Continuous Process: Implementing Low pH Viral Inactivation in a Continuous Process连续生产工艺：在连续生产时实施低pH病毒灭活
作者：Kakolie Banerjee ；Eric Karl Zimmerer ；Joelle Genevieve Johnson ；Ushma Mehta ；Elizabeth Goodrich ； Corinne MIller, PhD
Viral inactivation (VI) is a critical step in ensuring the safety of monoclonal antibody (mAb), Fc fusion, and recombinant protein therapeutics and it is typically an important component of an overall virus control strategy for downstream biotherapeutic production processes. Considerations for successful implementation of an inline VI process are discussed in this article.病毒灭活（VI）是确保单克隆抗体（mAb）、Fc融合和重组蛋白疗法安全性的关键步骤，通常是下游生物药生产工艺整体病毒控制策略的重要组成部分。本文讨论了成功实施在线VI工艺的注意事项。
Market drivers have motivated biomanufacturers to set cost reduction and faster production targets while maintaining viral safety and product quality. Transitioning from batch manufacturing to a continuous VI or inline VI (iVI) mode (Figure 1) enables manufacturers to better achieve these targets while providing other advantages, such as a reduced footprint and more streamlined production. Additionally, the smaller equipment facilitates transition to single-use flow paths, which eliminates cleaning and sanitization.市场驱动因素促使生物药生产商设定降低成本和加快生产目标，同时保持病毒安全性和产品质量。从批量生产过渡到连续VI或在线VI（iVI）模式（图1）使生产商能够更好地实现这些目标，同时提供其他优势，例如减少占地面积和更简化的生产。此外，较小的设备有助于过渡到一次性流路，从而消除了清洁和消毒。
Low pH VI is performed after protein A chromatography to deliver robust inactivation of enveloped viruses. Studies have shown that pH, time, and temperature are critical operating parameters. VI efficacy may also be impacted by protein concentration, isoelectric point (pI), ionic strength, presence of aggregates, buffer systems, and process impurities. The ASTM standard E2888-1 defines conditions that ensure ≥ 5 log reduction value (LRV) of model enveloped rodent retrovirus when a post–protein A eluate is incubated at pH ≤ 3.6 for > 30 minutes.1在蛋白A色谱后进行低pH VI以对包膜病毒进行稳健灭活。研究表明，pH、时间和温度是关键的操作参数。VI功效还可能受到蛋白质浓度、等电点（pI）、离子强度、聚集体的存在、缓冲系统和工艺杂质的影响。ASTM标准E2888-1定义了当后蛋白A洗脱液在pH ≤ 3.6下，培养>30分钟时，确保模型包膜啮齿动物逆转录病毒≥5个对数减少值（LRV）的条件。
Maintaining the feed at target VI conditions for the required duration while under continuous flow is achieved by passing through an incubation chamber. Because the kinetics of VI are rapid, typically achieving > 5 LRV at pH < 3.6 in under 30 minutes,2 , , manufacturers could benefit from shorter exposure times, which would enable the production of pH-sensitive biomolecules without compromising product quality. In this article, available incubation chamber designs are presented with a discussion on how industry can adopt these technologies to transition from existing batch processes to iVI.通过在连续流动下将进料保持在目标VI条件下，通过通过培养室来实现。由于 VI 的动力学速度很快，通常在 pH < 3.6 的 30 分钟内达到 > 5 LRV，生产商可以从更短的曝光时间中受益，这将使pH敏感的生物分子的生产成为可能，而不会影响产品质量。在本文中，介绍了可用的培养箱设计，并讨论了行业如何采用这些技术从现有的批处理工艺过渡到iVI。
INCUBATION CHAMBER DESIGN培养箱设计
Critical parameters of VI include incubation time and inactivation pH. Ensuring adequate incubation time in a continuous process is a key concern due to fluid dynamics that result in nonuniform residence times. Several technologies for iVI are being developed to narrow the residence time distribution (RTD) and guarantee a desired minimum incubation time.VI的关键参数包括培养时间和灭活pH值。由于流体动力学导致停留时间不均匀，因此确保在连续过程中有足够的培养时间是一个关键问题。正在开发几种用于iVI的技术，以缩小停留时间分布（RTD）并保证所需的最短培养时间。
