After the conjugation reaction, ADCs enter a critical downstream phase focused on purification and formulation. The core objective at this stage is to resolve impurities carried over from upstream and midstream processes — specifically, to remove residual free drug, organic solvents, and reaction by-products, as well as to eliminate potential aggregates. This ensures that the final product meets stringent quality standards. Achieving this involves not only applying precise separation techniques but also addressing special equipment challenges posed by the high potency of ADC components. Balancing purification efficiency with engineering safety is essential.

Ultrafiltration/Diafiltration (UF/DF)

UF/DF is the standard method for buffer exchange in biologics manufacturing and plays an indispensable role in ADC purification. It is particularly effective at removing residual solvents and free drug molecules. This method operates on the principle of molecular size exclusion and generally provides near-ideal separation efficiency. As shown in Figure 1, UF/DF demonstrates excellent clearance of various solvents and free drug impurities, with separation factors (S) close to 1, indicating highly efficient impurity reduction to acceptable levels.

Figure 1. Impurity clearance during the UFDF operation plotted against diavolume for the removal of (A) residual solvent and (B) free drug.

However, when impurity concentrations reach very low levels, UF/DF efficiency can decline due to the "rebound effect," where solvent molecules desorb from the membrane surface, or due to nonspecific interactions between solvents and membrane materials or system components. More complex challenges arise in removing free drug-related impurities. Experimental data in Figure 2 highlight this complexity: using 30 kDa nominal molecular weight cut-off (NMWCO) membranes for UF/DF, two free drug-related impurities of similar molecular weight (~2 kDa) exhibit drastically different clearance behaviors. Impurity A achieves over 2-log clearance after just one UF/DF step, while Impurity B remains largely uncleared even after ten diafiltration volumes. The underlying mechanisms are not fully understood but may involve self-aggregation of impurities in the aqueous phase, increasing their apparent size and hindering membrane permeation, or nonspecific binding to ADC molecules. When UF/DF alone cannot reduce critical impurities, especially certain free drugs and their derivatives, below quality thresholds, more selective and in-depth purification strategies must be employed.

Figure 2. Impurity clearance during UFDF is plotted against diavolume for free drug-related impurities: (A) impurity A and (B) impurity B.

Chromatography

To overcome UF/DF limitations, chromatography techniques that combine binding and elution modes offer powerful and selective separation, effectively supplementing the purification process. The core principle exploits significant physicochemical differences between large ADC molecules and small free drug molecules, such as charge, hydrophobicity, size, and specific affinity.

Typically, the conjugation reaction mixture is adjusted under conditions before loading onto chromatographic columns. The stationary phase selectively binds ADC molecules via ion exchange, hydrophobic interactions, or affinity, while free drugs and residual solvents, lacking binding affinity, pass through unretained. Notably, some chromatographic resins may exhibit nonspecific adsorption of free drugs, making resin screening and optimization of binding conditions critical. For example, cation exchange chromatography (CEX) has been successfully applied for effective free drug removal in ADCs. Careful design of loading, binding, washing, and elution steps enables the clear separation of ADC and free drug, with thorough washing steps being crucial for complete clearance of free drug.

Aggregate removal is equally important, particularly when conjugation results in aggregate levels exceeding acceptable limits. Traditional antibody purification methods like CEX can be adapted for ADC aggregates under certain conditions. For cysteine-conjugated ADCs (e.g., interchain cysteine or THIOMAB platforms) with noncharged linker-drugs, surface charge properties closely resemble unmodified antibodies. Experimental data (Figure 3) show similar CEX binding behavior between these ADCs and antibody intermediates, with only slightly stronger binding under some conditions. This suggests that CEX conditions optimized for antibodies can often be directly or slightly adjusted for ADC aggregate removal.

Figure 3. Batch adsorption contour maps of an antibody and its drug conjugate on cation exchange resin, showing binding strength (Log Kp) based on established methods.

In contrast, lysine-conjugated ADCs pose different challenges. Each lysine modification removes a positively charged group, significantly altering overall charge distribution and resulting in fundamentally different CEX binding profiles compared to unmodified antibodies. Hydroxyapatite chromatography has emerged as an effective alternative for aggregate removal in lysine-conjugated ADCs. Regardless of chromatographic mode, a crucial consideration during ADC purification development is whether chromatography affects the average drug-to-antibody ratio (DAR) or DAR distribution. Process monitoring must include aggregate levels and rigorous evaluation of these critical quality attributes' stability. Additionally, residual solvents and free drugs in process streams may impact resin performance and reuse stability, warranting careful assessment.

Special Requirements for ADC Production Equipment

Manufacturing ADCs—especially during conjugation—places exceptionally demanding requirements on production equipment due to the handling risks of highly potent cytotoxic drugs, extensive use of organic solvents, and the need for precise control over reaction parameters such as temperature, mixing, and reagent addition. These challenges extend from early-stage process development at small scale through GMP-scale production.

Figure 4. Comparison of small-scale equipment for ADC process development and validation.

Miniaturization Challenges in Process Development:

Early R&D phases require developing small-scale conjugation reactors that accurately simulate large-scale processes. Key difficulties include ensuring operator safety from potent compounds, solvent compatibility with conventional lab materials, and operating at minimal volumes (<2 mL to 50 mL) to conserve precious intermediates. Ideal microreactors integrate multiple functions: fully enclosed operation for safety, tolerance to high solvent concentrations, precise temperature and reagent dosing control, continuous and uniform mixing, inert atmosphere capability if needed, and cost-effectiveness with options for reuse or disposability. No single system perfectly meets all these criteria currently, leading to staged development strategies: initial screening in microcentrifuge tubes with water-bath temperature control to minimize material use; intermediate scale-up to pilot systems featuring continuous mixing and precise temperature control as intermediate supply grows; and final confirmation using automated reactors integrating mixing, temperature control, and data logging to ensure scalability. Downstream unit operations like chromatography and UF/DF have more mature small-scale models, generally following biologics scale-down principles but still requiring evaluation of solvent compatibility and containment for high-potency materials.

