Aggregation in antibody-drug conjugates: causes and mitigation

Advancing antibody-drug conjugate (ADC) candidates to the point of clinical trials, full-scale GMP manufacturing and commercialization can be a slow process. ADCs are more complex than traditional monoclonal antibodies (mAbs) and this complexity introduces new challenges when defining, optimizing and assuring the critical quality attributes (CQAs) of the final drug. These challenges include protein aggregation – the clustering of ADC molecules – which can impact the compound’s stability, efficacy and safety, creating significant barriers in both drug and process development. Protein aggregation stems primarily from the conformational and/or colloidal instability of a molecule.¹ Conformational instability can result in the formation of globally or partially unfolded states or a near-native state, whereas colloidal instability is the result of self-interaction between native, near-native, partially unfolded or unfolded states.

Fig 1. A typical ADC structure

Causes of aggregation in ADC molecules

There are numerous factors that can contribute to molecular instability and consequently to aggregation, with each requiring specific control measures.

Physicochemical properties of ADC components

The choice of antibody used – multispecific, bispecific or fragment – can impact the degree of aggregation seen in the final ADC product. Constructs that improve drug specificity often show a higher propensity to aggregation, complicating their characterization and formulation.² In contrast, smaller antibody fragments reduce the aggregation risk.³ Modification of an antibody through the attachment to hydrophobic or lipophilic payloads and linkers can increase the likelihood of aggregates that form to minimize their exposure of these species to the aqueous environment. These aggregate nuclei may then continue to grow and develop into higher molecular weight aggregates with reduced solubility, ultimately forming a precipitate^.4–6 The attachment of lipophilic linkers can also induce conformational changes in the antibody by exposing hydrophobic regions that are normally buried within its structure. This can modify the natural protein-protein interaction dynamics and promote aggregation. Drugs with higher drug-antibody ratios (DARs) frequently show increased aggregation rates and precipitation during formulation, and decreased in vivo efficacy.^7,8

Manufacturing conditions

In general, manufacturing conditions are optimized for conjugation chemistry, not for the stability of the mAb used in an ADC. For instance, higher concentrations, often preferred for more efficient manufacturing processes, can also increase the likelihood of aggregation, as they increase the probability of molecular interaction and clustering.⁹ ADCs are also often produced and stored using buffers that may not promote stability, and solvents used during conjugation to solubilize highly hydrophobic and poorly soluble payload-linkers can disrupt the extensive network of intramolecular and intermolecular bonds, leading to the formation of aggregates. The phase behavior of supersaturated aqueous drug solutions can also lead to the formation of colloidal drug aggregates through liquid-liquid phase separation, which is a precursor to crystallization.¹⁰

Shear stress caused by mixing during conjugation, and thermal stress caused by elevated reaction temperatures, can also lead to antibody denaturation, exposing hydrophobic regions that then promote intermolecular interactions and aggregation.⁹ Interestingly, shear stress-induced aggregates caused by mixing have been seen to promote higher internalization and receptor activation rates compared to thermal stress-induced aggregates.⁵ The mechanism behind this is, as yet, poorly understood, and highlights the need for further study.

Storage conditions

The storage and transportation of ADC formulations under conditions that can accelerate product degradation, such as exposure to thermal stress and shaking during transportation, may also lead to a higher aggregation propensity in certain compounds.^5,8 Even light exposure can excite the photosensitive functional groups contained in some payloads, triggering drug and protein degradation, and consequently causing the aggregation of ADC molecules.¹¹

Cause of aggregation	Examples
Physicochemical properties of ADC components	Antibody type Surface modifications DAR
Manufacturing conditions	ADC concentration Buffer Solvents Shear stress Thermal stress
Storage conditions	Thermal stress Shaking during transportation Light exposure

 

Table 1. An overview of the multiple factors that may lead to ADC aggregation during and after formulation.

