Lonza, a specialty contract development and manufacturing (CDMO) partner to the biopharma industry, will add new highly potent API (HPAPI) manufacturing suites at its site in Visp, Switzerland. The expanded capacity is for the specific support of antibody-drug conjugate (ADC) payload manufacturing.
By leveraging the company’s more than 100-year experience in chemistry, biopharmaceuticals and small molecule drug process development and scale-up, Lonza as developed a reputation in manufacturing and support of both clinical development and commercial licensing of antibody-drug conjugates (ADCs).
The expansion is based on a tailored business agreement with a major biopharmaceutical partner that ensures ADC payload supply continuity and flexibility at reduced cost of goods
This latest expansion includes two new manufacturing suites. These new suites enable handling of a variety of highly potent products with occupational exposure levels down to 1ng/m3 and strengthen the overall bioconjugation capabilities of the company.
The expansion underlines the strategic position of antibody-drug conjugates in the Lonza Pharma & Biotech portfolio, with the company developing and producing all components of this increasingly important cancer treatment: cytotoxic payloads, antibodies and the required linkers.
The first of the two new HPAPI suites specifically supports a global biopharmaceutical partner by securing the long-term supply of highly potent ADC payloads.
The second suite will be available to other customers for similar HPAPI and payload development and manufacturing programs. The expansion also increases Lonza’s capabilities in providing fully scalable HPAPI and ADC solutions from lab to commercialization, which supports the accelerated timelines that many drug programs in this category require.
“By ensuring critical supply for the treatment of cancer patients, we are supporting one of our global partners in the oncology field,” said Maurits Janssen, Head of Commercial Development of the API Business Unit at Lonza Pharma & Biotech.
“Oncology continues to be the leading indication in biopharma and the main driver for bioconjugates. We continue to increase capabilities and capacity to meet the HPAPI development and manufacturing needs of our partners,” Janssen added.
Lonza is an established partner in developing and manufacturing HPAPI, with more than 20 years’ experience in safely progressing more than 30 products from early-stage work to late-stage clinical or commercialization. The company has the capabilities in place to safely handle HPAPIs to exposure levels down to 100ng/m3 across all manufacturing scales. These new suites will extend the options for companies developing APIs with even higher potencies.
“Our customers developing highly potent medicines need a partner whom they can trust to handle these toxic substances and to deliver in sync with their needs, whether for clinical or commercial supply,” said Gordon Bates, President Chemical Division at Lonza Pharma & Biotech.
“Combined with our expertise in biologics development, manufacturing, bioconjugation and sterile fill/finish, this new capability will offer further solutions for companies developing complex therapies,” Bates concluded.
Future of ADCs
The expansion confirms Lonza’s belief in the growing global therapeutic potential of ADCs which is, according to experts expected to grow to US $ 4bn by 2023, with double digit approvals within 3-years.
As part of this growth, novel payloads that target tumor-initiating cells on third generation antibody-drug conjugates or ADCs could come to market in the next couple of years. Driven by a healthy late stage pipeline, experts confirm that they expect the market for antibody-drug conjugates to expand at around 19% compounded annual growth rate (CAGR) between 2017 and 2030.
These predictions are based on a new analysis published in the 2018 edition of the CPhI Annual Report – the complete findings of which were released earlier this month at CPhI Worldwide in Madrid, Spain, the global pharmaceutical event held October 9 – 11, 2018.
Lonza’s HPAPI and ADC payload expansion, which is, in part, based on these expectations, is expected to be on-line by the end of 2019.
Disclosure: Lonza is one of the underwriting sponsors of ADC Review | Journal of Antibody-drug Conjugates.
The technology behind antibody-drug conjugates (ADCs) has been around for many years, but so far is without widespread commercial success. Penelope Drake and David Rabuka of Catalent Biologics assess the history and progress to date, and look at what might be preventing ADCs from reaching their full potential.
Two decades ago, antibody-drug conjugates or ADCs were hailed as a major breakthrough, especially in the area of oncology therapeutics. The concept of delivering a potent drug payload directly to the site of the tumor for maximum effect with minimal damage caused to non-cancerous cells was viewed as, if not the Holy Grail of cancer treatment, at least a significant advance towards precision medicine. However, the concept has proved difficult to translate into clinical success.
The first ADC reached the market in 2000, but to date, the U.S. Food and drug Administration (FDA) has approved only four ADC therapeutics. The two most recent were granted approval in 2017, and could mark the start of a new era in which ADCs begin to realize their full potential.
