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Advanced Strategies for Balancing Yield, Stability, and Quality in ADC Production

Antibody–drug conjugates (ADCs) are a now-proven therapeutic approach that has added several commercial
products to the oncologist’s toolbox. Behind the scenes, ADC manufacturing continues to progress through innovations in conjugation chemistry, analytical methods, and formulation strategies. This past summer, I spoke with experts from three contract development and manufacturing organizations (CDMOs) experienced with ADC manufacturing about how they’re tackling some of the many practical complexities of these technologies. In a virtual roundtable, these leaders share insights on scaling up ADC processes, balancing yield with consistent drug:antibody ratios (DARs), and optimizing product stability.

Their perspectives highlight critical process parameters (CPPs) requiring careful monitoring during scale-up, advanced analytical techniques for comprehensive characterization, and formulation strategies to prevent degradation. As ADC technology matures, manufacturers also are developing sophisticated approaches to address challenges with hydrophobic payloads and ensure product quality across manufacturing scales.

Srivats Rajagopal is senior director of biology for Aragen Life Sciences in Bengaluru, India. After obtaining chemistry bachelor’s and master’s degrees in India, he earned both an MS and a PhD in chemistry from the University of Arizona in 2007, then did postdoctoral work at Drexel University College of Medicine in Pennsylvania. Rajagopal spent 12 years at Syngene International, then worked for a brief period at Perfect Day (a dairy-protein biotechnology start-up) before taking on his current role at Aragen.

Sridevi Khambhampaty is chief executive officer (CEO) at Shilpa Biologics Pvt. Ltd., also in Bengaluru. She holds an MS in biotechnology from Jawaharlal Nehru University and a doctorate in biological sciences from the National Center for Biological Science at India’s Tata Institute of Fundamental Research. Khambhampaty served in a postdoctoral fellowship at Stanford University School of Medicine before embarking on her career in the industry. After nearly a decade with Dr. Reddy’s Laboratories (moving from senior scientist through associate directorships to senior director of biologics development), she served as vice president first at Intas Pharmaceuticals and then Syngene. She’s been CEO of Shilpa for about a year.

Conor Barry is global head of bioconjugate and biologics R&D and vice president of research, development, and manufacturing at Piramal Pharma in Oxford, UK. He holds a PhD in organic chemistry from the University of Bristol and did postdoctoral work in strategy and innovation at Saïd Business School, part of Oxford University. Barry has worked in both academia and industry, notably serving for five years in a liaison role between the two at Oxford. He was head of analytical sciences at Spirogen (AstraZeneca), then chief scientific officer at EnzBond Ltd. before joining Piramal as head of development in 2021.

Scaling Up ADC Processes

I began our conversation by asking which process parameters require the most careful monitoring and adjustment when scaling up ADC conjugation reactions. I also wanted to explore what instrumentation or control systems are most effective for maintaining those parameters consistently across scales.

Khambhampaty: “There is no ‘one size fits all’ process; optimized reaction parameters need to be developed for each antibody, drug-linker, and conjugation type (e.g., lysine-targeted amides or cysteine-targeted thiol-maleimide) to achieve targeted DAR profiles. Thus, careful monitoring of reaction time and agitation rate is essential. My company uses a mixing vessel that allows for precise control of agitation speed, reaction temperature, and real-time monitoring of reaction duration.”

Barry: “Even with a well-developed process, scaling up can be challenging. It requires the right mix of expertise and pragmatism. Temperature-related steps will take longer to complete when a process is scaled up, so it is important to conduct appropriate hold-time studies during process development. Scaling up to larger vessel sizes also requires careful consideration of mixing rates. A useful approach is to ensure that agitation power input will be maintained. My company approaches [scale-up] using in-house factors developed through experience across a range of different process equipment.”

Rajagopal: “When scaling up ADC reactions, multiple CPPs must be considered, monitored, and controlled properly to ensure product consistency, safety, and efficacy. These parameters include reaction temperature, buffer pH, mixing/agitation rates, reaction duration, solvent composition, and the ratios of antibodies to payloads and reductants (see box, right). In-line process analytical technology (PAT) for real-time monitoring of DAR, aggregation, and free drug; design of experiments (DoE) to identify optimal parameter ranges during scale-up; and centralized control and data-logging systems are highly desirable for achieving consistency across process scales.”

Next I asked what strategies companies can implement to balance the trade-off between manufacturing yield DAR consistency during scale-up, particularly when dealing with hydrophobic payloads that tend to promote aggregation at higher concentrations.

