Adeline Siew is editor for Pharmaceutical Technology Europe. She is also science editor for Pharmaceutical Technology.

Adeline Siew, PhD

	Analytical testing, which deals with characterization of raw materials and finished dosage forms, plays an important role in pharmaceutical manufacturing and all phases of drug development. In this roundtable article, industry experts discuss how advances in analytical testing tools have helped address challenges in pharmaceutical analysis. Industry experts include: Guillaume Tremintin, market area manager, Biopharma, Bruker Daltonics; Lisa Newey-Keane, marketing manager, Life Science Sector, Malvern Instruments; Gurmil Gendeh, marketing manager for Pharmaceuticals at Shimadzu; Kyle D’Silva and Simon Cubbon, both Pharma and BioPharma marketing managers at Thermo Fisher Scientific; and Geofrey Wyatt, president of Wyatt Technology Corporation. 

: How have the tools for analytical testing of small molecules and biologics evolved over the past 10 years?

: Developments in analytical instrumentation for pharma have largely been addressing three key trends over the past decade. The first is the significant rise in small-molecule generic drug development. This development has increased the requirement for instruments that offer performance-relevant measurements and/or high informational productivity for deformulation and the demonstration of bioequivalence (BE).

	The second important trend is the shift towards continuous manufacture (CM). The potential ease of scale-up of continuous processes and the ability to file on the basis of full-scale experimental data makes CM particularly interesting for pharma, but its realization relies on effective monitoring and automated control. Here, the ongoing transition of core analytical techniques to fully integrated, online implementation is enabling rapid progress.

	Finally, in biologics, we’ve seen growing awareness of the importance and power of orthogonality when it comes to probing the complex nature of the proteins. Identifying the optimal set of biophysical characterization techniques is crucial to comprehensively elucidate critical aspects of behavior, such as stability. Significant progress has been made in this area, and there is now growing awareness of how to complement traditional techniques with newer/less well-established ones, such as differential scanning calorimetry (DSC) and Taylor dispersion analysis (TDA), to maximize understanding and add value.

: The tools have gotten more sophisticated, specific, and in some cases, more expensive too. In the never-ending quest to learn more about small molecules and biologics, the FDA has encouraged the use of orthogonal testing techniques. As a result, greater insight and understanding of these molecules has been possible.

: What we have seen evolve is the increased usage of high-resolution accurate-mass instruments (i.e., moving from instruments where you have a limited level of insight to higher resolutions that gives users the ability to confidently determine the identity of a target molecule). On an ultrahigh-resolution quadruple time of flight (QTOF) mass spectrometer (MS), such as the Bruker maXis II, the accurate-mass and the precise relative intensities of the isotopes allows derivation of a molecular formula or a protein monoisotopic mass. On the Fourier transform ion cyclotron resonance (FT-ICR) MS side, such as solariX XR, one can even resolve the hyperfine structure of the isotopic pattern and directly read the molecular formula of the ion being measured. Building confidence and getting deeper insights through high resolution has been the biggest change.

: Two key areas that have contributed to accelerated evolution of biologic characterization have been the widespread adoption of high-resolution accurate mass (HRAM) spectrometry and advancement of software tools that deliver meaningful answers to non-expert users. A decade ago, characterization of biologics demanded expert mass spectrometrists to both acquire and interpret data, typically done manually over days and weeks. Instrument variability and difficulty of operation meant that a high degree of skill was required for the results to be meaningful. Today, platforms such as the Thermo Scientific Q Exactive BioPharma with Thermo Scientific BioPharma Finder software have democratized mass spectrometry. Software tailored for drug quality attribute monitoring easily transfers this data to actionable knowledge with automated reports, reducing the demand for specialist operators. As well as being easier to use, the systems are also smaller and more affordable than a decade ago. Consequently, we see greater adoption of these technologies, not just in research laboratories, but also further down the drug pipeline in development; in chemistry, manufacturing, and control (CMC); and even in quality control (QC) environments as biologic manufacturers seek to implement multiple attribute monitoring (MAM) methods using HRAM mass spectrometry to replace a host of chromatographic testing methods performed for QC lot release.

