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Bioconjugation requires aseptic manufacturing and containment for cytotoxic payloads.
Antibody-drug conjugates (ADCs) comprise one of the fastest growing segments of the biopharmaceutical market. Consisting of antibodies linked to highly potent (cytotoxic) small-molecule “payloads,” ADCs are designed for the targeted delivery of therapeutic actives. The antibodies are engineered to bind to specific cells-most often tumor cells-and then release the active agent, avoiding the damage to healthy cells observed with traditional systemic treatments. This specificity has attracted significant interest.
Today there are nearly 200 ADCs in development, with 60% in the discovery and preclinical stages and 20 products approved or in late stages of clinical development (Phase II and above) (1). The global ADC market is expected to expand at a compound annual growth rate of 19.4% between 2017 and 2030. The newer ADCs are being developed with novel conjugation approaches, more potent warheads, and modified linker technologies. They are also being tested in combination with other novel therapies, such as immune checkpoint inhibitors and epigenetic modulators (1).
Initial conjugation strategies suffered from a lack of specificity. These issues have been addressed using a number of different approaches. Examples of third-generation ADC conjugation platforms include avoiding/limiting retro-Michael drug de-conjugation, cysteine re-bridging, enzyme-assisted ligation, glycan re-modeling, and ligation at the nucleotide-binding site of the antigen-binding fragment (1).
As more products are approved and other candidates move into later-stage clinical trials, the capability for large-scale production of ADCs is increasing. Because these complex therapies include both large- and small-molecule components and are highly potent, they present many manufacturing challenges.
Designing the correct process equipment for larger-scale clinical and commercial GMP bioconjugation of payloads to antibodies for ADC production must be based on the outcome of appropriate process development activities, according to Jyothi Swamy, associate director of ADC/Bioconjugation Contract Manufacturing at MilliporeSigma.
Understanding the critical process parameters drives the design of the process equipment. “Volume-range requirements, the order of addition, mixing needs, and in-process monitoring instrumentation are key factors in equipment design,” she notes. Multi-use fixed equipment must also be designed to ensure validated decontamination and cleaning.
"Development campaigns should begin with the final scale and equipment in mind, and all development trials should be designed accordingly,” Swamy adds. “If the development strategy has a process for controlling scale changes from the earliest batches, challenges should be minimal for commercial processes,” she says.
Assuming reaction stoichiometry, pH, buffer composition, temperature control, reaction time and fluid sheer rates, and mixing time are all under control, process hold times, protection of employees from cytotoxic raw materials, and product change-over for multiproduct facilities become the next series of challenges associated with scale-up, according to Thomas Rohrer, associate director of commercial development for bioconjugates at Lonza Pharma & Biotech. “Process hold times during the modification and conjugation reactions must be tested to determine whether holding the antibody-linker complex for a period of time following the antibody modification reaction causes any change in the reaction stoichiometry,” he explains.
A comprehensive occupational hygiene monitoring program that can detect cytotoxins in the 10-9 g range in room air and on process surfaces is also required to protect manufacturing personnel from residual cytotoxins following a manufacturing campaign. In addition, the cytotoxins used for ADC manufacturing may remain unchanged or may undergo degradation or deactivation during the product changeover cleaning, so a unique detection method and cleaning procedure may need to be developed, according to Rohrer.
ADCs for human clinical applications must be manufactured in an aseptic biological environment operating under cGMP. This aseptic environment must also isolate manufacturing personnel from exposure to cytotoxic chemicals. “The equipment used in ADC manufacturing must protect employees from exposure to cytotoxins and prevent microbial contamination of the process intermediates and drug product during the conjugation process,” Rohrer says.
Designing a facility (both clinical and commercial) that can meet both GMP and safety requirements is the key, agrees Swamy. “For ADC processing, a cleanroom environment is required while maintaining containment for potent compounds using engineering controls,” she says.
Both primary and secondary containment of the toxin are required. The primary containment strategy includes handling the unconjugated cytotoxin in a closed front isolator cabinet under negative pressure in a room with negative pressure in relation to surrounding cleanrooms, according to Rohrer. Hermetically sealed process vessels and equipment are then used to execute the process while protecting the valuable raw materials from contamination.
