Today, with seven commercially approved antibody-drug conjugates (ADCs) on the market and approximately 90 programs in clinical trials, simplifying the complex supply chain to make manufacturing efficient is a necessity. With approximately 70 percent of ADC projects outsourced to contract development and manufacturing organizations (CMDOs),¹ a transparent and integrated supply chain is critical for success. Now, this is more important than ever, with more clinical trials with combination therapy expected as more than 200 trials are registered and as ADCs are expected to gain prominence beyond oncology in the areas of anti-infection, anti-inflammatory, cardiovascular diseases and imaging and diagnostic agents.

The typical ADC supply chain is elaborate due to the numerous, specialized processes in their production and the logistical alignment needed between each step. The three main elements of an ADC are:

1. Antibody: specific for a tumor-associated antigen that has restricted expression on normal cells.
2. Payload: designed to kill target cells when internalized and released.
3. Linker: attaches the cytotoxic agent to the antibody. New linker systems are designed to be stable in circulation and release the cytotoxic agent inside targeted cells.

As separate steps, often in separate locations, custom linker and payload raw materials are made and then joined to produce the linker payload. Meanwhile, the monoclonal antibody (mAb) is produced elsewhere. Linker payloads and mAb are shipped to conjugation sites and, once prepared, the conjugated material is shipped to a drug fill/finish site. Finally, vials are often shipped for labeling and packaging to yet another facility. Customers often produce contingency batches requiring cold storage. Typically, a total of 5 to 10 CDMOs across the globe are involved in the supply chain.

The benefits of working with established CDMOs on ADC projects are numerous: experience with a variety of constructs and ADC technologies; efficient processes in place for tech transfer and manufacturing; manufacturing expertise, particularly with GMP batches; and understanding of the regulatory pathway for ADCs.

Interspersed with the above manufacturing steps, requisite quality control measures—such as mAb bioassays and conjugate stability testing—compound an already complicated pathway. Clearly, manufacturers that can simplify this supply chain are doing customers a great service.

ADC manufacturing then: small scale, high variability
Ten years ago, ADC manufacturing took place on a much smaller scale than today. Raw materials, such as mAbs, were only available in clinical rather than commercial-sized batches. Manufacturing was performed as an add-on to other procedures rather than as an optimized, stand-alone process, and few sites were capable of handling high-potency payloads.

Full-length mAbs were humanized IgGs, most often IgG1, conjugated randomly at cysteine or lysine sites, as shown in Figure 1. In these constructs, the payload could bond to the antibody in multiple locations, potentially affecting its activity. With an IgG scaffold containing over 80 lysines,² conjugation resulted in very heterogeneous ADCs with variable drug-to-antibody ratios (DARs). High DAR species in the final product can impact stability, solubility and cause problems in manufacturing and operations.


First-generation payloads included DNA disrupting agents such as calicheamicin, SN-38, duocarmycin and doxorubicin. These were linked to the mAb via monovalent, non-cleavable bonds or by acid-labile linkers, which weren’t reliably stable—a key consideration in selecting therapeutics. While these agents were an impressive first step, they had a much narrower therapeutic index than many had hoped.

Second-generation ADC development: site-directed conjugation
To solve the heterogeneity problem, research moved from native IgGs toward site-specific engineered mAbs, including mAbs with engineered cysteines, non-natural amino acids and sequence tags—all of which could be reacted to perform a more homogenous product. The goal was to manipulate the antibody, so it had specific, limited locations where the toxins were to bond. For instance, monoclonal antibodies containing engineered cysteine moieties limit conjugations to positions that do not disturb immunoglobulin folding or assembly or alter antigen binding.³ The structural representation of an ADC made with such an antibody is shown in Figure 2.


This type of construct first came to the clinic in 2013. Its tighter DAR distribution reflects a more homogeneous ADC final product with greater process control.