: We have seen an increasing adoption of ultra-high performance liquid chromatography (UHPLC) and sub-2-micron column chemistries for both small and large molecule analysis that allows faster, better, and higher resolution chromatography. As both small molecule and large molecule become more complex, we have also seen the development and increasing adoption of higher resolution technologies, for example, two-dimensional liquid chromatography (2D-LC) and liquid chromatography combined with mass spectrometry (LC/MS/MS). High-throughput, automated, and streamlined sample preparation that is fully integrated into analytical workflows has been adopted. Moreover, data systems have been adopted that not only integrate LC and LC/MS but the complete portfolio of analytical instrumentation, including spectroscopy, with seamless data analysis and reporting that are in full compliance to existing standards.

: What are the most significant recent advances in analytical testing tools that have helped speed up and improved the success rate of drug development? 

: The most significant recent advances have been high-resolution measurements that enable users to accelerate their protein characterization tasks. Because of the simplicity of the assay, customers can perform measurements upstream in their development. They have better chance of identifying problems on a lead candidate earlier in the development, which avoids spending too much money down alleys that will lead to failure or costly remediation. Users of maXis II have been able to use this type of measurement to detect incorrect glycosylation sites very early in the development at a stage when they are still able to send the molecule back to the molecular engineering department to optimize the molecule before advancing it any further. There also is a renewed interest in MALDI ionization to perform simple characterization tasks such identity testing of a recombinant product without the need for a chromatography modality.

: When developing biologics, the biopharmaceutical industry look to achieve full structural insight into their candidate molecules, with high confidence, as fast as possible. They look to fail candidates fast. Technologies such as ion exchange chromatography using pH gradients were first proposed by Genetech in 2009 for profiling of therapeutic protein variants. Since then, unique patented products such as Thermo Scientific CX-1 pH Gradients Buffer Kits have led to a 10-fold reduction in analysis time over conventional salt gradients, dramatically accelerating drug development time. 

	Understanding the primary structure of a biologic candidate is critical step performed early in the development process, but also at every stage after. This was traditionally a labor-intensive and time-consuming, 24-hour process to prepare the sample for analysis. Today, we see biopharmaceutical developers able to perform protein sample preparation in 45 minutes and achieve 100% coverage when mapping their drug candidates. The software technology for analyzing peptide digests has also increased its throughput dramatically. The time for biopharmaceutical comparative modification data interpretation can be taken down from two weeks to one day. 

: Mass spectrometers have definitely seen the biggest advances in terms of speed, resolution, and sensitivity gain over the past decade. The Shimadzu triple quadrupole mass spectrometer LCMS-8060, for example, delivers high sensitivity, fast scan speed, and fast polarity switching that enable collection of high-quality data and information. Having these features allows big pharma to fail candidates early, fast, and cheaply in preclinical studies, thus accelerating the pace of drug discovery and bringing safer and more efficacious drug candidates to market faster. 

: The tools we know best are those that we develop. Our DynaPro Plate Reader II (DynaPro PRII) is but one example. Until it came into being, the only way to make a dynamic light scattering (DLS) measurement was to use one-at-a-time, batch measurement techniques in small cuvettes. The measurement required an operator to prepare a sample, pipette it into a cuvette, then place the cuvette in the instrument, close a lid, make a measurement, open the lid, remove the cuvette, and, as the saying goes ‘lather, rinse, and repeat.’ 

	The DynaPro PRII has taken industry standard well plates of 96-, or 384-, or 1536-well formats and enabled them to be used as massively multiple cuvettes. An entire plate of hundreds or thousands of small molecules or other biologics can be pipetted into the wells, the well plate inserted in the instrument, and … that’s it. The technician can walk away and do other work while the instrument makes DLS measurements, ramps temperatures, etc. In fact, the DyanPro PRII enables researchers to perform tasks that were heretofore impossible. The daunting challenge of, for example, screening thousands of samples at different pH levels, or investigating promiscuous inhibitors, is no longer an impediment to making huge numbers of DLS measurements.

: Morphologically-directed Raman spectroscopy (MDRS) is a relatively new technique that combines the capabilities of automated imaging and Raman spectroscopy. It delivers particle size and shape data, along with chemical identification for individual particles in a blend, making it a powerful tool for deformulation as well as many other pharmaceutical applications. MDRS data was recently used to establish in-vitro bioequivalence, in lieu of clinical trial data, in the approval of a generic nasal spray application by Apotex, highlighting its value in the time-critical world of generic drug development.

