Antibody-drug conjugates (ADCs) represent a novel and promising class of cancer therapeutics. ADCs consist of three components: a tumor antigen-specific monoclonal antibody (mAb), a linker, and a cytotoxic payload. The antibody component facilitates the targeted delivery of cytotoxic drugs that are often unsuitable for patient treatment due to their narrow therapeutic window. Highly effective drugs must be conjugated to the antibody without compromising its biological properties. The significant differences in the biophysical characteristics of antibodies compared to cytotoxic payloads, such as size and hydrophobicity, present considerable challenges in the production of ADCs. Antibodies require careful handling, as they are sensitive to temperature fluctuations and high stirring rates. Additionally, cytotoxic drugs are frequently very hydrophobic, necessitating the use of organic cosolvents that are compatible with the antibody component.

Typically, the procedures for synthesizing new and promising ADCs are developed during the drug discovery phase. The importance of process development becomes evident in the later stages of a project. Establishing key process parameters before producing materials for toxicology and clinical studies can significantly reduce the risks associated with implementing process changes later in the product lifecycle. This approach ensures that materials of consistent quality are utilized at all stages.

When designing a process development plan, it is essential to clearly define the development goals. One primary objective is to ensure a reliable and safe process that integrates seamlessly with existing production facilities. Thorough control of ADC properties at the molecular level throughout the development phase is crucial for establishing a dependable and robust process. Key properties, such as drug-antibody ratio (DAR), monomer content, and attachment sites, must be meticulously controlled during the process. Furthermore, these analytically traceable characteristics can serve as critical indicators of product quality. Additionally, the ADC must exhibit biological activity and be recognized by the target antigen.

Typically, the development program begins with a familiarization phase to test and evaluate an initial set of process parameters, which are subsequently refined during the actual process development phase. In this phase, various process parameters are examined in detail, often utilizing a Design of Experiments (DoE) approach. The next step involves validating the optimized process parameters through multiple validation runs. If a representative scale-down model is available, all of this can be conducted at the milligram scale. Following this, the first scale-up of the antibody starting material to the gram scale can be attempted, and purification steps can be investigated. Subsequently, the process can typically be scaled up to over 100 grams to support toxicology and early clinical studies. As the process progresses toward commercialization, a comprehensive process characterization phase is essential for ADC production to meet all regulatory requirements, resulting in a well-characterized process that can be executed at a scale of 1 kilogram or higher.

Process Scale-Up

After identifying the critical parameters and defining and validating their set points to achieve the desired product quality, the process is ready for scale-up from milligram to gram scale. Consequently, additional data on process robustness and scalability must be obtained. At this stage, purification technology will be employed for the preparation of clinical material. Scalable technologies, such as tangential flow filtration (TFF), are typically preferred. TFF is a method used for concentrating and purifying proteins. It facilitates efficient buffer exchange and the clearance of small molecule impurities, which are often necessary during antibody modification. In particular, the removal of free drug derivatives is crucial for ADC processes to prevent any unconjugated residues of highly toxic drugs in high-dose ADC final products. The process solution volumes at the milligram scale are usually too small to implement a technology like TFF; however, the conjugation volumes at the gram scale are sufficiently large to effectively screen TFF parameters.

In a TFF system, the selection of operating parameters significantly influences process performance. In a coupled process, the objectives of the TFF step include optimizing flux, achieving efficient buffer exchange, and enhancing impurity clearance. To ensure reproducible and optimized process throughput and duration, various membrane types and materials can be evaluated, and key TFF parameters can be optimized early in the process development phase. To rapidly optimize these parameters, different feed flow rates can be tested while varying the transmembrane pressure (TMP) to gather actionable data that maximizes throughput. Higher throughputs are often desirable, as they facilitate shorter TFF processing times. Once the optimal TMP and feed/recirculation flow rates have been established, the impact of protein concentration on TFF performance can be examined. To determine the number of diafiltration volumes required in the TFF step, analytical methods are necessary to quantify the amount of target impurities. By analyzing samples collected from the recycle tank, the reduction in impurity levels can be monitored, allowing for the determination of the required number of diafiltration volumes for the process. The selected buffer volumes must ensure that the concentrations of these species remain below a specified threshold.

The objective of developing TFF purification is to identify the appropriate membrane type, feed/recirculation flow rates, TMP, and the number of diafiltration volumes. If the development indicates that TFF is not a suitable technology for removing impurities from the ADC process, or if it adversely affects product attributes to an unacceptable degree, alternative strategies such as chromatography should be considered. Generally, chromatographic purification is unnecessary unless protein aggregation cannot be controlled during the conjugation process, which is advantageous for both yield and product cost.

Clinical Material Supply Considerations

When manufacturing materials for toxicology or clinical studies, a robust bioburden control strategy must be implemented throughout the entire process. Bioburden control begins with the selection of high-quality raw material suppliers and the execution of rigorous release testing at the ADC manufacturing site. Choosing appropriate manufacturing equipment is essential to ensure that operations remain as closed and sterile as possible. Additionally, providing comprehensive bioburden awareness training for plant operators further minimizes the risk of contamination in ADC batches. Monitoring endotoxin levels and bioburden throughout the process via process control can yield valuable data and insights into potential sources of contamination. Furthermore, defining bioburden reduction measures within the process is critical to mitigating unnecessary risks.

