Monoclonal antibodies have dominated the biopharmaceutical market for over two decades. Few people doubt that their future success in fields such as oncology, inflammation, and autoimmune diseases owes much to the development of antibodies conjugated to cytotoxic drugs. Like all great innovations, this is a breathtakingly simple concept: Combine the targeting specificity of an antibody with a small-molecule drug as an effector component joined to that antibody by means of a small chemical or peptide linker, and you have a therapeutic strategy suitable for any indication in which cells produce disease-specific antigens. The resulting compound structures are known as antibody–drug conjugates (ADCs), and they direct cytotoxic effectors to specific disease-causing cells, such as cancer and hyperactive immune cells, which are subsequently induced to undergo apoptosis (1). By targeting cell-surface proteins such as receptors or adhesion molecules that are solely expressed on disease cells, but not surrounding healthy cells, the selective elimination of a disease at its source can be achieved without off-target effects (2).

Advancing the Science: Simple concepts are sometimes more difficult to put into practice than expected. It took years of unsuccessful testing before stable ADCs were suitable for clinical development and a small number of pioneering ADC products were eventually launched (3, 4). The first approval was for gemtuzumab ozogamicin (marketed by Wyeth as Mylotarg), an anti-CD33 antibody derivatized with calicheamicin. It was approved in 2000 by the US Food and Drug Administration (FDA) under the orphan drug program for treatment of acute myelogenous leukemia.

The number of investigational new drug submissions for this class of product has soared over the past decade as the industry has gained experience. By 2002, only five products had been approved, but that number had risen to more than 30 by 2012. The two most recent approvals are trastuzumab emtansine (Kadycla), a Genentech product indicated for HER2-positive breast cancer, which was approved in February 2013, and brentuximab vedotin (Adcetris), a Seattle Genetics product indicated for certain types of lymphoma, which was approved in May 2013. Kadycla uses the Herceptin antibody conjugated to an antimitotic agent — mertansine (also known as DM1) — whereas Adcetris combines an anti-CD30 antibody and up to five molecules of the antimitotic agent, monomethyl auristatin E.

Another 74 ADC candidates are currently in development, 70% of which are in the preclinical or early clinical development stages. Given normal attrition rates, we appear to be on the verge of a wave of second-generation ADC products that incorporate secondary metabolites such as mertansine and auristatin, which are significantly more potent than traditional chemotherapeutics.

The burgeoning interest in ADCs (evidenced by a full development pipeline) belies the immense challenges inherent in their manufacturing processes, which are considered in detail by this special issue of BioProcess International. One difficulty is that ADC manufacturing attempts to bring together two manufacturing concepts that have become separated since the birth of the recombinant DNA era. On one hand is manufacturing of the chemical payload, a conventional pharmaceutical manufacturing process, albeit requiring special handling due to the highly potent cytotoxic nature of such drugs. On the other hand is the antibody component, which is a well-understood biopharmaceutical product. The linker used to join those two components may be chemical or biological.

From Science to Technology

From a manufacturing perspective, ADCs are not usually regarded as constituting a self-defining product category but rather as drugs connected to biological entities or as antibodies with chemical conjugates (5). Both views fall short of the reality, which is that ADCs should be considered as a unique class of therapeutic entity with a rational manufacturing process designed from first principles. The current manufacturing framework for ADCs feels like an uneasy compromise in that the manufacture of each component and intermediate must satisfy the same regulatory requirements as the final drug substance itself.

Typical small-molecule drugs, antibodies, and even antibody fusion proteins (which are based on a similar concept to conjugates) are based on one type of starting material, but ADCs require three types of starting material: a master cell bank producing the antibody, a fermentation strain or chemical process producing the linker, and a source for the drug that acts as the effector molecule. Those are subject to the same strict regulatory considerations as the final conjugated product, and each is therefore subject to the same requirements in terms of structural characterization, activity testing, toxicology studies, and definition of an impurity profile as well as steps required to achieve a pure product (6). When the linker is added to either the drug or the antibody as the first part of the conjugation process, the resulting intermediate also must be considered separately.

In this context, coupling the cytotoxic agent can add another layer of microheterogeneity that must be considered as part of the characterization and release process. Site-specific conjugation may soon provide an advanced technical solution to that problem. Indeed, recent progress in development of heterobifunctional coupling agents that generate stable linkers to keep a payload attached to an antibody in a patient’s bloodstream — but control its release and activation once inside a target cell — have streamlined and simplified manufacturing by reducing the likelihood of nonspecific linkages.