Other second-generation developments included modulation of ADC hydrophobicity by using hydrophilic linkers; structure activity relationship (SAR) design relating a molecule’s structure to function; and enhanced analytical tools such as chromatography. Cytotoxic payloads with greater potency, such as auristatin and maytansine microtube disruptors, also came into play. All these developments helped improve the usefulness of these agents.

More sophisticated, specialized manufacturing techniques and special facilities were also required to handle these powerful toxins safely.

Second-generation linkers had slightly more functionality than earlier linkers. They were also monovalent, but some were cleavable, either enzymatically or via acid exposure inside cells or lysosomes. Examples include linkers based on proteases, hydrazine, polyethylene glycol (PEG) and disulfides. These linkers were expected to help the antibody release the toxin at the right place and the right time and also stabilize the ADC during preparation, storage and systemic circulation.

Third-generation ADC development: new expectations for linkers
Today’s researchers are exploring additional modes of action and ways to increase activity and specificity. Bi-specific MAbs, both IgG-like and non-IgG-like, contain two dissimilar binding sites. For example, a single ADC may deliver a toxin and activate natural killer cells. One recently constructed agent had four mechanisms of action. Obviously, the conjugation processes and analytics for these agents are non-trivial.

Another up-and-coming technology is utilizing Fabs (antigen-binding fragments) in place of intact mAbs. These are sections of antibodies that include sites for antigen binding and linkage. These Fabs are very stable, may be internalized more readily, are relatively easy to purify, and tend to be less immunogenic than larger ADCs.

Third-generation payloads—potent cytotoxins such as PBDs and tubulysin, which require special facilities and handling—are not that different than second-generation payloads.

The most unexpected aspect of third-generation development is the revolution in the general understanding of what linkers can do. SAR studies show that linkers change antibody properties, including changes in toxicity and pharmacokinetic profiles. It is now known that if a linker is altered, these parameters must be reevaluated.

New linker categories still include cleavable and non-cleavable, but they also encompass entities such as the Fleximer platform, a polyvalent and biodegradable molecule that can carry multiple payloads. Additionally, hydrophilic linker modulation such as pegylation can mask a larger molecule from the immune system and decrease renal clearance to increase longevity in the circulation. This is a very useful concept as PBDs are very hydrophobic and, once conjugated, are prone to aggregation.

Manufacturing today: A litany of challenges
Today’s complex, multifaceted ADCs place substantial demands on drug developers and manufacturers, and these challenges are compounded by this sense of urgency in a crowded field and need for life-saving therapies:

• Diverse strategies for engineering mAbs and fragments for site-specific conjugation to achieve controlled DAR distributions, stability and consistent pharmacokinetic (PK) and toxicity behavior necessitate numerous, sophisticated laboratory procedures.
• Aggregation must be controlled for drug safety and many trials are failing because of aggregation. Greater species uniformity and improved process design helps, but the widespread use of hydrophobic PBD payloads complicates this issue.
• Highly toxic payloads such as PBD require special facilities and diligent safe handling.
• Linker chemistries are becoming increasingly important and diverse. If hydrophilic, linkers can help with the aggregation problem. On the other hand, they complicate what was originally a simple function, requiring more steps, more R&D and more expertise.
• Additional drug mechanisms complicate manufacturing exponentially and demand increasingly sophisticated testing.
• SAR drug design, now de rigueur, requires a completely new category of expertise to help discover relationships between ADC structure, mechanism of action and efficacy.
• Expanded GMP manufacturing, thanks to multiple trials and combinations along with a better understanding of pre-clinical models; increased large-scale manufacturing of 1-3Kg/batch is expected.
• The above processes must be analyzed with increasingly sophisticated tools, such as automated surface plasmon resonance (SPR), immunoassays, capillary electrophoresis (CE) and liquid chromatography/mass spectrometry (LCMS). These advanced methods of characterization can also help with routine lot release and stability testing.