Current incubation chamber technologies for iVI belong to two categories: cyclical flow reactors (CFR) and packed bed reactors (PBR). Coiled tubing used in a CFR design decreases the RTD by inducing secondary flow motions.6 , Two CFRs have been developed for iVI: the coiled flow-inverter (CFI), developed by Bayer, and the serpentine tubular flow device (jig-in-a-box, or JIB), developed by Boehringer-Ingelheim. The CFI comprises a series of helical coils arranged at right angles, while JIB is designed with a flow path of alternating 270-degree turns.目前的iVI培养室技术分为两类：循环流反应器（CFR）和填充床反应器（PBR）。CFR 设计中使用的连续油管通过诱导二次流动运动来降低 RTD。已经为iVI开发了两种CFR：由拜耳开发的盘绕流量逆变器（CFI），以及勃林格-殷格翰开发的蛇形管式流动装置（盒中夹具或JIB）。CFI由一系列以直角排列的螺旋线圈组成，而JIB设计有270度交替旋转的流路。
Alternatively, the PBR uses a packed bed of nonporous, inert beads to minimize the RTD. The RTD is dependent on different critical design and operating parameters specific to each incubation chamber technology.4 , , Therefore, a mechanistic understanding of the parameters that affect RTD is necessary to guarantee robust performance. Chamber performance is characterized by injecting pulses of tracer molecules into a flow path and measuring the dispersion of the pulse exiting the chamber. This information is used to generate metrics for the chambers, as shown in Figure 2. Upon chamber commercialization, the data provided on RTD and VI will enable manufacturers to select a suitable chamber for their process needs.
或者，PBR 使用无孔惰性磁珠的填充床来最小化 RTD。RTD 取决于特定于每种培养箱技术的不同关键设计和操作参数。因此，必须对影响RTD的参数进行机理理解，以保证稳健的性能。腔室性能的特点是将示踪分子的脉冲注入流路并测量离开腔室的脉冲的分散度。此信息用于生成腔室的指标，如图 2 所示。在腔室商业化后，RTD和VI上提供的数据将使制造商能够根据其工艺需求选择合适的腔室。
LC I SYSTEM IMPLEMENTATIONLC I 系统实施
Manufacturers need to consider process-associated challenges and possible mitigation strategies for successful implementation of an iVI system.制造商需要考虑与流程相关的挑战和可能的缓解策略，以成功实施iVI系统。
Post–Protein A Process蛋白A后的工艺
Eluate from the protein A affinity capture may deliver a variable feed profile to the iVI process, which can be challenging to control because acid titration is dependent on both inlet feed concentration and pH. Titration methodologies must be established that enable the system to react precisely to the incoming feed conditions. A feedback loop could utilize inline pH and protein concentration measurement to adjust the acid injection rate dynamically as concentration changes in order to bring the feed to the target pH. Alternatively, installing a surge vessel post–protein A (Figure 1) would allow the pooling of the eluate to provide a more homogeneous protein concentration to be delivered for titration. Additionally, a surge vessel would mitigate the risk of negative impact caused by possible process interruption. 12 , , , 从蛋白A亲和捕获中洗脱出液可能会为iVI过程提供可变的进料曲线，这可能难以控制，因为酸滴定取决于进样口进料浓度和pH值。必须建立滴定方法，使系统能够精确地对进料条件做出反应。反馈回路可以利用在线pH和蛋白质浓度测量，随着浓度的变化动态调整酸注入速率，以使进料达到目标pH值。或者，在蛋白A后安装一个缓冲容器（图1）将允许洗脱液汇集，以提供更均匀的蛋白质浓度进行滴定。此外，缓冲容器将降低可能的过程中断造成的负面影响的风险。
“Manufacturers need to consider process-associated challenges and possible mitigation strategies for successful implementation of an iVI system.”“生产商需要考虑与工艺相关的挑战和可能的缓解策略，以成功实施iVI系统。”
pH Sensor Accuracy and StabilitypH电极的准确性和稳定性
Accurate pH measurement is required to ensure the feed reaches the target pH for VI. During operation, pH sensors can drift or lag, which may result in an incomplete VI. An incomplete VI is associated with pH inaccuracy.16 Strategies to minimize risk include well-characterized, robust sensors with defined operational limits and extensive pH stability performance data collected over extended periods of operation. Additionally, a system design that enables sampling for offline pH measurement would provide the capability to perform a single-point readjustment of the online pH reading. Periodic sensor recalibration will be an important operational requirement.需要精确的pH测量，以确保进料达到VI的目标pH值。在操作过程中，pH传感器可能会漂移或滞后，这可能导致VI不完整。不完全的VI与pH值不准确有关。将风险降至最低的策略包括具有明确操作限值的可靠、坚固耐用的传感器，以及在长时间操作中收集的大量pH稳定性性能数据。此外，支持离线pH测量采样的系统设计将提供对在线pH读数进行单点重新调整的能力。定期传感器重新校准将是一项重要的操作要求。
