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About Claire Scanlan
Claire Scanlan is a process development scientist at EMD Millipore's Biomanufacturing Science Network

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About Julien Zhao
Julien Zhao leads the emerging biotech initiative for the life science business of Merck KGaA, Darmstadt, Germany, which operates as MilliporeSigma in the U.S. and Canada. He drives the strategic development of emerging biotech market segment in Europe and Asia supporting integrated solutions, thought leadership and creative go-to-market programs. Julien joined MilliporeSigma in 2011 to develop and support the go-to-market strategy for the new solutions portfolio.

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About Mark Rogers
Mark leads the European Biotech Start-Up Initiative in Drug Development (R&D, Biodevelopment) and 2016 European Emerging Biotech Grant Program for the life science business of Merck KGaA, Darmstadt, Germany, which operates as MilliporeSigma in the U.S. and Canada. Mark has a background in molecular biotechnology and neurodegeneration research, where he worked on stem cell-based gene therapy, membrane-based yeast-two-hybrid assays, and embryonic stem cell-based transgenic model generation.

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About Ruta Waghmare, PhD
Ruta is the Global Director, Market Sales Development at the life science business of Merck KGaA, Darmstadt, Germany, which operates as MilliporeSigma in the U.S. and Canada. She has 16 years of experience in biotechnology in the areas of strategy, business development, and biomanufacturing. She joined MilliporeSigma in 2000 and has held several roles including Senior Scientist, Group Manager for Process Development Sciences, and as the Regional Sales Manager for Mid-Atlantic, Mid-West, and South-East US. Ruta has delivered over 65 scientific posters, papers and conference sessions worldwide, including a top feature article in BioPharm International as selected by readers.  She was also a co-innovator for the “run and spike” virus clearance strategy, a breakthrough approach to nanofiltration validation. Most recently, she launched and developed the strategic Emerging Biotech initiative at MilliporeSigma. Ruta received her doctorate in Chemical Engineering from Iowa State University and conducted Post-Doctoral research at the National Institutes of Health, Bethesda, Maryland on novel protein purification and crystallization methods. Ruta is passionate about science, and volunteers as the Chair- Manufacturing Sciences and Engineering Section) with the American Association of Pharmaceutical Scientists (AAPS).

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About Sabrina Xu
Sabrina Xu is the Associate Director for the Emerging Biotech Initiative for the life science business of Merck KGaA, Darmstadt, Germany, which operates as MilliporeSigma in the U.S. and Canada. This initiative’s purpose is to bring the full breadth of MilliporeSigma‘s capabilities to bear on the challenges of the Emerging Biotech market, and to offer holistic solutions for Emerging Biotech development. Prior to this position, Sabrina worked in sales at MilliporeSigma for 8 years, where she started as a key account manager and moved into a sales manager role in China, focused on account management and provide total solution for key accounts from drug development to GMP manufacture. Sabrina understands multinational companies’ quality systems, requirements and challenges for tech transfer(scale up/down).

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About Steve Cross
Stephen Cross is currently a Technology Manager with the life science business of Merck KGaA, Darmstadt, Germany, which operates as MilliporeSigma in the U.S. and Canada. He is based in the U.K. and manages a team of field based Process Development Scientists (PDS) offering scientific and technical support to customers and end users located in UK, Ireland, BeNeLux, Nordics and Russia, as well as occasionally supporting the extended EMEA region. Stephen started with MilliporeSigma in 2003 working in a team which helped develop the successful ProSep® Protein A chromatography range and then transferred to become a PDS in 2005. Prior to joining MilliporeSigma, Stephen completed a Ph.D. at University of Kent (Canterbury, U.K.) and worked for 9 years in scientific and technical roles for various small biotech companies in the U.K

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About Vera A. Katz
Vera Katz is leading the Segment Marketing Organization for Actives & Formulation focusing on ADCs for the life science business of Merck KGaA, Darmstadt, Germany, which operates as MilliporeSigma in the U.S. and Canada. Based in Switzerland, she leads a global team of Segment Marketing Managers supporting ADC customers. Starting in 2006 as a Product Manager for APIs, Vera held various roles in the Marketing department for Actives. Prior to joining MilliporeSigma, Vera completed a PhD at the University of Kaiserslautern (Germany) and worked 5 years in the strategy division of a management consulting company.