The two drugs approved most recently by the FDA are inotuzumab ozogamicin (Besponsa®) and gemtuzumab ozogamicin (Mylotarg®). Mylotarg, the very first marketed ADC, was originally approved in 2000 for treatment of CD33-positive acute myeloid leukemia (AML).
However, treatment-related toxicity concerns led to its withdrawal from the market in 2010, but it has now been re-approved with a lower recommended dose and altered dosing schedule.
Besponsa was approved for treatment of relapsed/refractory acute lymphoblastic leukemia (ALL).[1,2] They join brentuximab vedotin (Adcetris®), an anti-CD30 monomethyl auristatin E (MMAE) conjugate approved in 2011 to treat relapsed/refractory Hodgkin lymphoma and systemic anaplastic large cell lymphoma, and ado-trastuzumab emtansine (Kadcyla®), an anti-HER2 DM1 conjugate approved in 2013 to treat HER2+ metastatic breast cancer. Kadcyla is currently the only FDA-approved ADC for the treatment of solid tumors.
2.0: An Hybrid Entity
An ADC is very much a hybrid entity, combining both biologic and small molecule characteristics, and consisting of an antibody scaffold covalently modified with a variable number of small-molecule payloads, joined by a chemical linker. The antibody delivers the small molecule specifically to the intended cell type by targeting an antigen that is selectively expressed on tumor cells and internalizes upon antibody engagement. To be an effective therapy, all of these parts of the ADC must be optimized.
Changes to the linker can have a significant effect on the biophysical and functional performance of the ADC, and there are two main conjugation approaches for attaching linkers to antibodies, resulting in either heterogeneous or site-specific payload placement. Currently, the ADC clinical pipeline is still dominated by heterogeneous conjugates, although the functional and analytical advantages of site-specific conjugation  are now being recognized.
The average ratio of conjugated payload to antibody is referred to as the drug-to-antibody ratio (DAR) and this has a strong influence on both the efficacy and toxicity of an ADC. High-DAR ADCs can have poor biophysical characteristics that reduce efficacy and increase toxicity, but these effects can be mitigated using certain conjugation and linker technologies.
3.0 Clinically-tested Payloads
To date, the majority of clinically-tested ADC payloads are either antimitotic/microtubule inhibiting, such as auristatins, maytansinoids and tubulysin, or DNA alkylating (e.g., pyrrolobenzodiazepines, indolinobenzodiazepines, calicheamicins, duocarmycins), although a few other interesting payloads with novel mechanisms of action have been introduced (irinotecan derivatives and α-amanitin).
The past five years however, have seen a dramatic change in the ADC clinical pipeline as preclinical technological advances have started to feed into clinical-stage projects. In early 2013, of the 20 ADCs in the clinic, nearly 80% were heterogeneous conjugates with payloads of antimitotic drugs, namely auristatins or maytansinoids. But between 2013 and 2017, the number of ADCs in clinical trials more than tripled , with site-specific ADCs accounting for nearly 15% of the total. There has also been a trend away from antimitotic payloads towards more potent cytotoxic drugs, particularly DNA alkylators.
The proportion of antimitotic payloads fell from 80% to 65% overall, and accounted for only one-third of site-specific ADCs. This decline can be attributed in part to the unimpressive clinical results of ADCs bearing antimitotic payloads.
According to a recent review , nearly 40% of ADCs bearing maytansine, monomethyl auristatin E (MMAE), or monomethyl auristatin F (MMAF) that entered clinical trials were later discontinued, presumably due to lack of efficacy or (rarely) excessive toxicity.
However, the highly potent DNA alkylating payloads carry an increased risk to patients and the fine line between potency and safety is one that scientists and regulators are still striving to achieve. The first site-specific ADC to reach the clinic, vadastuximab talirine, is an anti-CD33 antibody conjugated through engineered cysteine residues in the heavy chain to yield a DAR 2 molecule and is the first clinical ADC to bear a pyrrolobenzodiazepine (PBD) payload, a highly potent DNA alkylator.
It began clinical phase 1 trials in mid-2013, but the phase 3 trial was recently terminated due to toxicity concerns, even though the drug showed a 70% complete remission rate for AML patients.
4.0: Mechanisms of toxicity
Meaningful improvements in ADC technology are expected to continue as preclinical studies focus on understanding the mechanisms of ADC toxicity, developing approaches for reducing off-target toxicities, and improving patient outcomes through changes in both ADC composition and clinical trial study design.
As yet, most clinical experience has been with ADCs carrying antimitotic payloads, which show prominent organ toxicities in the hematopoietic compartments and in the liver. Much less is known about the clinical effects of dosing DNA alkylators, although targeting of the hematopoietic compartments has been shown in clinical trials.