Implications of aggregation

The degree of aggregation can have an impact on various characteristics of the final therapeutic product, including efficacy, safety and stability. Remediation via aggregate removal may only be a partial fix and can impact the financial viability of the manufacturing process by reducing yield. Prevention is therefore preferable to removal, but elements of both may be necessary.

Efficacy

Aggregates have altered pharmacokinetics and biodistribution compared to a non-aggregated formulation. They may consequently be less target specific and can lead to narrower therapeutic windows due to faster plasma clearance. These factors reduce the cytotoxicity of ADCs to target cancer cells, hindering their therapeutic efficacy.^5–7

Safety

Several studies have found that ADC aggregates activate surface receptors, such as FcγR, on immune cells, eliciting immunogenic reactions and causing the ADC molecules to be internalized.⁵ Internalization into target-negative cells causes off-target toxicity and an increase in adverse side effects. Aggregation can also lower the solubility of ADCs and may inhibit the body’s specific clearance mechanisms, leading to an accumulation of the drug in the kidneys or liver. This may result in the non-specific killing of healthy cells in these organs, increasing the toxicity of the ADC.

Stability

The formation of ADC aggregates alters the physicochemical properties and degradation profiles of mAbs and creates a heterogeneous drug load distribution.¹² Product instability can reduce the shelf life of ADC drugs and complicate regulatory approval.

Process economics

Aggregation leads to the precipitation of the ADC, and the resulting precipitates require removal via chromatography or filtration. These processing steps can significantly add to the cost and time of manufacture but, more importantly, reduce the overall ADC yield, and any loss in yield impacts the economic viability of manufacturing.¹³

How to quantify aggregation

Detecting ADC aggregates during early process development can direct adjustments during later development. These can help to prevent the loss of product and enhance drug efficacy and safety during clinical trials and throughout the lifecycle of the drug. ADC characterization also reveals the stability of the formulation and helps define the drug quality. Several analytical methods can be used – either separately or in combination – to detect the presence and extent of aggregation, and to characterize these complex molecules at various stages of the development process. Each technique has its own set of advantages and disadvantages, all of which must be taken into consideration during study design.

Size exclusion chromatography (SEC)

SEC is the current industry workhorse for ADC aggregate assessment, quantifying different species based on differences in their hydrodynamic volumes. Advanced SEC methods, such as ultra-HPLC-SEC, SEC coupled with mass spectroscopy (SEC-MS) and SEC coupled with multiangle light scattering (SEC-MALS), have improved the analysis of protein aggregation in ADCs. SEC-MALS allows the determination of molecular weight and size distribution, which are critical for assessing aggregation levels. SEC-MALS can also detect changes in ADC formulations under different stress conditions, such as pH changes and temperature fluctuations. By monitoring these changes, researchers can gain greater insights into the stability and shelf life of ADC formulations compared to other analytical methods, such as sedimentation velocity analytical ultracentrifugation (SV-AUC).

SEC also enables high throughput testing with low material requirements. However, differences in the mobile phase required for the separation from the formulation buffer and dilution by the mobile phase may alter non-specific interactions and promote the formation of new aggregates. A typical SEC column may also be unable to resolve larger aggregates and can lead to inaccurate quantitation and characterization. As a consequence, SEC is frequently used in conjunction with flow or light scattering techniques or AUC.¹⁴

Flow imaging

Flow imaging techniques, such as macro-attenuated total reflection Fourier transform infrared (macro-ATR-FTIR) spectroscopic imaging and extensional and shear flow devices, can also be used to quantify the aggregation of ADCs. These methods provide valuable insights into the structural integrity and aggregation propensity of therapeutic proteins under the conditions encountered during bioprocessing and storage. The technique subjects proteins to pre-defined hydrodynamic forces that mimic real-world scenarios and can offer a sensitive approach to assessing the impact of different factors, such as temperature and stabilizing agents. Microflow-LC/high resolution MS (microflow LC-HRMS) can help better understand the pharmacokinetics and pharmacodynamics of ADCs in complex formulations. This type of flow imaging can be particularly useful to analyze the aggregation behaviors of mAbs and ADC molecules when under stress conditions, helping to prevent aggregation.