Antibody-Drug Conjugates: Manufacturing Challenges and Trends

Published on 21st March

Antibody-drug conjugates (ADCs), a form of Immuno-conjugate or bio-conjugate, are an emerging class of medicines designed for high-specificity targeting and destruction of cancer cells. The mechanism of action is targeted delivery of a cytotoxic agent to the cancer cell via monoclonal antibody targeting of a specific cell surface marker. [1][2][3][4]

Upon binding, a biochemical reaction activates internalization of the ADC into the cell cytoplasm, where the drug becomes active, killing the cancer cell. [5] The ultimate advance with ADC therapeutics is that targeting and release of the drug specifically within the cancer cell means that healthy cells are not adversely affected and cancer cells can be more effectively destroyed. Success in ADC therapeutics stems from a deep understanding around each of the trilogy ‘Antibody- Linker- Payload (drug)’ technologies, with a complementary optimization of all three to generate an effective and potent ADC. [3][4]

Whole IgG has been the gold standard in ADC targeting to date; however, innovation in the field is bearing new formats, including engineered and smaller antibody and antibody-like particles such as Fab fragments, single-chain Fv, and antibody-like scaffolds. [2] A crucial element in ADC complexes is the linking technology. The linker unit may be cleavable or non-cleavable from the drug unit, which affects drug activity and availability.

With non-cleavable ADCs, the linker unit remains attached to the drug, which mitigates externalization and the resulting side effect of the drug entering healthy neighboring cells. With cleavable ADCs, the drug is completely cleaved from the linker unit upon internalization, the antibody is degraded to its amino acid form, and the entire complex becomes active drug. Innovation in linking technology aspires to improve the coupling of payloads as well as to improve cleavage reactions, allowing improvements in payload delivery. The majority of ADC payloads are small molecules, which act via disruption of microtubules or inducing DNA damage. [2][4][6]

Market Insights
Although the first ADC was approved in 2001, it took almost a decade before the next ADC was approved. As of today, only Adcetris® and Kadcyla® are commercially available globally (Zevalin® has been approved in China only). Pioneers Pfizer/Wyeth withdrew Mylotarg® in 2010 after safety issues were observed during a comparative clinical trial.

The ADC market was worth approximately USD 900 million in 2015 with just two approved drugs [7], and its potential remains very large. There are 45 molecules in development, representing more than 300 projects in development: 42% in preclinical stage; 19% in Phase 1; 11% in Phase II; and 3% in Phase III. [8] Over the next decade, we believe that 5-10 new ADC commercial launches will occur, targeting diseases in the areas of oncology (predominantly solid tumors) [9] and immunology (90% of the pipeline).

With 45 ADC projects in development and their expertise in developing and launching Adcetris®, Seattle Genetics is expected to maintain leadership in this space. The company quadrupled the size of their ADC pipeline through multiple licensing deals. [8]

To date, 182 companies (108 in US; 53 in EU; 16 in Asia; 5 in ROW) are actively developing ADCs.[8] Despite this highly competitive market, however, the development of ADCs remains slow and will remain so in the coming years. An estimated 70%-80% of ADC manufacturing is outsourced. Considering the challenges in the development of linkers and payloads, [10] and the fact that only a few contract manufacturing organizations (CMOs) have the capabilities,  the market remains open for new players to emerge who can overcome the manufacturing challenges in this space.

Photo 1.0: Manufacturing scientist preparing for conjugation step used in ADC production.

Technical and Manufacturing Challenges
The biggest technical challenge associated with any drug manufacturing process is to provide consistently efficacious drug product, which is of the required purity and is safe from environmental and process related contamination. This challenge must be accomplished in a manner that protects employees, operators and the general environment from the harmful substances inherent in the process, and at a cost that makes the final drug marketable.

The starting point for ADC manufacturing is the parent monoclonal antibody (mAb). Often the supply of mAbs of suitable quality for therapeutic purposes is taken for granted due to the successful developments in purification templates and platform processes in previous years. [11][12]

It is not within the scope of this article to discuss future developments in mAb processing, but it is interesting to consider how growth trends in the ADC field will modify mAb manufacturing by raising a new set of priorities for mAb processes.  Many manufacturers already design and screen mAbs at an early stage for “manufacturability,” which is the set of physico-chemical properties that will make the molecule robust enough for the manufacturing environment, formulation and dosage requirements. [13] Such considerations include molecule pI, glycosylation and surface reactive sites as well as elimination of structural motifs known to potentiate aggregation or instability. The trend is for mAbs destined for ADC production to be further optimized for robustness against the more demanding process conditions inherent in ADC processes. Such enhanced requirements are likely to add to the existing cost and convenience drivers to stimulate further technological developments in mAb expression and processing.