	A relatively new addition to the biophysical characterization portfolio is TDA, which provides ultra-low volume, solution-based molecular size measurement. Technology that combines TDA with Poiseuille flow for relative viscosity assessment has been shown to be valuable for robustly identifying drug candidates with a poor developability profile early in the drug development pipeline, thereby saving time and money. TDA extends label-free measurements into highly complex solutions and is able, for example, to detect and size monomeric insulin, even in the presence of its hexameric form.

	Finally, our latest technology launch exemplifies the trend of enhancing instrumentation to make it easier for researchers to access robust data for secure and effective decision-making. The MicroCal PEAQ-DSC is a fully-automated differential scanning microcalorimeter that extends the use of DSC-the ‘gold standard’ for protein stability assessment-throughout the development cycle. Key features include: unattended 24-hour operation; automated data analysis; built-in automated cleaning; and self-validation protocols. 

: With so many features and functions on new analytical instruments (such as capabilities to measure various parameters using one equipment), does the industry or the people using them actually understand the concept behind these measurements, how the results are obtained, and how to interpret them correctly?

: This is a frightening and very realistic possibility. Too often vendors sacrifice ease-of-use for ‘black boxes’--instruments that simply produce numbers. The researcher has little indication of whether the numbers mean something or whether they may as well be random because there is something wrong in the measurement itself or with the sample preparation. At Wyatt Technology, our customers come to Santa Barbara, CA to participate in Light Scattering University, a course that demystifies light scattering by teaching some of the theory, practice, sample preparation, and data interpretation. The result is that we create a cadre of customers who have an understanding of the powers-as well as the limitations-of the measurements that the instrument makes.

: Given productivity pressures, well-documented skill shortages, and the ever-increasing number of analytical techniques routinely deployed, it’s important to consider what level of understanding is actually optimal. For example, if you are carrying out routine QC, then a highly automated, standard operating procedure-driven, modern instrument with integrated method and data quality checking software can eliminate any need for understanding or interpretation, and will effectively remove any subjectivity in data analysis. Techniques such as laser diffraction have already matured to this point. Indeed, it could be argued that for many industrial applications, modern systems can now be more effectively differentiated on their ability to make their performance accessible than on their performance itself. 

	Conversely, if you are investigating a more complex concept, such as protein stability, then greater understanding is vital, and orthogonal testing is key. Here, the analytical instrumentation expert understands best the merits and limitations of the technology, but the researcher knows their sample and what information is most useful, so collaboration is vital. Interaction with applications specialists can be incredibly helpful, and the best instrumentation companies invest heavily to develop relevant in-house expertise. However, embedding expertise in instrumentation software is also an important and growing trend that has the potential to greatly enhance the support available to industrial users at the benchtop. 

: It is the responsibility of the instrument manufacturers to deliver companion software solutions that harness the hardware capabilities and enable the users to gain the insights they need. For example, the recently released BioPharma Compass 2.0 software takes high-resolution protein measurements and translates them to actionable information such as a glycosylation profile or degradation levels. Similarly, the matrix-assisted laser desorption/ionization (MALDI) PharmaPulse solution takes 100,000s of measurements and derives possible active ingredients against a defined substrate. This substantially improves the library screening approach by providing high throughput while not requiring any labels.

: Analytical instrument companies such as Shimadzu continuously innovate not only new analytical instruments but also complete solutions that solve challenging real-world problems. Many of the early product and solution conceptualization and development are driven through partnership and collaboration with the industry. These new solutions are initially used by the industry partners or collaborators, who are often people that clearly understand the technology, the results, how to interpret the data, and more importantly, the value the data brings to their work. Over time, these new solutions are improved and packaged into ‘analyzers’ for the mainstream users. As an example, Shimadzu recently released one such ‘complete analyzer’ solution for the biopharmaceutical industry. The Shimadzu Cell Culture Profiling ‘analyzer’ combines optimized and validated UHPLC coupled with a triple quadrupole mass spectrometry method to simultaneously analyzes 95 compounds and metabolites in cell culture supernatant. The multi-parameter data from the LC/MS/MS platform is crucial in the cell culture process development and media optimization to promote cell growth; rebalancing media components and quantities by introducing new media components at variable levels to support continuous manufacturing; to monitor cell development to determine optimal harvest endpoints; or when it comes to development of cost-effective media.

: Traditionally, trying to understand a certain aspect of a complex biologic would require various techniques and painstaking analysis of any data-once you’d managed to acquire it, that is. Ensuring that manufacturers meet the ever increasing challenges that their customers face requires them to listen and develop instrumentation that simplifies analysis, alongside impactful software that drives scientists to their ultimate goal: results.