Dynamic light scattering (DLS)

DLS is a well-established technique for estimating the average size and size distribution of ADC aggregates and overall sample stability. Tracking size distributions over time using DLS can help reveal the onset and rate of aggregation.¹⁵ Researchers have used DLS with a machine learning algorithm (DLS-ML) and regression to model and predict the possibility of aggregation and the likely size distribution of aggregated particles in an ADC formulation.¹⁶ This rapid technique reduces the per sample cost of analysis, time of data acquisition and volume requirements of aggregation analysis, and is often used as an orthogonal method to SEC.

Polarized excitation emission matrix (pEEM) spectroscopy

pEEM spectroscopy is a sensitive, nondestructive and potentially fast technique for assessing aggregation and DAR in a single measurement. It provides Rayleigh scatter values for identifying aggregate/particle formation and fluorescence emission values to assess chemical and structural changes induced by conjugation. pEEM spectroscopy can also be closely correlated with size parameters obtained from DLS, highlighting its effectiveness in assessing ADC aggregates during manufacturing.¹⁷ This indicates the potential value of combining the two techniques to create a single measurement method that can assess multiple attributes of ADCs, such as size and aggregation.

Analytical ultracentrifugation (AUC)

AUC is a highly sensitive and powerful technique widely used in both industry and academia for the characterization and quantitation of molecular species, particularly for detecting the aggregation of ADCs in formulations. This method is especially sensitive to changes in molecular weight, making it an invaluable tool for identifying and quantifying aggregates. By comparing the sedimentation profiles of the ADC molecules, researchers can accurately quantify the extent of aggregation and gain crucial insights into the stability and quality of the formulation.

A variation on this technique, sedimentation equilibrium AUC (SE-AUC), focuses on quantifying the equilibrium concentration distribution of macromolecules, and provides thermodynamic information such as solution molecular mass, association constants, stoichiometries and solution non-ideality. However, another variation, sedimentation velocity AUC (SV-AUC), is often preferred for studying biotherapeutics such as ADCs despite having a limited dynamic detection range and requiring skilled analysts for accurate interpretation. The technique can monitor the movement of molecules over time, offering real-time insights into their size, shape and aggregation state. SV-AUC therefore enables the rapid and sensitive identification of even low levels of aggregation, providing a more comprehensive view of molecular weight distribution than other methods, for example SEC and DLS.¹⁴ SV-AUC therefore remains a cornerstone of research and industry for ensuring the quality and stability of therapeutic products.¹⁴

Imaged capillary isoelectric focusing (icIEF)

icIEF can be used to characterize heterogeneity in the charge isoforms of ADCs. Aggregation can often lead to changes in charge profile, and this information is crucial for detecting and quantifying the degree of aggregation in a formulation.¹⁸ icIEF can be coupled with high resolution mass spectroscopy (icIEF-HRMS) to provide a more comprehensive understanding of structural modifications that impact protein charge heterogeneity. This enables the detection and assessment of even trace levels of aggregation in complex ADC formulations.¹⁸ However, the preparation of samples for icIEF can be complex and time consuming, and the technique may not provide comprehensive information about the specific structural characteristics of aggregates, such as their molecular size or morphology. In addition, where multiple charged species are present, overlapping signals can complicate the interpretation of results, making it challenging to distinguish between different forms of aggregation using icIEF alone.¹⁹

Liquid chromatography-mass spectrometry (LC-MS)

LC-MS combines the separation capabilities of LC with the mass analysis capabilities of MS, providing detailed qualitative and quantitative insights into the composition, structure and aggregation state of ADCs.²⁰ Depending on the type of chromatography used, the primary separation is typically based on properties like size, charge or hydrophobicity. SEC is the most common choice as it allows monomers, dimers and higher order aggregates of ADCs to be resolved. Comparing the mass spectra of the different species provides data on the number of monomer units and any associated modifications, such as glycosylation or drug conjugation patterns, making it possible to identify and characterize aggregates and aggregation states within the ADC formulation. The relative abundance of different species can also be quantified based on the MS signal intensity, allowing the assessment of the extent of aggregation in the formulation. This quantitative data is critical for evaluating the stability of the ADC and assessing its suitability as a therapeutic product.

