Compatibility Considerations for Single-use Technology in ADC Coupling Process

Antibody-drug conjugates (ADCs) are a rapidly developing class of biotherapeutic cancer drugs in the past three years. ADCs use linkers to connect monoclonal antibodies (mAbs) with payloads (Figure 1). Using the specificity of monoclonal antibodies, ADCs can accurately target the target and reduce the toxic side effects of drugs on normal cells. ADCs combine the specificity, stability, and pharmacodynamic characteristics of antibody drugs with the small molecule toxin to target tumor cells. It is one of the hottest directions in the current research of anti-tumor drugs. So far, a total of 15 ADCs have been approved for marketing worldwide.

Figure 1: Schematic diagram of antibody-drug conjugate structure

Traditional ADC coupling reactions utilize the amino group of antibody lysine or the thiol group of cysteine obtained by opening interchain disulfide bonds. The amino group of lysine is connected to the activated carboxylic acid ester linker through an amide bond, and the thiol group of cysteine reacts with the maleimide group (Figure 2).

Figure 2 Schematic diagram of antibody coupling modification based on cysteine (upper diagram) and lysine (lower diagram)

The common cytotoxic drugs used in ADCs pose high risks in terms of health and safety, which poses unique challenges for their manufacture, especially the containment of coupling reactions and subsequent removal of uncoupled drugs, solvents, and unwanted product variants through various methods. The use of single-use "closed systems" can provide advantages: eliminating cross-contamination between batches, avoiding the generation of large amounts of toxic waste during cleaning, and minimizing operator exposure to toxic drugs, which are particularly attractive to ADC manufacturers.

The transition from traditional stainless steel or glass jars to single-use technology poses unprecedented challenges. During the ADC manufacturing process, solvents such as dimethyl sulfoxide (DMSO), dimethyl acetamide (DMAC), and dimethylformamide (DMF) are often used (Figure 3). The compatibility between process fluids and plastic/elastomeric materials used to manufacture single-use parts is clearly crucial and needs to be evaluated.

Figure 3 Coupling reaction process of ADC

To minimize costs and shorten the time to market, risk assessment is crucial for the implementation and qualification/validation of single-use systems in ADC production. The main risk factors include:

1. Chemical compatibility between process fluid and single-use system

2. Product composition

3. Material contact area

4. Contact time

5. Process temperature

6. Pre-sterilization method

7. Final container closure system

Chemical compatibility test and evaluation

BioLink research evaluated the chemical compatibility of DMSO, DMF, and DMAc with single-use consumables during the ADC coupling process.

Test method: Inject the reagent to be tested into a 1L single use bioprocessing bag, hang it at room temperature, and observe the changes of the reagent, bag, and tubing in the bag every day. After reaching the time node, test the film thickness, integrity, loading volume, drop and heat-sealing strength of the bag; for the joints and tubing, conduct visual inspection, thickness measurement, dimensional measurement, pressure resistance measurement, tangential torque test, pressure holding test, and torque test.

The results showed that at 30°C, the tubing had excellent resistance to contact with 100% DMSO or 100% DMAC solvents within 1 hour, and the membrane had excellent resistance to contact with 35% DMSO or 35% DMAc solvents within 24 hours, with no visual or functional defects observed.

Study for extractables

The ADC coupling process involves mixing, ultrafiltration, and chromatography, and the fluid tubing contains different components and material structures. The standard method based on the exposed surface area for extracting extractables is impractical, so BioLink has adopted a reduced model approach for dynamic extractable research, as shown in Figure 4.

Test method: After the reduced model was irradiated by 51.7-54.8kGy gamma, according to the actual use of customers, the extraction volume was 3L, and the extraction solvents water, 30% DMAc, 30% DMF, 30% DMSO were added respectively, and the extraction was carried out under the condition of dynamic cycling under the condition of water bath at 40℃ for 24h. After 24h, the samples were taken out and partitioned, and then subjected to the tests of LC-DAD-OTOF, GC -MS, ICP-MS test.

The results showed that the extract levels were within the PDE (daily exposure dose) range of the compound, indicating good extract levels and product safety.