A deeper understanding is needed of the absorption, distribution, metabolism, and excretion (ADME) and drug metabolism and pharmacokinetics (DMPK) fates of both the intact conjugate and its small molecule component. Knowing where the drug goes and how it is processed will enable connections to be drawn with commonly observed clinical toxicities.
A 2015 review of toxicity studies  concluded that ADC toxicity was not driven by target antigen but rather by linker/payload: ADCs sharing the same linker/payload composition tended to reach the same maximum tolerated dose, even when their target antigens showed endogenous expression in completely different tissue/organ compartments.
This sobering observation revealed how much progress still needs to be made to achieve specific cytotoxic payload delivery to tumor cells without damaging healthy tissues. But it also offers a possible explanation for the high failure rate of 2013 era ADCs.
It is likely that the lack of clinical benefit observed for some ADCs was the result of an inability to dose to an efficacious level due to off-target toxicities driven by the linker/payload.
If ADC off-target toxicity can be controlled, then it is likely that the maximum tolerated dose can be increased, perhaps leading to better clinical response to treatment.
This work is published by InPress Media Group, LLC (ADCs – The Dawn of a New Era?) 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.
Antibody-drug conjugates or ADCs are complex immunoconjugates. They are designed to selectively deliver a small-molecules cytotoxic payload to cancer cells. Directed to specific tumor antigens, antibody-drug conjugates consist of a monoclonal antibody linked via a molecular linker to a cytotoxic agent. 
In addition to the targeting monoclonal antibody, the linker technology is crucial. The linker needs to be sufficiently stable in circulation to allow the payload to remain attached to the antibody while, at the same time should allow efficient release of an active cell-killing agent after the antibody-drug conjugate is internalized.
After binding to a specific antigen on the surface of cancer cells, the ADC is internalized where, inside the cell, the cytotoxic payload is released to kill the malignant cell. Today, these cytotoxic payloads include two microtubule-disrupting agents maytansinoids and auristatins as well as a DNA-targeting antibiotic, calicheamicin.
These payloads are included in a number of antibody-drug conjugates approved by the U.S. Food and Drug Administration (FDA). These agents include brentuximab vedotin (Adcetris®; Seattle Genetics) for Hodgkin and anaplastic large cell lymphoma, ado-trastuzumab emtansine (Kadcyla®, also known as T-DM1; Genentech/Roche) for HER2-positive metastatic breast cancer, gemtuzumab ozogamicin (Mylotarg®; Pfizer) for acute myeloid leukemia and inotuzumab ozogamicin (Besponsa®; Pfizer) for the treatment of acute lymphoblastic leukemia.
In addition, nearly 180 other agents are in development – from early stage discovery to advanced stages of clinical development. These novel agents including sacituzumab govitecan for breast cancer, mirvetuximab soravtansine for ovarian cancer, rovalpituzumab tesirine (Rova-T) for lung cancer, depatuxizumab mafodotin for glioblastoma, and oportuzumab monatox for bladder cancer.
While today four antibody-drug conjugates are successfully implemented in clinical strategies, the majority of these ADC are used in liquid, hematological, cancers. The number of antibody-drug conjugates in the treatment of solid, non-hematological, tumor is limited. Most ADCs focusing on solid tumors have not progressed beyond Phase I clinical trials, suggesting that there is an unmet need to optimize additional factors governing translational success.
The first approved antibody-drug conjugates were approved for the treatment of hematologic malignancies. Gemtuzumab ozogamicin is an anti-CD33 antibody conjugated via an acid–labile linkage to calicheamicin. The second approved antibody, brentuximab vedotin, included an anti-CD30 antibody conjugated via a cleavable valine-citrulline (vc) dipeptide linker to the microtubule-disrupting agent monomethyl auristatin E (MMAE).
The first antibody-drug conjugate to be approved for the treatment of non- hematologic, solid tumors was ado-trastuzumab emtansine. This antibody-drug conjugate was developed by conjugating the sulfhydryl group of maytansinoid DM1 to lysine amino groups of the anti-human epidermal growth factor receptor 2 (HER2) antibody, trastuzumab, via reaction with the bifunctional non-cleavable linker, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).
While antibody-drug conjugates have been successfully included in therapeutic strategies for the treatment of patients with various forms of malignancies, there is a growing number of agents for which clinical development programs have been discontinued because of insufficient activity at the maximum doses that can be tolerated upon repeat administration. This is especially the case in solid, non-hematological, tumors.