LC-MS is a highly sensitive and specific technique and it is able to detect and quantify very low levels of aggregated species in ADC formulations that might be missed by other methods. This detailed aggregation profiling helps to optimize formulations to enhance the shelf life and therapeutic effectiveness of the final product. The technique's robustness and high sensitivity make it ideal for monitoring pharmacokinetic parameters in complex formulations, even for low dose, highly potent drugs.

Capillary electrophoresis sodium dodecyl sulphate (CE-SDS)

CE is a highly efficient separation technique that resolves ions based on their electrophoretic mobility, making it suitable for characterizing biotherapeutics like ADCs.²¹ Combining CE with SDS can separate proteins based on their size and charge, which, in turn, allows the differentiation of monomeric forms and aggregated species.²² The presence of aggregates can be identified as additional peaks in an electropherogram, which can be quantified against the monomer peak to providing the quantitative analysis that is vital for regulatory compliance.

The CE-SDS technique involves preparing samples under both reduced and non-reduced conditions. Reduced conditions allow a clearer view of the protein's subunits and aggregates, whereas non-reduced conditions maintain the native structure, which is essential for understanding the aggregation state in its functional form. CE-SDS is often used in conjunction with other techniques, such as icIEF, which provides additional insights into charge heterogeneity, for more comprehensive characterization. It can also be paired with SE-HPLC to monitor low molecular weight protein aggregation.

A particular advantage of CE-SDS is its high sensitivity and resolution, which allows the effective separation and detection of protein aggregates that may not be adequately resolved using other techniques alone. Other strengths include the method’s ability to provide detailed insights into protein behavior under various formulation conditions, as well as its reduced sample preparation time and potential for analyzing multiple samples simultaneously.

Aggregate mitigation and solutions

Both the intrinsic protein properties of ADCs and the extrinsic solution characteristics of their manufacture must be regulated in order to keep proteins in their native states and minimize aggregation.¹ The prevention and reduction of aggregation during ADC manufacture therefore requires a holistic design of experiment (DoE) approach, comprising a suite of control measures at each step of the process. The primary goal is to optimize the manufacturing process by identifying the relationships between various input variables and the resulting output responses. The bullets below outline the major steps involved in a DoE approach to aggregation mitigation.

1. Define objectives: clearly state the goals of the experiment, such as minimizing aggregation.
2. Identify CQAs: determine the key properties of the ADC that need to be controlled to ensure product quality, such as potency, purity, stability and safety.
3. Select factors and levels: identify the input variables (factors) that are expected to influence the CQAs. These factors could include temperature, pH, concentration of reactants, types of linkers and reaction time. Define the levels of each factor to be tested.
4. Choose experimental design: select an appropriate experimental design based on the number of factors and levels. For example, full factorial design, fractional factorial design and response surface methodology.
5. Conduct experiments: perform the experiments according to the chosen design under controlled conditions.
6. Collect and analyze data: measure the output responses (CQAs) for each experiment. Use statistical methods to analyze the data and determine the main effects, interactions and optimal conditions for each factor.
7. Develop a model: establish a mathematical model that describes the relationships between the factors and the responses to better understand and predict the outcomes under different conditions.
8. Optimize and validate: use the models to identify the optimal conditions for the manufacturing process. Validate the findings by conducting additional experiments or scale-up studies to confirm that the optimized conditions lead to the desired product quality.
9. Implement and control: implement the optimized conditions in the manufacturing process. Establish control strategies to monitor and maintain the process within the identified optimal range to ensure consistent product quality.