Barry: “Equipment limitations will come into play at some point in ADC manufacturing process scale-up. To overcome them, process intensification becomes the goal. Often such limitations hit first at the tangential-flow filtration (TFF) or chromatography steps. As TFF and column loadings increase, it is important to ensure that suitable hold-time limits are established to allow for the longer processing times that result. We must ensure that critical quality attributes (CQAs) such as monomer and free-drug content are maintained within specifications. Having robust process optimization and understanding of product stability profiles from the get-go provide a solid basis for derisking both scale-up and intensification.”

Khambhampaty: “A number of strategies help us balance yield and DAR consistency during scale-up for hydrophobic payloads. Optimized conjugation conditions help to control pH, solvent mixing, and reaction times to limit aggregation while achieving target DARs. Controlled payload addition through gradual feeding in a predissolved form will minimize localized aggregation in a reactor. Concentration and buffer management help to moderate protein concentration and stabilize excipients, further reducing aggregation risk. And optimized purification processes use chromatographic methods to control aggregates and DAR species.”

Rajagopal: “Dealing with hydrophobic payloads is one of the most difficult challenges in ADC process development because hydrophobic molecules can promote aggregation, reduce final yield, and compromise product quality. Successful processes require a multipronged strategy to balance manufacturing yield and obtain consistent DAR levels. During ADC process development at Aragen, we use preformulation screening for optimized payload solubilization, optimization of conjugation conditions, in-line PAT, and DoE as part of that strategy. We also combine batches with complementary DAR profiles to meet specifications without compromising yield. Additional methods such as surface-plasmon resonance (SPR) binding analysis, melting-temperature calculations, in vitro/in vivo functional testing, and pharmacokinetic/ pharmacodynamic (PK/PD) profiling also can help to ensure the ADC quality, DAR acceptance criteria, and functionality.

“Preformulation screening of solvents and micelle-forming agents (e.g., polysorbates) helps us optimize payload solubilization. Conjugation parameters that can be optimized include buffer conditions, temperature, pH, antibody/payload molar ratios, reaction duration, and the need for stabilizing agents (e.g., glycerol, histidine, arginine, and/or phosphates). In-line PAT tools help us detect turbidity and aggregation early, so we can use reaction quenchers to terminate reactions if needed. Our DoE can plan and conduct experiments to identify optimal parameter ranges for scaling up each process step. It all helps to produce high-quality ADC with both high-yield recoveries and desired DAR levels.

“To minimize the DAR variability, we prefer a site-specific conjugation strategy over nonspecific lysine conjugation reactions. Cysteine targeting, enzymatic conjugation (e.g., using transglutaminase, sortase, or SpyLigase), and N-glycan engineering are our preferred strategies. Thiomab engineering — genetically modifying antibodies to introduce a specific cysteine residue that enables site-specific conjugation — can incorporate multiple cysteine residues to meet specified DAR targets. And we can modify linker chemistry (e.g., multiarm linkers and dual-payload technology) for reactivity tuning to control conjugation kinetics.”

Analytical Methods

In-Process Testing: Product characterization is vitally important in process development, so analytical technologies are vital to success in ADC manufacturing. With that in mind, I asked what combination of in-process test methods provides for the most comprehensive real-time monitoring of conjugation efficiency and product-quality attributes. And I wondered where in a process such analyses should be implemented for optimal process control.

Khambhampaty: “Ultraviolet (UV) spectroscopy can be used for real-time DAR monitoring to track conjugation progress, providing data to guide us in quenching a reaction to achieve a desired DAR. We apply this kind of real-time monitoring to assess conjugation efficiency.”

Rajagopal: “During ADC process development, a multimodal analytical strategy is essential to achieving comprehensive real- time monitoring of conjugation efficiency and product-quality attributes for manufacturing. We integrate a different analytical strategy at each stage of our ADC workflow.

“In antibody preparation, we use UV–visible (UV-vis) spectroscopy for concentration determination and pH/conductivity meters to ensure buffer suitability. A TFF system is implemented for buffer exchange. For payload solubilization, UV-vis and liquid chromatography with mass spectrometry (LC-MS) characterization helps in payload identity verification, concentration determination, solvent screening, and so on.

“For conjugation reactions, we use dynamic light scattering (DLS) analysis and turbidity sensors to monitor aggregation during the process. In-line PAT (e.g., UV-vis and HPLC) helps us track conjugation kinetics, and combining in-line pH/temperature sensors with automated regulators manages optimal condition during the process.

“After conjugation and before purification, we use hydrophobic- interaction (HI) or reversed-phase (RP) HPLC for DAR distribution analysis, size-exclusion (SE) HPLC for aggregation detection, and LC-MS analysis for DAR analysis and conjugation-site identification. In purification development, we use SEC and TFF techniques for removal of unconjugated payloads. UV-vis helps us monitor recovery and concentration, sodium dodecyl-sulfate– polyacrylamide gel electrophoresis (SDS-PAGE) and SE-HPLC provide information about purity and aggregates, and HI/RP-HPLC and LC-MS measure DAR distribution.