Alternatived to current technologies
Current antibody-drug conjugate-technologies focus on large, whole immunoglobulin formats. Many of these ADCs have been developed with site-specifically conjugated payloads with a DAR (drug to antibody ratio) of 2 or 4.
As discussed above, the majority of ADCs have not had much success in the treatment of solid, non-hematological, tumors. As result, leading researchers are now exploring alternatives, smaller formats-drug conjugates, including single domain antibody fragment–drug conjugates, single-chain formats such as the scFv, diabodies (head-to-tail dimer of a scFv) and small immuno-proteins (SIPs-scFvs dimerised using a CHε4-domain, approximately half the size of an monoclonal antibody), from 80 kDa to around 1 kDa in total size, which have better penetrating properties as well as more rapid pharmacokinetics (PK).
Discussed in a review by Mahendra P. Deonarain, Gokhan Yahioglu and colleagues, working for Antikor Biopharma, in Stevenage Herts, United Kingdom, and the UK Department of Chemistry, Imperial College London, London, United Kingdom, published in the June 2018 edition of Antibodies (Volume 7, Issue 2), both practical studies and theoretical reviews support the idea that smaller antibody fragments may have faster diffusion and extravasation coefficients and penetrate tumors more rapidly than monoclonal antibodies.
In general, these alternative agents are potent in vitro, particularly the more recent ones incorporating auristatins or maytansinoids. However, due to the more rapid clearance, the potency profile of these smaller compounds changes when being tested in vivo. Strategies to manipulate the PK properties, while, at the same time, retaining the more effective tumor penetrating properties, may, as being discussed by Deonarain and colleagues, make small-format drug conjugates viable alternative therapeutics to the more established antibody-drug conjugates.
Antibody drug conjugates (ADCs) are a relatively new type of drug that combines the targeting ability of a biologic with a highly potent cytotoxic agent.
This powerful combination promises to become a game-changer in the fight against cancer—potentially replacing broad spectrum chemotherapies with more specific, less damaging options. At the same time, because ADCs’ cell-killing drug payloads are thousands of times more toxic than conventional treatments, safety concerns are proportionally amplified. That makes gaining regulatory approval for first-in-man studies far more demanding than with a traditional biopharmaceutical.
While it’s natural for pharmaceutical developers to focus on toxicological and pharmacological findings from animal studies, far too often, early stage ADC developers underestimate the importance of their filing’s Chemistry Manufacturing and Controls (CMC) section. This may result in regulatory requests for additional information or unanticipated studies, which can delay or even permanently derail a promising program.
This white paper discusses a pragmatic approach to helping ADC developers ensure IND success. It highlights two main challenges:
Complexity of the ADC molecule
Insufficient CMC data
This publication outlines strategic and analytical approaches that can save time and effort, and help ensure that regulatory requirements for CMC data are satisfied. It suggests that the best way to accelerate the regulatory path to first-in-man studies is to focus the CMC development plan on three areas:
Critical Quality Attributes (CQA)
Frequently overlooked studies
1.0 Antibody-drug Conjugates and the IND Process
Before human clinical trials can commence in the United States, new drugs must go through a complicated and time-consuming Investigational New Drug (IND) application and approval process. An IND application must demonstrate complete pharmaceutical or biopharmaceutical analyses. In addition to extensive data from animal pharmacology, toxicology studies, clinical protocols and investigator information, it must include detailed Chemistry, Manufacturing and Controls (CMC) information on the manufacturing and stability of the clinical trial material (CTM).
When it comes to clinical studies with ADCs, additional scrutiny of CTM is to be expected. The inherent instability of biologics, together with the level of toxicity associated with an ADC’s small molecule payload have grave implications on patient safety. It is not surprising, then, that CMC data requirements and the level of analytical support needed to support an ADC program are substantially greater than with more traditional therapies.
According to the editors of ADC Review / Journal of Antibody Drug Conjugates, “One of the most critical aspects is to address all the unique issues involved in the submission of an IND completely, correctly, and in a timely fashion…” 
Incomplete or incorrect information can result in requests for additional studies, delaying the filing of a successful IND or worse—the financially motivated end to an otherwise promising program. But with a well-planned approach to testing and diverse technical/analytical expertise on your team, ADC developers can avoid these pitfalls and help ensure a seamless path to the clinic.
2.0 Why ADC development is so hard
According to the 2016 Nice Insight CDMO Outsourcing Survey, 57% of companies surveyed said they were developing ADCs, compared to 51% who said they have naked monoclonal antibodies (mAbs) in development. Another source states that 182 companies around the world have ADCs in their pipeline. Despite this surge, only four ADCs have been licensed to date. Plenty of examples exist of drugs that showed potential in early pre-clinical stages, but didn’t progress, and were terminated. Many of these failures were due to toxicity or incomplete characterization data.