ADC components

Aggregation-prone regions within mAbs are believed to contribute to thermodynamic stability and are often located in the antigen binding region, which is essential for effective binding to the target antigen. Recently, research has outlined a novel approach where variants of mAbs can be rationally designed to reduce their aggregation propensity by introducing artificial aggregation gatekeeper residues.²³ These residues allow the antibodies to maintain their binding capabilities while decreasing aggregation, consequently improving ADC formulation stability.

Modulation of antibody hydrophobicity by using hydrophilic linkers also reduces the likelihood of aggregation between antibodies. The use of hydrophilic linkers containing polyethylene glycol (PEG) groups, pyrophosphate diester groups or negatively charged sulfonate groups reduce the likelihood of ADC aggregation.⁶ Alternatively, inserting hydrophilic spacers – such as PEG and cyclodextrins – into the linker can also counteract the hydrophobicity of an ADC molecule and improve solubility, preventing aggregation and boosting the drug performance. Multiple active cytotoxic molecules can then be loaded onto each mAb without triggering the antibody aggregation usually associated with higher DARs._⁷

Glycosylation has also been found to stabilize proteins and protect against aggregation through steric hindrance, reduced structural dynamics and effective moisture management.^8,24 The attached glycans create a physical barrier around the protein, preventing the molecules from coming too close to each other, effectively reducing the likelihood of aggregation. In addition, the use of glycoside payloads in ADCs contributes to their hydrophilicity, reducing aggregation and improving stability. This increases the stability and safety of the ADC to non-target cells, and significantly improves cytotoxicity by enabling bystander efficacy, leading to improved performance against target antigen-expressing tumors.

Higher DARs typically lead to greater aggregation rates, and developing an ADC ideally delivers a careful balance that can maximize drug efficacy and minimize the likelihood of aggregation.⁷ Payload design also has a significant impact on aggregation, and analysis has indicated that novel hydrophilic payloads enable the synthesis of ADCs with high DARs that are resistant to aggregation.⁴ Another major focus of research in this area is the effect of cross-linking in the payload. Cross-linking can lead to unwanted side effects and reduce drug effectiveness, and it is thought that developing DNA-linking payloads without cross-linking could lead to better therapeutic outcomes.²⁵

In addition, research has shown that miniaturized antibodies and antibody fragments reduce the risk of aggregation due to their smaller size, enhancing tissue penetration and minimizing off-target toxicity compared to traditional ADC drugs.³ Other studies indicate that higher concentrations of antibody fragment may reduce the likelihood of aggregation even further, potentially due to a crowding effect or an undefined self-interaction between protein molecules, although this needs to be investigated.¹

Conjugation conditions

Aggregation can be minimized by fine-tuning different aspects of the conjugation chemistry and formulation conditions, such as using stabilizers – including sugars or surfactants – and optimizing the pH and ionic strength of the buffer. Reducing thermal and physical stress during formulation can also be very effective, however, keeping antibodies separate from each other during conjugation is the most effective way to prevent aggregation right from the start and avoid the need for purification steps later on. This can be achieved through immobilizing the antibodies on a solid-phase support, such as an affinity resin, while conjugation of the payload-linker is performed. Preventing the occurrence of aggregation using affinity resins ensures the final product is free of contaminants like excess payload-linkers and solvents, minimizing any potential negative effects for patients.¹³

Process controls and monitoring

Implementing robust controls and monitoring during the manufacturing process enables the remediation of aggregation and helps to ensure ADC yield and quality. For instance, high shear forces during manufacturing can lead to denaturation and aggregation, so optimizing conditions to minimize shear stresses experienced in steps including reaction mixing and ultrafiltration can help to maintain protein integrity by preventing denaturation.²⁶