“Apart from that stepwise strategy during development, real-time process control in manufacturing depends on integrated, centralized data acquisition and control systems (SCADA/DCS) with feedback loops for adjusting feed rates, temperature, and pH. Each step of the process is crucial, and all CQAs and CPPs need to be monitored throughout.”

Advanced Characterization Techniques

Analytical technologies continue to advance along with biological science and instrumental engineering. Next-generation analytical techniques such as native mass spectrometry and multiattribute monitoring (MAM) are making inroads for many biologics, so I asked whether they offer promise in characterizing ADCs beyond traditional DAR analysis. How might the resulting insights inform a manufacturer’s decision-making in process optimization?

Khambhampaty: “During optimization, we use the LC/MS method to assess DAR distribution patterns and as a characterization technique for ADC molecules. UV spectroscopy provides a population-averaged DAR showing the complete heterogeneity of conjugated molecules as well as the DAR distribution. Monitoring shifts in the latter parameter during process optimization is valuable for understanding the critical limits of different process parameters of a conjugation reaction.”

Rajagopal: “At Aragen, we typically use both intact and reduced LC-MS for DAR analysis as well as peptide mapping to identify conjugation sites, SDS capillary electrophoresis (CE) for fragment analysis, and SE-HPLC for monomer purity.

“Native mass spectrometry (nMS) is increasingly useful. In this technique, a sample is ionized in the gas phase by preserving noncovalent interactions, which helps us investigate protein stoichiometry and interactions with ligands and other proteins. Native MS can identify preferred conjugation sites and elucidate the impact of conjugation on antibody stability. Such information can be used further for linker–payload design and minimizing off-target conjugation. Native MS also resolves intact ADC species with different DAR and thus is helpful for detecting low-abundant species affecting product efficacy. That information is useful in guiding further purification development to remove those low-abundant species.

“In MAM with LC-MS, we can analyze multiple CQAs (e.g., DAR, oxidation, deamidation, and glycosylation) of mAbs and ADCs at once. All those attributes are critical for tracking product consistency across batches, so MAM helps to shorten quality control (QC) turnaround times.”

Stability Optimization

Excipient Selection: Turning to the topic of product stability, I asked about formulation excipients. Which such ingredients are most effective in preventing payload hydrolysis and protein aggregation during long-term ADC storage? And how do companies optimize their concentrations?

Barry: “If you look at the formulation compositions of the ADCs approved for market so far, a number of excipients and properties are common among them. That being said, the individual formulation compositions are actually quite diverse in how they combine those excipients — similar to what is seen now in clinical development, where the design spaces for formulations are broadly defined. However, certain conditions and excipients perform better with certain bioconjugate and payload–linker formats. At Piramal, we apply an initial high-throughput screening of a focused panel of excipients and buffer formats based on our experience of developing lyophilization-compatible formulations. Using that approach, we typically can identify good prospective formulations in a single round of screening.”

Khambhampaty: “Key excipients for ADC stability, after appropriate buffers are selected for pH control, include trehalose and/or sucrose to prevent aggregation, polysorbate 20/80 to reduce surface adsorption, arginine to suppress aggregation, and ethylenediaminetetraacetic acid (EDTA) and/or methionine to prevent hydrolysis and oxidation. Optimization often depends on linker type; for example, maleimide requires an acidic environment. Lyophilization is the preferred formulation strategy for ADCs due to its ability to preserve conjugate integrity and minimize premature release of payloads during storage.”

Rajagopal: “Long-term storage of ADCs is difficult and requires formulations tailored to linker chemistry, payload hydrophobicity, and mAb behavior. Some commonly used excipients include histidine to stabilize antibodies and linkers; citrate to prevent metal-ion–dependent hydrolysis of payloads; sugars and polyols to prevent aggregation and stabilize both mAbs and hydrophobic payloads in lyophilized formulations; surfactants to prevent aggregation and surface adsorption; chelators to prevent metal-ion–catalyzed hydrolysis of maleimide linkers; amino acids (e.g., l-arginine and l-glycine to prevent aggregation, reduce viscosity, and enhance solubility and stability; and antioxidants (e.g., methionine and ascorbic acid to scavenge reactive oxygen species and improve stability. We combine defined amounts of those excipients in a final formulation buffer. In development, we’ll run DoE formulation screening and stability studies under different excipient concentration conditions to determine the best combination for a given linker, payload, and antibody.”

Process-Induced Degradation: Turning back to process optimization, I asked about stress-induced degradation pathways that can affect ADC stability and efficacy. What modifications to conjugation and purification processes can minimize them?