This white paper deals with two of the most common challenges relating to IND approval for ADCs. These are:
The complexity of the ADC molecule itself, which is critical, as analysis of this complex structure informs decisions about its design and manufacture.
Lack of necessary CMC data on the clinical trial Material
2.1 Challenge #1 – The complexity of the ADC molecule
The analytical challenges unique to ADC development are numerous, but chief among them are the complexity and stability of the mAb, the very difficult synthesis and characterization of the small molecule payload (cytotoxic agent) and linker, the chemical linking chemistry, and different conjugations that may be involved. 
Understanding the structure and behavior of biologically derived molecules–and interpreting analytical findings to inform development decisions—requires a myriad of analytical techniques and experienced biopharmaceutical scientists.
Few Contract Manufacturing Organizations (CMOs) have the breadth of testing services required for full biopharmaceutical analysis. Not surprisingly, an estimated 70%-80% of ADC analysis is outsourced.
ADC analysis also requires expertise handling highly cytotoxic compounds. Because the potency of ADC payloads is much greater than biologic drugs, it is crucial to truly understand the role that each part of the ADC – mAb, linker and cytotoxic agent – plays in the toxicity, stability and safety of a new drug.
Linkers: improvements in linker design focus on serum stability and drug-to-antibody ratio (DAR). The overall concern with linkers is to produce more homogenous ADC populations by studying the conjugation between linker and mAb.
Payloads: choosing the right payload involves certain basic criteria, such as solubility, stability, and the likelihood of conjugation. But ascertaining the correct drug potency also has proven to be a critical factor. According to McCombs et al, “poor clinical efficacy of first-generation ADCs is attributed to sub-therapeutic levels of drug reaching the target.”
The IND analytical package must include not only assays and purity analyses, but also the drug-to-antibody ratio (DAR) and site(s) of conjugation. Only advanced biopharmaceutical analysis can supply this information.
Selecting the right analytical techniques is critical. Valliere- Douglass et al. suggest that conventional analytical methods used for standard biopharma characterization are not sufficient for ADCs. They outline the latest methods in mass spectrometry that have helped scientists fully characterize ADC drugs when conventional techniques fall short.
A list of analytical services and techniques necessary for ADC characterization is given in Part 4 of this white paper.
2.2 Challenge #2 – Failure to provide sufficient CMC data
One of the primary reasons IND submissions for new ADCs are delayed is because the biopharma company (or their contract service provider) fails to perform analyses in accordance with Chemistry, Manufacturing and Controls (CMC) guidelines.
This is because nine times out of ten, the drug developer lacks a clear plan for meeting CMC data requirements when mapping the development process.16 In fact, a key factor in streamlining your IND submission for a new ADC is finding a development partner who can help you articulate a well-planned CMC strategy early in the project.
Complete structural characterization, physico-chemical testing, and biophysical analysis of the antibody-drug conjugate are required. This includes the parent monoclonal antibody, as well as analysis of biological activity, toxicity, and stability of the drug product. Table 1 on the following page shows the structural analysis needed for the mAb intermediate.
As already mentioned, ADC analysis is more complex than traditional biopharmaceutical analysis. Multiple biopharma studies and analytical methods are required, as well as concurrent expertise in performing these techniques and interpreting the data.
Table :Necessary analysis of mAb to meet CMC guidelines, and corresponding analytical techniques
Bottom line: you may find traditional techniques used for biopharmaceutical analyses are quickly becoming obsolete. New, highly sensitive and specific technologies are becoming the standard, and are indispensable if you are to progress through the clinic ahead of your competition.
3.0 Why traditional approaches fall short
The complexity of the ADC molecule and lack of emphasis on CMC development strategy are the primary causes for delays in ADC IND approvals. But since most early stage developers lack internal analytical resources, they must partner with consultants or CROs who understand regulatory guidance and can help them navigate the IND process. They also need access to a full suite of cGLP and cGMP-compliant analytical testing services. But it can be difficult to find a partner with the experience and capabilities necessary to step into this role.
There are two primary reasons why the choice of outsourcing partners can be especially critical for ADC developers:
Older techniques are unable to provide the analyses necessary for ADC molecules – the stability of specific molecules cannot be determined, and a deep understanding of the molecule may not be possible.