How CDMOs must rise to the occasion
To ensure the CDMO-customer relationship is a success, it needs to be built on transparency and trust. Regular meetings should be held to share data with an integrated, core team made up of experienced professionals across project management, development, manufacturing, quality and regulatory.

Established CMDOs in the ADC business will need to keep pace with future technological advances in this fast-growing industry. Companies that will be successful in the bioconjugation space are providing dedicated facilities for high-potency biologicals, establishing platform operations and developing a workforce with the advanced and specialized expertise to meet the expectations of customers and regulatory agencies. Next-generation bioconjugation will not only be challenged by new and novel chemical unit operations, but will also require novel analytical technologies to provide a more granular understanding at the molecular level. Techniques and tools will need to provide answers for the control strategy of complex products and will need to evolve to support sophisticated release strategies for on-line and at-line in-process testing.

Along with comprehensive laboratory services and expert assistance, the following technologies are particularly helpful for successful ADC manufacturing:

Single-use systems (SUS)
SUS technologies add simplicity and are available for all steps throughout the ADC manufacturing process. Having a complete, single-use process in place can make a big difference: no cleaning studies are needed, reducing costs. The components are designed to be scalable. Extractables and leachables documentation is available to meet regulatory requirements. Operator safety is increased, particularly when handling highly potent ingredients. Implementation of single-use reactor along with a full line of single use equipment for all unit operations is expected be the preferred platform of choice for all bioconjugation processes.

Process and analytical technology (PAT)
The advancement of PAT allows real-time testing during a GMP process to gather rich, real-time data about the physical and chemical parameters during active processes. It can be used to ensure the process is going as planned and it can also be used to monitor trends in process iterations.

Chromatography
Purification strategies have been expanded to include large-scale chromatography and an increased number of constructs have chromatography. The major reasons to integrate chromatography include: to clear lipophilic drugs that are not amenable to TFF clearance; to remove aggregates and to refine conjugated species distribution, as in removing unconjugated mAb; and to ensure the best possible ADC therapeutic index and specificity, an important function since 60% of constructs require this type of purification.

Potential customers are also evaluating services beyond preparation of the materials, such as formulation support and studies to support the regulatory filing.

Complexity is the new normal
Ten years ago, ADCs were a relatively simple concept: use an antibody to target a cell and precisely deliver a biologically active agent. Now, the mission is much more complicated. CDMOs with long-range plans for ADC manufacturing are setting up processes to handle challenging supply chains and investing in facilities and processes to ensure efficiency, quality and security for their customers. Companies that can help deliver multiple constructs to enable a well-designed clinical program are providing customers the opportunity to advance in the field at a fast pace. CDMOs are uniquely positioned to see a wide variety of best practices and can provide solutions based on what has been observed in the industry. In addition, as more commercial products enter the market, there is an acute need for companies that understand how to execute late-stage studies to support a filing strategy. The growth of ADCs in the clinical and commercial API space is a testament to the ability of manufacturers to evolve the technologies required to handle complex molecules.

Whether it’s understanding the structure-activity relationships around the antibody, linker and drug, or managing a complex supply chain, a CDMO with experience should have the skills and tools needed to help ADC developers navigate these challenges. 

Sources

1. HPAPIS and Cytotoxic Drugs Manufacturing Market,” Roots Analysis online, last modified August 6, 2014, https://www.rootsanalysis.com/reports/view_document/hpapis-and-cytotoxic-drugs-manufacturing-market/64.html
2. Chari RV. “Targeted cancer therapy: conferring specificity to cytotoxic drugs,” Accounts of Chemical Research 41, no. 1 (2007): 98–107. doi: 10.1021/ar700108g.
3. Jagath R. Junutula et al. “Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index” Nature Biotechnology 26, (2008): 925-932. https://doi.org/10.1038/nbt.1480.

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Lisa McDermott is director, process and analytical development at MilliporeSigma.

Jyothi Swamy is associate director, ADC/bioconjugation contract manufacturing at MilliporeSigma.