的稳定性，同时还有利于粉饼外观。

控制复溶时间的方法：Luoma and Lim developed a method to shorten the reconstitution time of a high-concentration IgG1 subclass monoclonal antibody. They explored how reconstitution time is influenced by the vacuum level in the vial's headspace. They observed a 3-fold decrease in reconstitution time at lower pressures, with times dropping from 75 minutes at higher pressures to about 20 minutes at lower pressures. On the other hand, reducing headspace pressure from 200 Torr to 0.1 Torr cut reconstitution time by at least half in all tests, achieving over 50% reduction in time at pressures below 50 Torr. 有研究表明，降低产品顶空的压力可以大幅降低复溶时间。In addition to lowering headspace pressure, Zhang and colleagues suggested adding an annealing step during the lyophilization process to shorten the reconstitution time of lyophilized high-protein concentration drug products. They found that including a −3°C annealing step in the lyophilization cycle could decrease reconstitution time by 38% compared to processes without annealing. 有研究表明，增加退火温度也可以缩短高浓度蛋白产品的复溶时间。Kulkarni and colleagues reported that crystalline mannitol can significantly decrease the reconstitution time for high concentration protein formulations. It improves the wettability of the cake solids, aids the penetration of reconstitution liquid into the cake’s interior, and makes cakes easier to disintegrate. 晶型甘露醇也可以显著缩短复溶时间。