	The Thermo Scientific Q Exactive BioPharma platform, for example, allows scientists to analyze complex biotherapeutics using a single system: from native, intact, and sub-unit mass analysis through to peptide mapping, with minimal training and maintenance. Combined with Thermo Scientific BioPharma Finder integrated software, you have a data collection and interpretation workflow that streamlines scientists’ access to their results, allowing them to make rapid, informed decisions, rather than spend time setting systems up and learning complex software platforms. So, provided scientists have the right tools (no matter how complex they may be), the industry is in good stead to be able to acquire and interpret those all-important results.

: What areas are still lacking that make analytical testing challenging and what developments can we expect to see in this field over the next 5 to 10 years?

: Where you see the most movement is in terms of sample preparation and data processing. The big changes will be bringing more automation to streamline the sample preparation and make it more consistent and less dependent on the skill of the operators. On the back end, it would be to have smarter software that is able to process data faster, more effectively, thus allowing companies to compare larger data sets and derive trends and have feedback on the processes based on analytical results. Additionally, chromatography remains a challenge for some compounds, and so the improvements in ion mobility will add an additional dimension of separation that can help solve problems that challenge conventional chromatography techniques. For example, the Bruker trapped ion mobility separation (TIMS) has enabled the separation of isomers that until now could not be separated. 

: Biologics are large and complex molecules, and sample preparation for their characterization and bioanalysis (e.g., absorption, distribution, metabolism, and elimination [ADME]/toxicology) has always been a challenge. We can expect to see more innovation and advancement not only in the automation but also in innovative chemistries to simplify the sample preparation workflows. The Perfinity platform from Shimadzu is one such example that fully automates tryptic digestion and brings sample preparation online with LC-MS. Shimadzu also recently introduced a nano-technology based antibody bioanalysis sample preparation solution called the nSMOL Antibody Bioanalysis Kit. This kit performs selective proteolysis of the antibody Fab region and greatly simplifies and streamlines antibody bioanalysis sample preparation. We expect to see many more similar innovations in nano-technology and micro-fluidic based sample preparation for the biopharmaceutical industry in the next five to 10 years.

: For biologics, the informational need is still being refined, particularly when it comes to biosimilars and the demonstration of BE; and analytical requirements are evolving in tandem with this. Getting more information from each sample remains crucial as this enables information gathering earlier in the drug development pipeline. Robustly identifying the best candidates as early as possible clearly cuts the time and cost of biopharmaceutical development. 

	The application of orthogonal techniques is bringing significant challenges in terms of data handling. The quantity of data is one issue, but a more taxing one is how to handle what can appear to be divergent data sets. Software platforms that integrate and rationalize data from an optimal set of techniques will be the way forward. 

	In terms of online implementation, we already have techniques such as laser diffraction particle size analysis that have successfully completed this transition but, over the coming years, others will follow suit. Such instrumentation will need to deliver exemplary reliability as CM matures, but will also be judged on ease of integration as automated control becomes a more routine feature of pharmaceutical processing.

: The list of molecular characterization assays demanded by regulators for biologic drugs is extensive. Typically, more than 30 individual chromatographic or immunoassays are performed to test for critical quality attributes of drug candidates by biologic manufacturers, which is both labor- and time-intensive. 

	Over the next five years we will see technologies emerging to amalgamate multiple assays into one and simplify the process. An area where this is already being implemented in the area of multiple attribute monitoring (MAM) using HRAM-a single workflow capable of replacing multiple individual steps. Several companies already have drug candidates in late-stage development using this workflow. In 10 years, this technology will move from a laboratory process off-line, to being beside the bioreactor at/on-line to inform the manufacturer immediately of the structural properties of their candidate molecules. 

: I believe artificial intelligence (AI) will lend its benefits to our industry in the not-too-distant future. With more data being generated, it may become harder to distill information from the data. AI holds the promise of aiding the data interpretation and becoming wiser, the more data to which it’s exposed.

Articles

Technology improvements in analytical testing tools have helped increase understanding of molecules and speed up development processes.

Evolution of Analytical Tools Advances Pharma, But Challenges Remain

	Antibody-drug conjugates (ADCs) are targeted therapies that combine a specific antibody or antibody fragment linked to a drug. This drug targeting approach has enabled better tumor penetration with less side effects in the treatment of cancer. Developing ADCs, however, requires careful selection of drug, antibody, and linker. David Simpson, CEO of Glythera, and Ian Evetts, commercial director of Glythera, spoke to 

 about the challenges involved in optimal ADC design. 