Future ADCs are likely to utilize novel biological components, such as minimized antibody derived binding moieties (scFv, nanobodies, domain antibodies etc) and molecules with bispecific target binding, which require adaption of the conventional mAb DSP platform or the adoption of new purification templates. [14]

Photo 2.0: Manufacturing scientist working in a glove box to weigh HPAPIs used in ADC production.

ADC process development is complicated by the need to optimize additional process steps that are not present in conventional mAb manufacturing e.g. the antibody-drug conjugation reaction and subsequent drug substance purification. The drug-antibody-ratio (DAR) is a critical quality attribute for the ADC as it defines potency and therapeutic index. [15] The extent of the derivatization of the mAb can also, in extreme cases, adversely affect its biological and pharmacological properties, leading to poor tolerance, less effective targeting and/or stability problems. The choice of conjugation chemistry and linker are critical, and a thorough optimization of the reaction parameters is necessary.  Clinically approved ADCs have used conjugation chemistries with broad group specificity, targeting naturally occurring amine (lysine) or thiol (cysteine) amino acid side chains. [16] Thus, multiple and variable drug incorporation into the ADC is possible, but will have to be controlled to maximize efficacy and to meet the regulatory requirements regarding drug entity definition. [17]

Additional considerations for the control and optimization of the conjugation chemistry include the possible generation of mAb aggregates and drug/linker side reactions, which could lead to subsequent purification and analytical challenges. Many reaction conditions can be controlled and it is necessary to have a thorough understanding of the critical parameters and how their possible interactions affect DAR and final ADC quality.  It is common to assess the critical reaction parameters using a statistical design of experiments . High-throughput screening methods are also advantageous, but require additional capital investment and the provision of complementary high-throughput analytical facilities to ensure maximum efficiency.  DAR can be monitored using RP-HPLC, HIC or analytical IEX, and size exclusion chromatography can be used to detect oligomer and aggregate formation.  More recent innovations point toward improvements in site-specific conjugation chemistries targeting well-characterized and unique sites within the mAb. Such sites may be designed into the mAb structure de-novo by incorporating non-natural amino acids into the protein structure. [18]

Photo 3.0: Purification of a mAb at the Biodevelopment Center in Martillac, France.

The manufacturing challenges inherent with ADCs are attributable to the additional process, safety and analytical requirements conferred by conjugation of the biologic to the highly active cytotoxic component.

An ADC must be manufactured in a Current Good Manufacturing Process (cGMP) aseptic environment whilst also ensuring containment of the highly toxic drug compounds to protect operators and the wider environment, which presents significant operational difficulties. The highest risk of operator/environmental exposure comes with the use of powdered cytotoxic reagents. These operations require the use of low pressure isolators and advanced personal protection equipment with some operations being designated as Safebridge® containment Category 4. [19] Integration of these precautions with the aseptic manufacturing environment requires integrated facility and equipment design/engineering and a multi-disciplinary approach. [20]

Manipulation of the cytotoxic materials in liquid format is facilitated by the use of closed systems where all product contact surfaces are single use and disposable. Not all single use components will be routinely tested for resistance against the organic solvents used to solubilize the hydrophobic drugs used in ADC manufacture and thus dedicated extractable and leachables testing is typically required.

ADC manufacturing facilities require high capital investment and extensive specialized training of operators, which explains the trend towards its domination by specialized CMOs.[21] As ADC pipelines advance, there may be advantages in developing sites where mAb production and conversion to an ADC can be closely combined.

An additional process development and manufacturing challenge includes the subsequent purification of the required ADC sub-population from the post conjugation reaction mixture.  The reaction mixture will include ADC variants with a range of DAR, unincorporated drug, spacer derivatives and organic solvents. Primary purification can be achieved by utilizing the size differential conferred by the mAb. Tangential flow filtration (TFF) can be used to retain the high molecular weight species whilst the underivatized reaction components can be removed by diafiltration.  This process also offers the opportunity to concentrate the ADC and reduce volume for subsequent downstream steps.  The UF/DF purification will likely require additional optimization (compared to the parent mAb process) because the addition of hydrophobic drug moieties to the mAb may result in reduced stability or solubility and an increased propensity to aggregate. Further purification can be achieved using conventional chromatography modalities, with cation exchange having the potential to resolve both mAb-derived aggregates and ADC species showing extremes of DAR.