In the ADC process flow, the naked antibody is initially transferred through a filter into a closed reaction vessel. If the reaction time is extended, additional in-process filtration may be necessary. A final filtration will be conducted before the formulated ADC solution is stored. Furthermore, buffers may be filtered after preparation and/or at the point of use, and the acceptable storage duration and temperature for these solutions must be rigorously assessed. After establishing the process parameters and purification development, the production concept is defined to scale up the process to tens of grams, ensuring that the production materials can support toxicology studies and early clinical phases. The product quality of different batches should be consistent with that previously achieved in the laboratory.

Challenges in Developing a Commercial Process

When a process enters the clinical phase, it must be further characterized to ultimately achieve validation, which is essential for producing ADCs for commercial supply. During the process design phase, the parameters of the future commercial process are defined based on insights gained from additional laboratory experiments and scale-up activities related to early clinical phase ADC production. At this stage of the process lifecycle, employing DoE methods enables the acquisition of in-depth process knowledge through a relatively limited number of experiments.

After identifying important process parameters in the early stages through an initial factor screening DoE to ensure the desired product quality, these factors can be further optimized during the phase of a more comprehensive DoE. If desired, the curvature of the output observed in the initial screening DoE can be further examined using response surface models for nonlinear systems. This approach allows for the development of a model that accurately describes the behavior of the conjugation process and visualizes the interactions among all relevant variables. Consequently, a design space can be defined for the process parameters. Within the boundaries of this design space, the process performs as expected. This can be validated through additional experiments that demonstrate the robustness of the conjugation process. These studies will facilitate the identification of critical process parameters (CPPs) and the establishment of acceptable ranges for all relevant process parameters that must be adhered to in order to achieve consistent quality production for each batch of ADC.

Since changes in commercial manufacturing, compared to early clinical stages, typically involve further scale-up, any potential impact on product quality must be considered. Factors such as power input through stirring, the tip speed of the impeller, and the duration of homogenization when reagents are added are variables that are often challenging to maintain at a constant level. Therefore, it is essential to ensure that the selected values for these variables do not adversely affect any ADC properties. If the feed contact material changes during the scale-up process, additional laboratory investigations may be necessary to demonstrate its compatibility with the process fluid stream. Furthermore, filter size testing experiments are crucial for selecting the appropriate filter size for the commercial process. With all the information collected and the in-depth knowledge gained through production, ADC products for commercial applications can be produced more effectively and safely on a large scale.

Duoning Biotech offers specialized bioprocessing technologies for ADC products, addressing the requirements of various stages from process development to commercial production. We provide a comprehensive single-use product supply platform, which includes liquid storage equipment and associated fluid management solutions, ranging from laboratory to large-scale production. Additionally, we can verify material chemical compatibility and assess extractables/leachables for specific organic solvents utilized in the process.

Duoning DuoMix®/Mini DuoMix® mixing systems support a wide range of applications, from 2-20 L benchtop setups to 3,000 L production-scale operations. This series features powerful mixing capabilities that can quickly achieve homogenization. Additionally, the system offers various stirring impeller designs, and its optimized configuration minimizes shear stress on the target product. Beyond basic functions such as stirring and weighing, the system can be equipped with monitoring elements for pH, temperature, and conductivity, as well as auxiliary peristaltic pumps. The DuoMix®/Mini DuoMix® series is designed with single-use mixing bags made from Duofilm® 001, which can be customized to fit the specific hardware system or process being utilized. This customization ensures that application requirements are met effectively, including buffer preparation and pH adjustment, bulk homogenization in chromatography, virus filtration, serving as a recirculation vessel in tangential flow filtration, and functioning as a conjugation reaction vessel under specific conditions.

Mini DuoMix® single-use benchtop mixer.

To better support end users in process development and scale-up, and to ensure that the mixing time, shear stress, and other conditions of the selected equipment meet product and application requirements, we can provide targeted application cases and computational fluid dynamics (CFD) data for the equipment. This will make your work easier and more efficient.

CFD simulation diagram of 2,000 L mixing equipment. For more detailed information, please contact our technical support team.

Due to the presence of small molecule toxins, cleaning validation can pose challenges in the use of process equipment, particularly purification equipment. To address this issue, Duoning offers single-use flow paths, including single-use insert plates, for ultrafiltration, which prevent contact between the drug solution and the holder, thereby simplifying the cleaning validation process. Additionally, Duoning provides a complete set of tubing assemblies designed for use with single-use ultrafiltration and chromatography systems, ensuring that the entire liquid contact flow path for the purification process is ready-to-use and disposable. This approach enhances the safety of personnel and the environment.

Schematic Diagram of Single-Use Ultrafiltration Tube Set and Single-Use Chromatography Tube Set

At the same time, we provide comprehensive filtration solutions. Our offerings encompass cartridge filters, disc filters, capsule filters, and hollow fiber filter modules made from various membrane materials. Additionally, for the downstream purification of ADC, our chromatography resins, which are based on different working modes, can be used in combination. These modes include anion/cation exchange and hydrophobic interaction chromatography resins. This product series employs a specialized matrix and modification technology platform to ensure the robustness and repeatability of the chromatography process.