In ADC development, what are the key considerations when selecting the drug, antibody, and linker?

 ADCs are generally designed to incorporate highly potent, normally intolerable, anticancer cytotoxics; assuming, of course, that the resulting ADC construct is a stable entity with no de-drugging potential. De-conjugation of the drug–linker component from the antibody outside of the cancer cell is the main driver of an ADCs toxicity and adverse event profile. ADC stability is solely controlled by the linker attachment chemistry. With stable attachment chemistry, highly potent payloads can be selected with confidence, enhancing cell kill potential. Although there is also premise to combine antibodies to mid-potency agents, especially where the antibody itself has anti-tumor effects, the use of mid-potency payloads is generally uncommon amongst ADC innovators.

 Preclinical studies aligned with commercial considerations suggest that criteria for the ideal cytotoxic should include:

		Sensitivity of the cancer type to the class of the cytotoxic (e.g., antitubulin agents are frequently used in breast cancer)

		Pan-tumor activity, especially relating to solid tumors

		Activity in multi-drug resistant (MDR) models

		Ability to induce cell death (i.e., high toxicity).

	The cytotoxic also should be chemically tractable and stable while bound to the monoclonal antibody (mAb). In addition, drugs must contain a suitable functional group for conjugation and need to be stable under physiological conditions. It is important that therapeutically appropriate amounts of the cytotoxic are linked to the mAb to optimize the potential effectiveness of the ADC once it reaches the target cancer cell. 

	In general, we should avoid drugs that are associated with neurotoxicity (e.g., platinum-based therapies or vinca alkaloids and first-generation taxanes).

	The drugs currently being used to construct ADCs generally fall into two categories: microtubule inhibitors and DNA-damaging agents, although other drugs such as polymerase II inhibitors are also under investigation. Microtubule inhibitors bind tubulin, destabilize microtubules, and cause G2/M phase cell cycle arrest. DNA-damaging agents include anthracyclines, calicheamicins, duocarmycins, and pyrrolobenzodiazepines (PBDs). These drugs function by binding the minor groove of DNA and causing DNA stand scission, alkylation, or cross-linking. The cytotoxins are highly potent, with free drug IC

 Tolerability of an ADC is also affected by the specificity of antigen expression in cancerous tissue vs. normal tissue; non-specific expression results in toxicity and reduced efficacy due to a reduction in the dose of conjugate available to the tumor. Therefore, the ideal tumor antigen must be specifically localized to the tumor cell-surface to allow ADC binding and, preferably, display differential expression between tumor and normal tissue, with increased expression in cancer cells. 

	Antigen choice is also dictated by its ability to internalize upon ADC binding. Internalization of an ADC occurs through receptor-mediated endocytosis followed by ADC degradation in the lysosome, and this leads to free drug release for effective cell killing. However, endocytosis is not guaranteed for all cell-surface antigens, and the rate of internalization can vary considerably. Optimal drug release into the cell requires minimal recycling of the ADC to the cell surface as well as enhanced delivery of an internalized antigen/ADC to the lysosome. The ideal tumor antigen, then, should be cell-surface expressed, highly upregulated in cancer tissue, internalized upon ADC binding, and able to release the cytotoxic agent inside the cell.

 Of course, antibody selection also plays a critical part in the success of an ADC. Of a number of key attributes, high specificity for the tumor antigen is essential because an antibody that binds non-specifically or cross-reacts to other antigens can be taken up in normal tissues, inducing toxicity as well as elimination of the ADC before it can reach the tumor. Efficiency of uptake requires high affinity binding to the target antigen (KD < 10 nM), and antibodies should also be minimally immunogenic. Ideally, antibodies should also possess optimal pharmacokinetic properties including relatively long half-lives and slower clearance in plasma.

 We should clarify the role of chemistry of the linker and the chemistry used for attachment of the drug–linker adduct to the antibody. Linkers are generally selected based on the preferred method for drug release from the antibody (i.e., depending on whether cleavable or non-cleavable linkers are used). However, the attachment chemistry used to conjugate the drug–linker adduct to the antibody determines the overall stability of the ADC and is, therefore, crucial in maximizing efficacy and minimizing intolerability. So, although the selection of linker to attach to a drug is an important factor in the manufacture of an ADC, the ADC stability and, hence, clinical performance is wholly governed by selection of appropriate attachment chemistry to attach the drug–linker adduct to the antibody. Selecting the right linker and attachment chemistry can ensure maximum cell-killing activity by avoiding de-drugging and off-target tolerability. 

