Putting it all together

Producing the ADC requires both biologic-based and small-molecule manufacturing. "One of the biggest challenges in manufacturing ADCs is controlling all the components that go into the final conjugation step," says Boldt. "Namely, the three main components that make up an ADC (e.g., antibody, linker, and payload) are all manufactured in very different ways. For example, it is not uncommon for these components to be manufactured by synthetic chemistry and mammalian cell culture. Thus, there presents a challenge in ensuring all these components have been manufactured under cGMP, and subsequently bringing them all together to generate the final ADC under cGMP, as well."

The biologics portion of the ADC and the high-potency API require very different handling methods, and manufacturers must make sure that handling requirements for both are met. "It is imperative that manufacturers emphasize the protection of the product from workers as well as the protection of workers from the product," says Jason Brady, head of business development, conjugates and cytotoxics at the CMO Lonza. Clinical ADC manufacturing is executed in an aseptic biological manufacturing environment to protect the product from contamination, explains Brady. Once conjugated with the high-potency API (which is manufactured in a high-containment environment), the resulting ADC also is handled under high-containment conditions. The level of containment is determined by occupational exposure limits for the high-potency API and resulting ADC. The environment must provide manufacturing personnel with isolation from cytotoxic chemicals in the occupational exposure range of 5 ng/m3 of air. Also important is that facility design includes design of equipment and process contact surfaces that permit clean-in-place and steam-in-place to remove minute traces of residual drug contamination during both interbatch and product changeover cleaning, according to Brady.

As ADCs advance in the clinical pipeline so does the technology to manufacture ADCs to control certain product and process conditions. "New technology that can limit the heterogeneity of ADC products is something that will be important in the future," says Brady. "ADCs made via current technologies are heterogenous mixtures. Heterogeneity can be controlled and measured by robust and reproducible manufacturing processes and proper analytics, but new technologies will likely emerge to influence and improve ADC manufacturing," he explains. Some fraction of the finished drug product consists of unconjugated antibody. The remaining portion of the finished drug product contains conjugated antibody with a variable number of the cytotoxic small molecules conjugated at different sites on the antibody. Controlling the number and location of cytotoxic molecules conjugated to the antibody is being pursued as a means to create a more uniform product and as a way of being able to explore structure–function relationships by varying the site of attachment of the cyotoxin.

One strategy for controlling the site of attachment has been developed by researchers from Genentech, a member of the Roche Group. They describe precise site-specific conjugation of human IgG1 to MMAE by replacing Ala114 at the junction of the CH1 and the variable heavy-chain domain with cysteine to create an engineered antibody called a THIOMAB. This site was chosen because it does not participate in antigen binding or effector functions. According to Jagath Reddy Junutula, senior scientist at Genentech, the process for creating a THIOMAB differs only slightly from that of a conventional mAb. The THIOMAB is subjected to partial reduction to remove cysteine and glutathione adducts. The partial reduction also breaks interchain disulfide bonds, which must be reformed by a reoxidation step. After reoxidation, the engineered cysteine residues are available for conjugation.

Genentech researchers used this process to conjugate MMAE to a THIOMAB version of an antibody against MU16, a cell-surface protein expressed in ovarian cancer cells. The THIOMAB conjugate was shown to be homogenous and to contain a single drug molecule attached to each heavy chain, for a total of two MMAE molecules per ADC. The THIOMAB–MUC16 was found to have comparable efficacy to a conventionally produced ADC and to be better tolerated in two preclinical species (7). In a subsequent study, a different cytotoxin, DM1, was conjugated to a THIOMAB version of trastuzumab. Results were similar, with the THIOMAB T–DM1 displaying comparable efficacy and better tolerability in preclinical species than its conventionally produced counterpart (8).

According to Junutula, the reoxidation step is the only thing that distinguishes manufacture of a THIOMAB drug conjugate from that of a conventional ADC. "We can make up to grams scale without any difficulty. And the results are huge—you have a homogenously conjugated cytotoxic drug to the antibody," he says.

While the THIOMAB uses the substitution of one amino acid for another to control the site of conjugation, several groups are working toward incorporating nonnatural amino acids into the mAb for to control the site of conjugation and also to provide an expanded repertoire of functional groups that could be used for linker chemistry. The biopharmaceutical company Ambrx has developed expression systems in E. coli, yeast, and Chinese hamster ovary (CHO) cells that can be used for such substitutions and which can be scaled up to volumes required for commercial manufacturing. Ambrx's expression systems contain engineered transfer RNAs that will read through a stop codon called amber, as well as engineered tRNA synthetases that will aminoacylate the orthoganal tRNA with an Ambrx nonnatural amino acid. The expression system will insert a nonnatural amino acid whenever the amber stop codon is encountered (9).

Sutro Biopharma, a provider of protein-synthesis technology, also is developing a platform for introducing nonnatural amino acids, but in a cell-free translation system that is reported to be scalable to commercial production volumes (10). The system is based on an extract of E. coli, and because it is an open system, the tRNA charged with a nonnatural amino acid can be added directly to the reaction mix as a reagent.