As well as these measures, certain excipients, such as polyoxyethylene sorbitan, can be incorporated into reconstitution media during manufacture to prevent and reduce peptide and protein aggregation. Non-ionic surfactants – for example Tween 20, Tween 80 and Polysorbate 80 – are also effective in preventing protein surface adsorption and aggregation due to their low critical micelle concentrations.²⁶ On the other hand, some excipients – including the antimicrobial preservative benzyl alcohol – can inadvertently increase aggregation, and attention should be paid to the specific substance used. Research has found that shorter or less ethoxylated excipient compounds can form hemimicellar clusters at the protein surface, which is beneficial for stability and the prevention of aggregation. Optimal excipient design therefore incorporates many short chains of five to 10 PEG units with some hydrophobic content, such as medium-length aliphatic chains.²⁷ In addition, molecular docking simulations can aid the understanding and prediction of excipient-protein interactions, guiding the selection of excipients to minimize aggregation in lyophilized formulations.²⁸ By employing a combination of molecular dynamics modeling, quantitative analysis and formulation design strategies, manufacturers can optimize the selection and application of excipients to enhance the stability of ADC formulations and reduce aggregation, ensuring the efficacy and safety of the final drug product.

Purification of ADC formulations

The removal of ADC aggregates has been shown to mitigate off-target cytotoxicity for several cell lines and ADCs. There are numerous methods available for removing impurities, such as aggregates and unconjugated payloads and antibodies, from the ADC formulation, with each technique offering distinct advantages. The choice between these methods often depends on the specific context of an ADC's properties and the desired purity level.

Chromatographic techniques for the purification of ADCs

Chromatography-based methods make use of physiochemical differences between different particle types, and so can be used to remove host cell proteins, viral particles and aggregates from ADC formulations during antibody downstream processing. They have also shown promise in supporting payload clearance.²⁹ A number of chromatography ligand chemistries can be used for the purification of ADCs: hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEC) and multimodal chromatography (MMC). IEC provides the ability to keep the DAR within a target range. It is scalable, incurs low production costs and carries less risk of product loss than other purification methods,³⁰ but may not be able to differentiate between molecules with similar charge characteristics. HIC provides an alternative mechanism of separation based on differences in hydrophobicity and MMC resins exploit differences in charge as well as hydrophobicity, potentially providing greater selectivity for highly efficient purification and aggregate removal.

Filtration techniques

While chromatography offers precise separation and analysis capabilities, filtration methods can provide efficient solid-liquid and size-based separation. A combination of both techniques may yield optimal results in resolving precipitation in ADCs. Tangential flow filtration (TFF) is commonly employed in ADC processing to concentrate and diafilter conjugates, effectively removing unreacted small molecules and solvents. This method also helps to eliminate aggregates, ensuring the ADC formulation meets purity standards.³¹ Single-pass TFF (SPTFF) can also be very effective at separating ADCs from unreacted components and by-products.³² Critically, the absence of the recirculation required for traditional TFF can simplify process design and potentially reduce shear stress, helping to reduce aggregation and maximize yield.

Process controls and monitoring

Implementing robust controls and monitoring during the manufacturing process enables the remediation of aggregation and helps to ensure ADC yield and quality. For instance, high shear forces during manufacturing can lead to denaturation and aggregation, so optimizing conditions to minimize shear stresses experienced in steps including reaction mixing and ultrafiltration can help to maintain protein integrity by preventing denaturation.²⁶

As well as these measures, certain excipients, such as polyoxyethylene sorbitan, can be incorporated into reconstitution media during manufacture to prevent and reduce peptide and protein aggregation. Non-ionic surfactants – for example Tween 20, Tween 80 and Polysorbate 80 – are also effective in preventing protein surface adsorption and aggregation due to their low critical micelle concentrations.²⁶ On the other hand, some excipients – including the antimicrobial preservative benzyl alcohol – can inadvertently increase aggregation, and attention should be paid to the specific substance used. Research has found that shorter or less ethoxylated excipient compounds can form hemimicellar clusters at the protein surface, which is beneficial for stability and the prevention of aggregation. Optimal excipient design therefore incorporates many short chains of five to 10 PEG units with some hydrophobic content, such as medium-length aliphatic chains.²⁷ In addition, molecular docking simulations can aid the understanding and prediction of excipient-protein interactions, guiding the selection of excipients to minimize aggregation in lyophilized formulations.²⁸ By employing a combination of molecular dynamics modeling, quantitative analysis and formulation design strategies, manufacturers can optimize the selection and application of excipients to enhance the stability of ADC formulations and reduce aggregation, ensuring the efficacy and safety of the final drug product.