Barry: “In-process degradation is typically a manifestation of inherent product-specific degradation pathways. Understanding your product from a developability perspective — as early as possible — is key to cutting off those pathways. Thus, we can design processes to limit such degradation. For example, if a product is known to aggregate at certain pH conditions, then the process can be designed in such a way as to prevent those unfavorable conditions. That might involve compromising on lower reactivity rates in one process step (supported by hold-time data) to allow us to use milder pH conditions.”

Khambhampaty: “Stabilizers can be used in the conjugation process to minimize stress-induced degradation. Furthermore, effective optimization (typically using a DoE study) focuses not only on correct DARs, but also on preserving other molecular quality attributes such as purity. Optimization also ensures that fragmentation and instability are minimized and controlled. By applying an appropriately optimized purification process, we can remove unwanted degraded products.”

Rajagopal: “Both the ADC conjugation reaction and purification steps, when performed under suboptimal conditions, can lead to product degradation by inducing methionine oxidation, asparagine deamidation, and disulfide scrambling. These stress-induced degradation pathways negatively impact ADC efficacy. Another aspect of methionine oxidation arises within antibodies from enzymatic conjugation using N-glycan engineering of the N297 residue within the crystallizable fragment (Fc) domain and the N-glycan on that residue. Oxidation from NaIO₄ can broaden DAR value distribution due to low antibody conversion.

“We need to perform extensive process optimization runs under different conditions to minimize stress induced by buffer components. Example solutions to the problem include adding antioxidants or using only mildly oxidating buffers; minimizing light exposure; controlling temperature and pH; limiting shear during mixing; adding stabilizing agents; and using a controlled amount of reducing agent or removing it completely after the reduction step with a rapid purification protocol. Stress-induced degradation can be monitored with in-line PAT tools, MAM, LC-MS, and SE-HPLC.”

Site Specificity for Conjugation

Finally, I asked the experts to compare new enzymatic conjugation methods with the more established engineered-cysteine methods in terms of manufacturing robustness, scalability, and product quality.

Khambhampaty: “There are pros and cons for each. Enzymatic conjugation (e.g., using transglutaminase or sortase) offers very high site specificity and homogeneity (typically a DAR of 2), but the process is only moderately robust, with limited scalability due to enzyme cost and complexity. Engineered cysteine conjugation provides high homogeneity in a robust and scalable process used in some commercial ADCs. Typically, the native mAb cysteines have been used, so molecular instability is less of a concern in such cases. However, Genentech (Roche) results have shown that stability of the resulting ADCs can depend on mutations in the heavy or light chain. DAR variability is more of an issue here than it is for enzymatic conjugation.

“Basically, enzymatic methods offer better precision, and cysteine- based conjugation methods are more scalable and manufacturing- friendly. Note that all approved ADCs currently are generated by traditional (cysteine- or lysine-based) conjugation methods that are not site specific but have provided consistent DARs and composition profiles. Thus, enzyme-based conjugation may be overrated.”

Rajagopal: “Enzymatic conjugation and cysteine methods offer distinct advantages and trade-offs in terms of manufacturing robustness, scalability, and final product homogeneity. Both conjugation strategies are site specific. However, enzymatic conjugation requires production of highly efficient and pure enzymes. Inefficiencies can lead to compromised ADC batch consistency. Also, enzymatic conjugation requires additional purification steps after conjugation. Engineered-cysteine conjugation is a simpler approach with higher batch consistency.

“Engineered-cysteine methods also are more scalable due to their more straightforward chemistry and fewer dependencies. Enzymatic methods can be scaled but require additional process development steps. Engineered-cysteine methods can be scaled up rapidly and are cost-efficient; enzymatic methods have complex linker-payload chemistry.

“Enzymatic methods do offer superior homogeneity and site specificity, leading to tightly controlled DARs, which is ideal for regulatory and clinical consistency. Engineered-cysteine methods are slightly more variable but still highly controlled and consistent.”

Driving Toward Efficiency

The evolution of ADC manufacturing reflects a growing sophistication in balancing precision with scalability. Although traditional conjugation methods remain the foundation of approved products, enzymatic approaches could offer enhanced site specificity — but for now they come with scalability issues that remain to be addressed. Experts emphasize multimodal analytical strategies including advanced methods and automation as essential for comprehensive process control. Formulations are optimized using tailored excipient combinations to address stability challenges during shipping and storage. As the field advances, manufacturers continue to refine their approaches to quality and process parameters, implementing real-time monitoring systems and developing robust purification strategies to ensure consistent DARs while maximizing product yields. Such improvements will drive the next generation of not only effective, but also manufacturable and cost-efficient ADC therapeutics.

Sourse – BioProcess International