 Developed linkers have all suffered from stability issues largely due to use of maleimide chemistry for attachment of the drug–linker conjugate to the antibody, and reductions in drug: antibody ratios (DAR) over time have been shown for both cleavable and non-cleavable linker formats. In this situation, the drug–linker adduct is cleaved before entering the tumor cell, reducing the potency of the treatment and releasing the cytotoxic drug into systemic circulation. 

	Several groups have focused on enhancing the stability of ADCs through stabilizing conjugation, although most have concentrated on next-generation maleimide chemistry approaches, primarily via catalysis of ring hydrolysis. Through controlling the microenvironment around the succinimide thioether, stability of maleimide conjugation can be improved, because solvent accessible sites slow down retro-Michael reactions. If cysteine is close to positively charged entities, rapid succinimide hydrolysis occurs, preventing further linker-maleimide exchange. Also, modifying maleimide with the insertion of an amine group can shift the reaction through hydrolysis. However, these approaches don’t necessarily lead to the desired reduction in adverse events or to an increase in 

 efficacy, at least for cleavable linker systems, for which less obvious increases in exposure have had no real impact on ADC efficacy. Although ring-opening stabilizes the conjugate, the long-term stability of the ring-opened product is not precisely known. High pH conditions required for mild hydrolysis methods of stabilizing conjugation induce uncontrollable de-drugging and asparagine deamidation.

 What recent advances have you seen in linking technologies? 

 A recent development of linker systems has actually been focused on the conjugation platform with a need to improve stability due to significant advances in toxin potency and the potential for off-target toxicity. In addition, our improved understanding of the stoichiometry associated with maximal ADC efficacy has re-focused innovation to alternative conjugation solutions and, in particular, site-directed technologies.

	A focus area for many major pharma companies has been to improve the tolerability of ADCs by reducing the de-drugging potential of highly potent toxins from the antibody, which results in free and active drug capable of circulating to alternative and off-target sites. Recent developments have included modification of existing maleimide-based technologies to reduce the potential for the retro-Michael reaction 

, to the development of fundamentally changed, novel chemistry solutions that no longer employ the maleimide component, resulting in significantly improved stability profiles and tolerability.

	The ongoing innovation in toxin development has improved both potency and broad tissue type efficacy resulting in the increased use of engineered cysteines to overcome the instability issues associated with many of the approved platforms. In addition, we see the development of entirely novel approaches through alternative conjugation targets, many of which focus on the use of sugar-based conjugation to the antibody which, by definition, reduces the potential drug loading and, therefore, reduces the risks associated with high levels of free drug and tolerability.

 Can you elaborate on the manufacturing challenges for ADCs?

 Bringing together biologics and toxic payloads has presented major challenges through the history of drug conjugates and more so now with the rapid expansion of target products entering human trials, which require cGMP manufacturing. Drug substance manufacturing to date has generally been achieved through the combined efforts of multiple partners for the production of both the biologics and toxin chemistry components as drug substance and then their combination safely in a single facility. As the clinical pipeline has grown, with more lead candidates requiring cGMP manufacturing, many contract manufacturing organizations (CMOs) have been quick to adapt to the market needs through expansion of their own biologics or chemistry capabilities with additional conjugation suites. However, as with all contract manufacturing models, the ability to undertake multiple manufacturing campaigns using different toxins and antibodies presents its own challenges in terms of control and segregation.

	With the advent of increasingly potent cytotoxins used in the development of ADCs, facility design and the containment systems have required significant redesign to be able to cope with scale, demand, and operator safety. Working procedures have been a keen focus to reduce operator risk of exposure to these toxins through either inhalation or transdermal contamination. .

Safe and effective drug targeting with ADCs requires careful selection of drug, antibody, and linker. 

Targeting Drug Delivery with ADCs

Antibody-drug conjugates (ADCs) are targeted therapies that combine a specific antibody or antibody fragment linked to a drug. This drug targeting approach has enabled better tumor penetration with less side effects in the treatment of cancer. Developing ADCs, however, requires careful selection of drug, antibody, and linker. David Simpson, CEO of Glythera, and Ian Evetts, commercial director of Glythera, spoke to 

 about the challenges involved in optimal ADC design. 