Most ADCs in development primarily rely on cleavable hydrazine, disulfide, or dipeptide linkers or noncleavable thioether linkers that rely on antibody degradation to release a payload drug. Recent innovations among ADCs in preclinical development include antibodies and drugs functionalized with specific molecular handles that can be linked using “click” chemistry. It is important to understand that ADCs are highly toxic substances that can be handled by only a few specialized contract manufacturers. Specific segregation requirements must be met, and cross-contamination risks need to be assessed. Integrated production concepts are under discussion. Single-use manufacturing solutions that offer complete containment seem to provide a good starting point for addressing some of these challenges (3).

Unlike the typical manufacturing process for a monoclonal antibody — which begins with a production cell line and progresses through filtration and chromatography steps to capture and polish the final drug substance — an ADC manufacturing process involves additional steps of linker coupling and toxin coupling followed by ultrafiltration/ diafiltration to remove excess toxin and linker. The antibody, linker, and toxin each must be characterized both structurally and in terms of impurities before they can be added to the process. That must ensure the absence of antibody-degradation products, critical contaminants, and drug/linker-related impurities such as degradation products, residual solvents, and heavy metal ions.

Critical process parameters will be based on the molar ratio of drug to antibody because the reaction stoichiometry can be used to control the number of effector molecules linked to an antibody — and thereby control the potency of an ADC (7). All components must pass tests of physical and chemical stability, photostability, and freeze–thaw behavior. And the testing process must take into account the abundance of so-called conjugatable and nonconjugatable impurities: contaminants that are more or less likely to influence the purity of a ADC by undergoing aberrant linking to form unwanted byproducts.

Both the facility for preparing a cytotoxic component and the conjugation unit must be fully contained because such toxins are often lethal at doses as low as one molecule per cell, which presents an extreme exposure risk to manufacturing personnel. Similarly, once an ADC has been processed to remove contaminants, the waste must be inactivated and incinerated on site. The ADC itself then must pass a nearly identical series of tests to those used for its components, although if the antibody involved is well-characterized, then some release tests may not be necessary. The drug/antibody ratio and the drug-loading distribution (homogeneity of the ADC population) are important considerations in addition to the amounts of free antibody, drug, and linker remaining at the end of the manufacturing process.

A Promising Future

ADCs are more potent than antibodies and more targeted than small-molecule cytotoxins, so they can achieve greater therapeutic efficacy while limiting systemic toxicity. However, as discussed elsewhere in this special issue, the manufacturing of ADCs remains a significant challenge at every step, from the unusually stringent demands of the raw material supply chain to the infrastructure for ensuring a safe working environment and the regulatory framework that requires the components, intermediates, and products to be treated as separate entities and tested accordingly.

ADCs are already successful, but they are still an emerging technology. We can look at their ancestors to see what is in store for this product class. The histories of both antibody manufacturing and chemotherapy have shown that novel technologies take time to mature — not only because technical barriers need to be overcome, but also because the most efficient platform processes evolve hand in hand with the regulatory framework as industry and regulators learn from each other. ADCs surely have a bright future, but it will take the combined efforts of innovators in research and manufacturing as well as regulatory agencies before these remarkable products can truly fulfill their potential.

References

1 Train PA. Antibody Drug Conjugates As Cancer Therapeutics. Antibodies 2(1) 2013: 113–129.

2 Teicher BA. Antibody Drug Conjugates. Curr. Opin. Oncol. 26(5) 2014: 476–483.

3 Gottschalk U. Antibody–Drug Conjugates: The Success of Failure. Pharmaceut. Bioproc. 1(4) 2013: 311–313.

4 Wu AM, Senter PD. Arming Antibodies: Prospects and Challenges for Immunoconjugates. Nature Biotechnol. 23(9) 2005: 1137–1146.

5 Rohrer T. Consideration for the Safe and Effective Manufacturing of Antibody Drug Conjugates. Chem. Today 30(5) 2012: 76–79.

6 Miksinski SP, Shapiro M. Regulatory Considerations for Antibody–Drug Conjugates. Academy of Applied Pharmaceutical Sciences Conference 18 October 2012, Toronto, Ontario, Canada.

7 Ducry L, Stump B. Antibody–Drug Conjugates: Linking Cytotoxic Payloads to Monoclonal Antibodies. Bioconjug. Chem. 9(9) 2010: 665–667.

Uwe Gottschalk is pharma/biotech chief technology officer at Lonza Ltd., Muechensteinstrasse 38, 4002 Basel, Switzerland; 41-61-316-8111; [email protected].