Operator and environmental protection is also critical during the purification operations and, wherever possible, closed systems and single use components are used.  The availability of pre-packed scalable chromatography columns and ready to use, single use TFF capsules both improve convenience and reduce risk. Final processing stages will include further TFF (UF/DF) to adjust final formulation and conventional sterile filtration to meet regulatory guidelines.

Photo 4.0: A process engineer packing a chromatography column.

ADC manufacturing presents an exciting challenge to the industry. Progress is dependent not only upon innovative research in medicinal chemistries and biologics, but also on the related support industries that supply partnership in engineering, devices and consumables to ADC manufacturers.  The progress and future promise for ADC therapies is validation of the synergy between these fields.

Future Trends

Manufacturing
A robust pipeline exists for ADCs, with over 45 molecules in development. Seattle Genetics and its partners have applied their ADC technology to more than 200 antibodies and have licensed their technology to several companies. [22]

Key areas for future development include new cytotoxic agents as well as new linkers that are adequately stable and at the same time can be cleaved efficiently to deliver the cytotoxic drug. [23] There also continue to be developments in the areas of manufacturing and scale up for this technology, given the cytotoxicity of the drug and the challenges associated with manufacturing the antibody variants. [14]These capabilities are only owned by a handful of CMOs, particularly the conjugation services.  The CMOs who do offer conjugation and linkage services are heavily reliant on single-use technology and continue to push the industry for advances in that area. [24] As ADC technology advances and continues to gain footing and funding in the biotechnology and financial sectors, the CMO marketplace will likely expand.

Supply Chain
The supply chain of an ADC is highly complex, combining development and manufacturing capabilities in pharma and biopharma, and an utmost analytical skill and capability set. But as most companies/CMOs are specialized on a precisely defined niche, such as high potent or linker technology, the ADC developing company must manage a highly complex supply chain with often up to seven or more partners. Therefore, ADC developing companies are demanding a more integrated supply chain solution with one partner covering the majority – or all – of the supply chain. An increasing number of collaborations, strategic alliances and acquisitions during recent years confirm this trend. Further, CMOs who already offer part of the ADC supply chain are entering the market with investments in new conjugation facilities. [25]

Chemistry
To further advance the efficacy of ADCs, new drug platforms and new or improved linker technologies are in development. Today’s drug conjugation strategies yield heterogeneous conjugates, resulting in a relatively narrow therapeutic window. The cell-killing effects of ADCs are obviously highly dependent on the drug to antibody ratio (DAR); therefore, controlling the drug to antibody ratio is a major focus of the development of new linker technologies. [26][27]

ADCs with improved homogeneity will help to balance cytotoxic effects against the side-effects of a therapy.[28] Finally, companies such as Macrogenics also are focusing efforts on more specifically targeting tumors via dual targeting of tumor antigens through bi-specific ADCs.


February 20, 2017 | Corresponding Author: Julien Zhao | DOI: 10.14229/jadc.2017.21.03.001

Received: February 20, 2017 | Accepted for Publication: March 7, 2017 | March Published online March 21, 2017 |

Last Editorial Review: March 20, 2017

Featured Image: Photo of scientists researching in laboratory. Courtesy: © Fotolia. Used with permission.

Photo 1.0, 2.0, 3.0 and 4.0 Courtesy of Merck KGaA, Darmstadt, Germany © 2017 Merck KGaA, Darmstadt, Germany. All rights reserved.

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This work is published by InPress Media Group, LLC (Antibody-Drug Conjugates: Manufacturing Challenges and Trends) and is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Non-commercial uses of the work are permitted without any further permission from InPress Media Group, LLC, provided the work is properly attributed. Permissions beyond the scope of this license may be available at adcreview.com/about-us/permission.


Copyright © 2017 InPress Media Group. All rights reserved. Republication or redistribution of InPress Media Group content, including by framing or similar means, is expressly prohibited without the prior written consent of InPress Media Group. InPress Media Group shall not be liable for any errors or delays in the content, or for any actions taken in reliance thereon. ADC Review / Journal of Antibody-drug Conjugates is a registered trademarks and trademarks of InPress Media Group around the world.


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