Absence of a Plan
All too often, early stage developers lack a defined CMC strategy. When this is the case, archived samples often aren’t set aside, validation reports and studies are inconclusive, and compatibility studies are overlooked—all of which can lead to delays and/or insufficient data. In the absence of a clearly defined testing strategy, analytical methods are not in place to ensure the identity, strength, quality, purity and potency of the drug. These are required for every New Drug Application (NDA).
Finally, according to an article by Amer Alghabban in Pharmaceutical Outsourcing: “The way a pharmaceutical company contracts CROs/ CMOs has a critical and direct impact on a company’s realization of its goal”
Alghabban states that many manufacturers – 45.6% in one survey–have reported quality problems with their vendors, inexperience with regulatory requirements, and 49.1% of vendors were not able to keep their promises.
Ultimately, current practices fail to overcome the two challenges outlined in section 2 because ADC developers partner with the wrong CRO.
4.0 Three ways to streamline the IND Process for ADCs
There are proven ways to increase your chances of successfully filing an IND for a new ADC, and at the same time reduce the amount of effort and expense involved.
Complete characterization and protein analysis play the most important part in this process. This means characterizing attributes such as the drug-to-antibody ratio (DAR) and sites of conjugation. DAR is a critical factor for ADCs, because it represents the average number of drugs conjugated to the mAb. The DAR value influences the drug’s effectiveness, as low toxin loading lowers potency, and high toxin loading can negatively affect pharmacokinetics (PK) and toxicity. Sites of conjugation are important, because improving site-specific drug attachment can result in more homogeneous conjugates and allow control of the site of drug attachment.
There are several considerations that can accelerate time-toclinical trials for an ADC. These include:
Analyzing critical quality attributes, or CQA
Developing a defined testing plan to ensure no necessary studies are overlooked, such as compatibility and residual solvent analysis—and a schedule that ensures the most efficient and timely completion
Adopting platform approaches to ADC development
The following sub-sections will address each of these in turn.
4.1 Conduct Detailed Studies of Critical Quality Attributes
Critical quality attributes (CQA) are biological, chemical and physical attributes that are measured to ensure the final drug product maintains its quality, safety, and potency. The precursor to defining CQAs is complete characterization of the drug product and intermediates.
Currently, characterization of the mAb intermediate is already well defined, and includes studies such as:
Mass Analysis — Intact, reduced, deglycosylated
Peptide Map (UPLC–UHR QTof MS): sequencing, Post Translational Modifications (PTMs) and disulfide linkages
N-Glycan Profile Site, extent and structure of glycosylation
Differential scanning calorimetry
CQAs (relating to safety and efficacy of the drug) for an ADC product also include the following additional assays:
Appropriate analytical techniques
Drug-to-antibody ratio (DAR)
Drug load distribution
Peptide map-UPLC-UHR QToF
Peptide map-UPLC-UHR QToF
Linker payload structure
FTIR, UPLC/MS/MS, NMR
Table 2:CQAs for an ADC relating to safety and efficacy, and corresponding analytical techniques
Additional attributes considered CQA, due to their impact on health and efficacy include:
Free drug concentration
Free Drug Concentration
As mentioned earlier, the FDA is concerned primarily with human safety in regards to an IND submission. With ADCs, this means they are concerned with the concentration of free drug (toxin) in the final product — both on release and on stability. While the main advantage of ADCs is their targeted specificity, any free toxin introduced into the bloodstream is a serious threat to human health and safety. Therefore, any assay used to measure free drug concentration must be exceptionally sensitive (≤1 ng/ mL). This is typically performed by UPLC/MRM/MS.
Antigen binding is vital to the efficacy and specificity of an ADC. Non-specific binding results in the death of healthy cells and toxicity. Techniques to measure binding include:
Enzyme-linked immunosorbent assay (ELISA) – a biochemical technique for detecting and quantifying peptides, proteins and antibodies. Multiple formats can be utilized, but all incorporate binding of an antibody to the analyte resulting in a subsequent signal (UV, fluorescence, phosphorescence).
Electro-chemiluminescence (ECL) – a detection method based on luminescence from electrochemical reactions. ELISA and ECL can be used interchangeably, but ECL’s greater sensitivity allows it to be used in other studies, streamlining the IND process.
Surface Plasmon Resonance (SPR) – a label-free method used to monitor noncovalent molecular interactions in real time. Generally considered a poor candidate for antigen binding, due to poor inter-day precision.