If the primary containment should fail, the secondary containment reduces the exposure of manufacturing personnel to the cytotoxin by exchanging the room air in the process suite more than 30 times each hour. The high-efficiency particulate (HEPA) filters that serve the manufacturing area are designed as safe-change filters to protect personnel during preventive maintenance. Personnel working in the manufacturing area wear one-way over-gowning that is removed before exiting the protective air locks.
Specific modifications for production equipment used in bioconjugation processes include double mechanical seals and overflow trays, which strengthen the primary containment envelope, according to Rohrer. In addition, all waste generated during the manufacturing process that may contain cytotoxin must be inactivated. If inactivation conditions are not compatible with stainless steel, Rohrer points out that incineration of all liquid and solid process waste is recommended to prevent introduction of cytotoxic substances into the environment.
Other important considerations for larger commercial-scale bioconjugations noted by Swamy include the use of larger amounts of toxic payloads and typically large volumes due the dilute nature of most conjugation chemistry.
The need to understand containment practices is just one type of expertise required by operators performing bioconjugations to produce ADCs. Knowledge of both small-molecule and biologic manufacturing is needed, because aspects of both are involved, according to Swamy. Lonza has found that site manufacturing personnel with aseptic or high-potency operations experience are ideal candidates for ADC bioconjugations operators. “Employees with this base level of experience can rapidly transition into ADC operations with some additional ADC-specific training,” Rohrer says.
The linkage of small-molecule cytotoxic payloads to larger biomolecules creates some additional challenges with respect to the stability of the products. All protein therapeutics made for clinical or commercial applications may harbor high-molecular-weight variants (i.e., aggregates) that have the potential to reduce the potency of the drug when scale-up is performed, according to Rohrer. “This issue is magnified in ADCs because many of the cytotoxic drugs conjugated to the antibodies are hydrophobic, thus increasing the chances of aggregate formation during the manufacturing of the drug substance and subsequent storage of the drug product,” he explains. “Analytical methods used to detect high-molecular-weight variants and residual amounts of free drug must have the precision, sensitivity, and selectivity to detect these process-related impurities,” he adds.
All analytical methods used during the bioconjugation process must be robust and suitable for validation, according to Swamy. The most appropriate analytical methods for a given ADC depend on the properties of the drug, the linker, and the choice of conjugation sites, notes Rohrer. “The clinical efficacy of an ADC reflects the target site specificity and binding properties of the antibody, the in-vitro and in-vivo stability of the linker, and the potency of the drug payload. These unique properties require an in-depth understanding of the physicochemical properties of the individual and combined elements of the ADC, so appropriate analytical and bioanalytical methods must be selected to monitor the functional attributes of the resulting drug substance,” he observes.
On-line and at-line PAT testing can be added at later stages of the development process to enable better monitoring-and hence understanding-the progress of a bioconjugation process, according to Swamy. These techniques must, however, be verified against existing methods. “As the bioconjugation process matures, the addition of data-trending and automated processing can also reduce the off-line testing required,” she comments.
Because bioconjugations processes for ADC production are complex and require specialized expertise in the handling and processing of cytotoxic materials-combined with the need for aseptic manufacturing and expertise in biologic and chemical APIs-many pharmaceutical companies outsource these activities to contract development and manufacturing organizations (CDMOs). CDMOs operate multiproduct facilities, which present additional challenges for ADC bioconjugations.
“One of the biggest challenges with ADC manufacturing is cleaning and decontamination of the processing equipment. This challenge is mitigated by the use of complete single-use technology, including the processing equipment,” Swamy asserts. She adds that the use of complete single-use technology also provides operator safety. Rohrer agrees: “When reaction conditions permit, introduction of single-use equipment into the ADC manufacturing paradigm can help with product change-over.”
Notably, problems associated with the introduction of new cytotoxic drugs that are hydrophobic and therefore difficult to remove from product contact surfaces using traditional cleaning strategies can be solved by using disposable equipment. “Single-use equipment is well-established at the laboratory scale, and the lower operating/validation cost at manufacturing scales combined with the development of polymer films engineered to meet the solvent requirements of ADC manufacturing has encouraged wider use,” notes Rohrer.
1. Roots Analysis, Antibody Drug Conjugates Market (4th Edition), 2017-2030, Oct 27, 2017. www.rootsanalysis.com/reports/view_document/antibody-drug-conjugates-market-4th-edition-2017-2030/180.html.