Incubation Chamber Size培养室尺寸
The iVI system must provide a residence time that ensures exposure of the feed to the target pH for the required incubation time. Because lower incubation times risk incomplete VI, the chamber size should be adjusted to factor in the shortest residence time and desired safety margin.2 In addition to these design considerations, biomanufacturers will need to assess their process for minimum and maximum low pH exposure time, feed characteristics, and flow rate to define a chamber size that delivers the appropriate RT with a safety factor.iVI系统必须提供停留时间，以确保饲料在所需的培养时间内暴露于目标pH值。由于较低的培养时间存在VI不完整的风险，因此应调整腔室尺寸，以考虑最短的停留时间和所需的安全裕度。除了这些设计考虑因素外，生物制造商还需要评估其工艺的最小和最大低pH暴露时间，进料特性和流速，以确定可提供具有安全系数的适当RT的腔室尺寸。
VI VALIDATION IMPLEMENTATIONVI 验证实施
Although chamber design studies have established theoretical considerations for designing an iVI system, implementation in a manufacturing environment requires process-specific validation. With iVI, two possible strategies for validating efficacy of the VI process include:尽管腔室设计研究已经确立了设计iVI系统的理论考虑因素，但在制造环境中实施需要针对特定工艺的验证。对于iVI，验证VI过程有效性的两种可能策略包括：
Perform validation studies in the inline operating mode using a small-scale chamber the range of design and operating parameters of process scale. Data from spiking VI. System-induced virus loss from mechanisms such as shear or adsorption loss 使用小型腔室在在线操作模式下执行验证研究，该腔室的流体流动曲线相当于工艺规模声称的LRV可以完全归因于低pH值。
If the iVI process uses an incubation chamber with a flow profile that has been well combined with a knowledge of the experimentally-determined minimum residence timethe supplier to characterize the MRT for the range of conditions under which the work by the supplier to prove an equivalent LRV in iVI compared to static VI at the same exposure times.4 , 7 , 9如果iVI工艺使用的培养箱的流动曲线已得到很好的表征，则可以使用已建立的静态保持需要供应商进行初步工作，以证明在相同曝光时间内，iVI中的LRV与静态VI的等效性。
CONCLUSION结论
The biopharmaceutical industry is innovating to meet increasing production demands while assuring patient safety. Transitioning from batch manufacturing processes to continuous operating modes embodies this objective, enabling flexible and streamlined manufacturing. As a critical piece of the overall virus control strategy for biotherapeutic production, low pH virus inactivation can be successfully integrated into a continuous processing train by ensuring that the critical parameters of pH and incubation time are robustly controlled.生物制药行业正在不断创新，以满足不断增长的生产需求，同时确保患者安全。从批量生产流程过渡到连续操作模式体现了这一目标，实现了灵活和简化的制造。作为生物治疗药物生产整体病毒控制策略的关键部分，通过确保 pH 值和培养时间的关键参数得到稳健控制，可以将低 pH 病毒灭活成功集成到连续处理序列中。
Major challenges of implementing continuous processing operations include robust sensing and maintenance of operational requirements, managing heterogenous feed inputs, and process validation. Recent advances in incubation chamber design, along with an increased understanding of key considerations for robust design and control of iVI systems, are big steps toward facilitating the adoption of fully continuous biomanufacturing to improve patient access to life-saving therapies.实施连续生产的主要挑战包括对操作的可靠检测和维护、管理异构进料输入和工艺验证。培养室设计的最新进展，以及对iVI系统稳健设计和控制的关键考虑因素的更多理解，是促进采用完全连续生物药生产以改善患者获得救生疗法的重要一步。
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