While all of the physico-chemical analyses (CE, icIEF, SEC, etc.) provide an idea of the purity and stability of a single aspect of an ADC, they do not provide a measure of the functional stability of the entire molecule. Cell bioassays are the ultimate measure of an ADC’s activity, stability and 3-dimensional structure, as they measure the effect of all degradation pathways. Bioassays, by their very nature, are variable and are technique-dependent, making them difficult to utilize as part of your IND submission. While research quality bioassays are sufficient for drug development; a qualified, accurate cell bioassay is an absolute requirement for an IND application. Optimizing these assays to make them precise and robust requires expert and experienced scientists. They provide a method that can be confidently used for stability and post-IND formulation development. Upon IND approval, these studies should be initiated immediately, shortening formulation/ process optimization.
4.2 Perform Studies that are often overlooked
A successful IND depends on multiple studies – particularly relating to toxicology – that are often overlooked, or even neglected. This is due to a lack of planning early on in the process. And these oversights can result in delays of several months.
A number of overlooked studies should be performed prior to initiation of toxicology and other early clinical tests. These include:
Infusion set/syringe compatibility
Toxicology studies are performed at low doses and require greater sensitivity than release/stability assays. As required by the FDA, dose formulations must be assayed for toxicology studies, to ensure the correct dose is being delivered. The typical approach is ECL or ELISA. If ECL is developed for release, it is easily adapted to these studies, streamlining the overall IND process.
Infusion Set/Syringe Compatibility
Concern has been raised about the occurrence of critical incidents related to infusion sets. Every drug developer and CRO needs to establish a set of procedures to evaluate infusion sets from their vendors, particularly in terms of drug loss to surfaces. This includes filters, pre- and post-IV bags, and tubing. Multiple concentrations and durations should be tested.
According to the FDA: “The purpose of in-use stability testing is to establish a period of time during which a multiple-dose drug product may be used while retaining acceptable quality specifications once the container is opened.” 
The FDA recently announced a draft GIF #242 entitled “In- Use Stability Studies and Associated Labeling Statements for Multiple-Dose Injectable Animal Drug Products”. The draft will outline how to design and carry out in-use stability studies to support the in-use statements, for multiple-dose injectable drug products.22 While this focuses on animal and multi-dose studies, the draft also reflects the importance the FDA places on in-use stability for human trials, and yet they are often neglected during the IND process.
Multiple stability-indicating assays are required, including:
Size Exclusion Chromatography (SEC)
Micro Flow Imaging (MFI)
The linkage of the payload to the monoclonal antibody is an organic chemical event involving many of the typical solvents and catalysts. Therefore, similar to traditional pharmaceuticals, both residual solvents and heavy metals must be monitored on release of the drug substance. Typical assays include:
4.3 Adopt a “Platform’ Approach
The basic idea behind a platform approach is to leverage “prior knowledge” to reduce the effort needed to start clinical trials. It begins with identifying a class of molecules that show comparable characteristics, such as physico-chemical properties and stability profiles.
New candidates with characteristics that match known molecules can be treated as a “next-in-class” candidates. Once comparable characteristics are validated, developers can focus additional testing on areas of difference between the new candidate and historical likenesses—reducing testing requirements and at the same time further adding to the body of shared knowledge related to the platform, and increasing the platform’s robustness. Adopting a platform approach can significantly streamline IND testing requirements, accelerating time to clinic and reducing costs. According to Bradl et al., the platform approach enabled biopharmaceutical development for toxicological studies within 14 months after receiving DNA sequences.  After another six months, material from GMP facilities was provided for clinical studies. This resulted in a time requirement of 20 months from DNA to Investigational Medicinal Product Dossier.
Of course, a key element is actually identifying those molecules that match the definition of a “next-in-class” candidate. Careful planning in regards to methods, data, and documentation will provide a universal approach applicable to other antibody drug conjugates.
Standardization of instrumental parameters, data collection and data manipulation can speed up characterization. The necessary studies include:
1. QToF – An ultra-high resolution Quadrupole Time of Flight MS, coupled to a UPLC can provide the vast majority of characterization data. Powerful QToF software, designed specifically for proteins, deconvolutes complicated mass spectra, simplifying data interpretation. The QToF can determine:
Post translational modifications
Payload linkage sites
2. Release and Stability:
The majority of assays are similar for all ADCs: SDS CE, icIEF, SEC, UV, and DAR. Generic assays can be qualified directly and only modified/optimized if qualification criteria are not met.
Design method qualifications appropriate to Phase I and template protocols
Binding assays should all utilize ECL. The sensitivity of this technique allows it to be used for toxicology and compatibility studies, as well as release and stability.