GMP Production Equipment Selection: Single-Use vs. Reusable Systems:

Choosing equipment for clinical or commercial ADC production hinges on fixed reusable reactors (stainless steel/glass) versus single-use systems (plastic membranes, bags, tubing). Single-use technology is increasingly favored in biopharma due to enhanced manufacturing flexibility, minimized cross-contamination risk, and elimination of cleaning validation burdens—particularly advantageous for ADCs, given their potent small molecule components and extremely low residue limits. Developing sensitive and specific cleaning and residue testing methods is costly and complex, making single-use systems attractive for bypassing batch-to-batch cleaning challenges, thus accelerating development and reducing costs.

However, single-use ADC production faces unique challenges: organic solvents necessary for solubilizing small molecules may be incompatible with single-use plastics/membranes, potentially causing leachables that compromise product quality. Comprehensive compatibility studies are mandatory to evaluate interactions between process fluids and single-use materials, ensuring leachables remain within acceptable limits without adverse effects on critical quality attributes. Regardless of system choice, meeting process control requirements—including stringent temperature regulation, precise reagent addition, aseptic operation, effective mixing, and optional atmospheric control—is essential for consistent, high-quality ADC manufacture.

Integrated System Engineering Throughout the Process

From impurity removal after conjugation, through advanced purification techniques, to stringent equipment selection and validation, the downstream processing and manufacturing of ADCs constitute a highly complex, interconnected system engineering challenge. Success depends on a deep understanding of impurity physicochemical properties and removal mechanisms, judicious selection and optimization of purification strategies combining UF/DF and chromatography, and scientifically driven, risk-based matching of safe, reliable equipment solutions across development phases and scales. Only through such integrated approaches can the carefully prepared ADC intermediates be efficiently and safely transformed into final drug products that meet the highest quality standards and ultimately benefit patients.

Creative Biogene offers global pharmaceutical companies and innovative drug developers comprehensive end-to-end CDMO solutions for protein and conjugated drug development, from preclinical stages to commercial launch. Our core services include Payload-Linker synthesis, conjugate drug developability studies, cell line construction, and more, ensuring tailored support for your ADC projects with scientific rigor and operational excellence.

FAQ

Q: How to solve small molecule precipitation issues during the ADC ultrafiltration process?

A: In ADC (Antibody-Drug Conjugate) production, ultrafiltration is one of the common purification steps. However, during this process, precipitation of small molecules such as Payload-Linker often occurs. To address this issue, it is first necessary to analyze the possible causes, which mainly include the following two categories:

I. Analysis of Precipitation Causes:

1. Conjugation Process Design Issues: When using the "two-step method" for ADC conjugation reactions, the first step typically involves conjugating the Linker to the antibody, and the second step involves conjugating the Payload to the Linker. To improve the conjugation efficiency between Payload and Linker, excess Payload is usually added to ensure the final target DAR (Drug-to-Antibody Ratio) value is achieved.

However, this approach cannot guarantee 100% conjugation efficiency. For example, if a Linker has 3 active sites but only 2 successfully conjugate with Payload, the unreacted Payload, due to its strong hydrophobic nature, tends to aggregate on the membrane surface during ultrafiltration, leading to precipitation and forming small molecule impurities.

2. Enhanced Hydrophobicity Due to High DAR Values: When ADC products have relatively high DAR values, their overall hydrophobicity increases, making them prone to enrichment or deposition on the membrane surface during ultrafiltration. This causes small molecule precipitation, affects filtration efficiency, and may even clog the membrane, reducing the flux and yield of the entire ultrafiltration process.

II. Solutions:

To address the above issues, the following three optimization strategies can be considered:

Solution 1: Optimize Conjugation Process and Control Payload Loading. Control small molecule precipitation at the source by optimizing the conjugation platform period, precisely controlling the timing and amount of Payload addition. While ensuring DAR compliance, minimize excess Payload loading to reduce the risk of unconjugated Payload residue and precipitation.

Solution 2: Adjust Organic Solvent Ratio During Ultrafiltration. For products that have already experienced precipitation, try appropriately increasing the content of organic solvents (such as ethanol, DMSO, etc.) in the ultrafiltration system to enhance the solubility of Payload-Linker, reduce deposition on the membrane surface, and improve system stability. This solution is effective for most products with low to moderate hydrophobicity and is relatively easy to implement.

Solution 3: Staged Ultrafiltration with Organic Solvent Dialysis System. For products with extremely strong hydrophobicity, severe precipitation issues, and sensitivity to process conditions, a more complex but more thorough process pathway can be adopted. During the ultrafiltration stage for removing Free-Drug (free small molecule drugs), introduce a buffer system containing organic solvents to allow small molecules to remain stable in solution. Once their concentration decreases to a certain threshold, transfer to the final formulation buffer system for complete buffer exchange.

However, this solution significantly extends the ultrafiltration cycle and increases costs. It is not suitable for molecular systems sensitive to shear forces or organic solvents, and its use should be carefully considered in actual projects.