Purification of ADC formulations

The removal of ADC aggregates has been shown to mitigate off-target cytotoxicity for several cell lines and ADCs. There are numerous methods available for removing impurities, such as aggregates and unconjugated payloads and antibodies, from the ADC formulation, with each technique offering distinct advantages. The choice between these methods often depends on the specific context of an ADC's properties and the desired purity level.

Chromatographic techniques for the purification of ADCs

Chromatography-based methods make use of physiochemical differences between different particle types, and so can be used to remove host cell proteins, viral particles and aggregates from ADC formulations during antibody downstream processing. They have also shown promise in supporting payload clearance.²⁹ A number of chromatography ligand chemistries can be used for the purification of ADCs: hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEC) and multimodal chromatography (MMC). IEC provides the ability to keep the DAR within a target range. It is scalable, incurs low production costs and carries less risk of product loss than other purification methods,30 but may not be able to differentiate between molecules with similar charge characteristics. HIC provides an alternative mechanism of separation based on differences in hydrophobicity and MMC resins exploit differences in charge as well as hydrophobicity, potentially providing greater selectivity for highly efficient purification and aggregate removal.

Filtration techniques

While chromatography offers precise separation and analysis capabilities, filtration methods can provide efficient solid-liquid and size-based separation. A combination of both techniques may yield optimal results in resolving precipitation in ADCs. Tangential flow filtration (TFF) is commonly employed in ADC processing to concentrate and diafilter conjugates, effectively removing unreacted small molecules and solvents. This method also helps to eliminate aggregates, ensuring the ADC formulation meets purity standards.³¹ Single-pass TFF (SPTFF) can also be very effective at separating ADCs from unreacted components and by-products.³² Critically, the absence of the recirculation required for traditional TFF can simplify process design and potentially reduce shear stress, helping to reduce aggregation and maximize yield.

Post-manufacture storage and transport

Aggregation tends to increase at higher temperatures, so maintaining a cold chain during shipping and storage is another control measure to take into consideration. Studies have shown that products stored and transported at cooler temperatures – for example at 5 °C ,remain stable for longer without undergoing aggregation than those stored or transported under conditions that can accelerate degradation, such as 40 °C.⁸ Similarly, some formulations are more likely to aggregate under sub-zero conditions, and so require the addition of stabilizing reagents. For example glycerin, polyethylene glycol 300 or propylene glycol can be added to formulations that contain aluminum salts to produce a large steric repulsive region between particles and hinder particle-particle interactions. This prevents aggregation and preserves immunogenicity, even after the formulation is repeatedly subjected to −20 °C.³³

Another approach to protecting formulations from thermal stress involves turning aluminum-adjuvanted formulations into a solid state using advanced lyophilization methods. For example, researchers have atomized liquid formulations with aluminum hydroxide or phosphate and various excipients – including mannitol, glycine, dextran and trehalose – into liquid nitrogen, then freeze-dried them into a powder. This can help prevent clumping and improve the immunogenicity of formulations.³³

Summary

Managing aggregation throughout the manufacture of an ADC requires a multi-layered approach. Antibody, linker, payload and formulation design can all help prevent the formation of aggregates. In the early stages of research and development, these design choices are often secondary to those that relate to efficacy, however the stability of any intermediate and the final formulation may be intrinsically linked to these same choices. Unfortunately, even the best molecule design is unlikely to completely guard against the formation of aggregates, but effective analytical methods to identify and quantify aggregates at all stages help inform development and are critical when defining the CQAs. These help identify the critical control points in the process and the target operating space for each of the manufacturing unit operations. When aggregates form, or have the potential to form, their removal through chromatography or filtration helps build a robust control strategy and deliver a final substance that achieves the target CQAs.

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