Other investigations typically include prophylactic studies in anticipation of agency questions. While they are not necessarily required for the IND filing, having data to support responses to agency questions will prevent delays. By preparing data in an IND-ready format, you’ll ensure “drag and drop” of the data, greatly facilitating the process in the typical last minute rush to complete the IND.
5.0 Buyer’s Guide: Choosing the right CRO for Fast IND Submission and Approval
According to a report by Global Industry Analysts, Inc., the global biopharma market is estimated to reach U.S. $ 306 billion by the year 2020.25
With this continued market expansion, including antibody drug conjugate development, there is a greater need for contract lab support. Not only this, but there is a critical need for high-quality contract laboratory partners who understand the regulatory guidelines, can perform required risk assessments, and can develop, validate and execute challenging analytical procedures.
If you’re looking for help from a CRO to reduce risk, and increase your chances of a successful IND submission, here’s what you need to look for:
True loyalty and partnership
You need a CRO that will take complete ownership of your product, and not just treat it like another sample. A CRO that partners with you closely – and isn’t simply a vendor – means they form a core part of your team, and have a personal stake in your success. They’re hands-on, and keep you updated every step of the way. Whatever CRO you choose, be sure they make their experts available to you at all times. They should take part in meetings, telecons, kickoff calls, and be involved in every stage of the process.
Significant scientific expertise in biopharmaceutical development and biopharma services is a must. A large proportion of the CRO staff should be made up of Ph.D. scientists and biopharma veterans. The CRO should assign scientific advisors that act as connections between your team and theirs. Their expertise and scientific background means they can accurately map out the entire process, from development to IND submission.
The right experience
Ideally, your CRO should have experience supporting successful IND submissions under tight deadlines. They should also have a solid track record of working on multiple biopharma products over several years. These drugs should span a wide range, from monoclonal antibodies and antibody-drug conjugates, to biosimilars and pegylated proteins. All projects need to be backed by an exceptional regulatory record.
Flexibility is important when the unexpected happens. Your CRO needs to work closely with you to determine the best analytical approaches. Their flexibility (and scientific expertise) means the CRO can think outside the box when things don’t go according to plan. They can quickly identify alternative ways of getting things done. In fact, finding novel ways to characterize and understand biopharmaceutical behavior is often necessary to file a successful IND.
Full range of analytical biopharma services. The complexity and heterogeneity of ADCs mean they are exceptionally challenging to characterize. A full suite of analytical services is necessary to do this. Be sure to ask your CRO about their capabilities, and what biopharma services they offer. As mentioned in this white paper, you need to be sure your CRO won’t overlook anything, and can help you meet CMC regulations. Their scientists should be experts in these techniques and interpretation of their data. At a minimum, these techniques should include cell-based bioassay development and analysis by ultra high resolution QToF, as well as routine release and stability testing.
6.0 Case Study: CMC Suport for ADC Development Situation
Virtual client had very aggressive timelines for submitting INDs for two antibody drug conjugates within 12 months. The Client requested complete chemistry support for the CMC section of the IND
In collaboration with the client’s scientists, EAG proposed a fast-tracked method development and validation program to meet their timelines. EAG scientists performed complete characterization of the mAb and drug product, including complete sequencing, PTMs, and glycan analysis. Developed and validated multiple methods for release and stability including: icIEF, ELISA, cell bioassay, DAR, free drug, N-linked Glycan, SEC, CE-SDS, and HCP
All data was delivered to the client within the deadline, and both INDs were submitted on schedule
Both INDs were successful, and the FDA had no observations/ remarks regarding the EAG’s portion of the IND. Our client’s priorities changed during the study, requiring additional studies beyond the scope of the original project. We were able to accommodate these changes and still meet their deadlines. EAG scientists were fully involved in project kick-offs.
Finding a CRO who can partner with you to accelerate your antibody drug conjugate IND submission is challenging. It’s not easy to determine which CROs can truly partner with you to help you achieve your objectives.
This white paper has outlined two critical challenges with ADC development. Specifically, these challenges relate to successfully filing an IND. They are:
The complexity of the ADC molecule
Failing to meet CMC regulations
Given these challenges, there are 3 ways to streamline the IND process:
Characterize all critical quality attributes
Perform studies that are often overlooked
Adopt a platform approach
ADC, antibody drug conjugate; DAR, drug-to-antibody ratio; CMC, Chemistry Manufacturing and Controls; IND, Investigational New Drug; ELISA, Enzyme-linked immunosorbent assay; ECL, Electro-chemiluminescence; SPR, Surface Plasmon Resonance.
ADCs, Antibody-drug Conjugates, Characterization, Chemistry Manufacturing and Controls (CMC)
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