:
	In ADC development, what are the key considerations when selecting the drug, antibody, and linker?

: ADCs are generally designed to incorporate highly potent, normally intolerable, anticancer cytotoxics; assuming, of course, that the resulting ADC construct is a stable entity with no de-drugging potential. De-conjugation of the drug-linker component from the antibody outside of the cancer cell is the main driver of an ADCs toxicity and adverse event profile. ADC stability is solely controlled by the linker attachment chemistry. With stable attachment chemistry, highly potent payloads can be selected with confidence, enhancing cell kill potential. Although there is also premise to combine antibodies to mid-potency agents, especially where the antibody itself has anti-tumor effects, the use of mid-potency payloads is generally uncommon amongst ADC innovators.

. Preclinical studies aligned with commercial considerations suggest that criteria for the ideal cytotoxic should include:

: We should clarify the role of chemistry of the linker and the chemistry used for attachment of the drug-linker adduct to the antibody. Linkers are generally selected based on the preferred method for drug release from the antibody (i.e., depending on whether cleavable or non-cleavable linkers are used). However, the attachment chemistry used to conjugate the drug-linker adduct to the antibody determines the overall stability of the ADC and is, therefore, crucial in maximizing efficacy and minimizing intolerability. So, although the selection of linker to attach to a drug is an important factor in the manufacture of an ADC, the ADC stability and, hence, clinical performance is wholly governed by selection of appropriate attachment chemistry to attach the drug-linker adduct to the antibody. Selecting the right linker and attachment chemistry can ensure maximum cell-killing activity by avoiding de-drugging and off-target tolerability. 

	ADC stability. Developed linkers have all suffered from stability issues largely due to use of maleimide chemistry for attachment of the drug-linker conjugate to the antibody, and reductions in drug: antibody ratios (DAR) over time have been shown for both cleavable and non-cleavable linker formats. In this situation, the drug-linker adduct is cleaved before entering the tumor cell, reducing the potency of the treatment and releasing the cytotoxic drug into systemic circulation. 

	Several groups have focused on enhancing the stability of ADCs through stabilizing conjugation, although most have concentrated on next-generation maleimide chemistry approaches, primarily via catalysis of ring hydrolysis. Through controlling the microenvironment around the succinimide thioether, stability of maleimide conjugation can be improved, because solvent accessible sites slow down retro-Michael reactions. If cysteine is close to positively charged entities, rapid succinimide hydrolysis occurs, preventing further linker-maleimide exchange. Also, modifying maleimide with the insertion of an amine group can shift the reaction through hydrolysis. However, these approaches don’t necessarily lead to the desired reduction in adverse events or to an increase in in-vivo efficacy, at least for cleavable linker systems, for which less obvious increases in exposure have had no real impact on ADC efficacy. Although ring-opening stabilizes the conjugate, the long-term stability of the ring-opened product is not precisely known. High pH conditions required for mild hydrolysis methods of stabilizing conjugation induce uncontrollable de-drugging and asparagine deamidation.

: What recent advances have you seen in linking technologies? 
	

: A recent development of linker systems has actually been focused on the conjugation platform with a need to improve stability due to significant advances in toxin potency and the potential for off-target toxicity. In addition, our improved understanding of the stoichiometry associated with maximal ADC efficacy has re-focused innovation to alternative conjugation solutions and, in particular, site-directed technologies.

: Can you elaborate on the manufacturing challenges for ADCs?

: Bringing together biologics and toxic payloads has presented major challenges through the history of drug conjugates and more so now with the rapid expansion of target products entering human trials, which require cGMP manufacturing. Drug substance manufacturing to date has generally been achieved through the combined efforts of multiple partners for the production of both the biologics and toxin chemistry components as drug substance and then their combination safely in a single facility. As the clinical pipeline has grown, with more lead candidates requiring cGMP manufacturing, many contract manufacturing organizations (CMOs) have been quick to adapt to the market needs through expansion of their own biologics or chemistry capabilities with additional conjugation suites. However, as with all contract manufacturing models, the ability to undertake multiple manufacturing campaigns using different toxins and antibodies presents its own challenges in terms of control and segregation.

	With the advent of increasingly potent cytotoxins used in the development of ADCs, facility design and the containment systems have required significant redesign to be able to cope with scale, demand, and operator safety. Working procedures have been a keen focus to reduce operator risk of exposure to these toxins through either inhalation or transdermal contamination. 


	Volume 41, Number 8
	August 2017
	Pages: 24-27

	When referring to this article, please cite it as A. Siew, “Targeting Drug Delivery with ADCs,” 

Safe and effective drug targeting with ADCs requires careful selection of drug, antibody, and linker.

Targeting Drug Delivery with ADCs

	Evaluating a drug’s efficacy in the body requires taking into account factors that influence the internal environment of cells as most drug targets are located inside the cell. One of the main challenges in early drug discovery is achieving sufficient drug levels within the target cells. The ability to measure the amount of drug locally available to bind intracellular targets is, therefore, crucial for identifying a lead candidate. However, methods for determining intracellular drug levels are lacking.

 a new label-free method for quantifying intracellular bioavailability, which could be used to predict exposure of small molecules to the target cells and hence the drug’s efficacy. The method is based on measuring the concentration of unbound drug available to exert a therapeutic effect inside the cell. It takes into account drug molecules that bind to various cell components, which are then unavailable and unable to exert its intended effect. The researchers demonstrated that by using this new method to measure intracellular bioavailability, it is possible to predict the effects of the drug in various more advanced cell models for specific therapeutic areas, such as cancer, inflammation, and dementia. 

	The method is also being investigated for its potential to predict drug efficacy in the body. Systemic bioavailability, defined as the amount of drug in the blood, may not accurately correlate to a therapeutic effect because the proportion of drug entering the target cell could be higher or lower than that in blood. For example, if the drug molecules bind to other cellular components, get metabolized, or are transported out of the cell, the available fraction of drug inside the cell will be reduced (i.e., intracellular bioavailability will be lower and there are less free drug molecules available to exert its pharmacological action). Correction factors must, therefore, be introduced in pharmacokinetic studies.

	“Our preliminary studies show that replacing the correction factors with a simple determination of local bioavailability in the cells seems to be possible,” Professor Per Artursson, who is heading the study, commented in a 

. “But more experiments are required before we know how applicable our principle is at tissue and organism level. Clearly, intracellular bioavailability is on the way to becoming an important early instrument in pharmaceutical drug research.”

The new label-free method for quantifying intracellular bioavailability can be used to predict drug exposure to target cells and hence its efficacy.

Scientists Develop New Method for Determining Intracellular Bioavailability

	When referring to this article, please cite it as A. Siew, “Advances in Analytical Testing Tools for the Bio/Pharma Industry,” 

Industry experts discuss how advances in analytical testing tools have helped address challenges in pharmaceutical analysis.

Advances in Analytical Testing Tools for the Bio/Pharma Industry

	This year marks the 40th anniversary of our sister publication 

 and it is our tradition to reflect on how far the industry has come over the past four decades. The global pharmaceutical market continues to grow, but one thing that stands out is the increasing need to balance cost and value. Despite criticisms on Pharma’s ability and willingness to adapt to change, the industry has made manufacturing improvements to meet market demands for cost efficiency while ensuring that product quality is not compromised. 

	Anil Kane, executive director, Global Head of Technical & Scientific Affairs at Patheon, notes that the shrinking drug discovery pipeline has led to significant changes in dosage form development and manufacturing strategies. Existing molecules are being evaluated for newer indications in the same or different therapeutic categories, he says. Drug developers are exploring the possibility of combining new drug entities with off-patent drug candidates to produce synergistic effects or other clinical benefits.

	Biotech has contributed to the discovery and manufacture of large-molecule drugs and the development of biosimilars, observes Mike Arnold, board chair of the International Society for Pharmaceutical Engineering (ISPE). He adds that medicines are becoming more personalized to the benefit of the patient. 

	Technology has made a significant impact on the pharmaceutical industry, Jim Agalloco, president of Agalloco & Associates, says. Automation and computerization of systems have reduced manual labour requirements and the potential for mistakes, he says; and data can now be assembled, analyzed, and used in ways not previously possible.

	In reviewing the history of contract manufacturing and outsourcing trends, Matthew Moorcroft, vice-president, global marketing for Cambrex, reflects how services and capacities have evolved in response to changing pharma models--from small-scale boutique operations specializing in individual technologies or chemical processes, to the rise of one-stop shops and low-cost providers in India and China. 

	These are just some of the trends shaping the industry. You can access the complete content of 

	When referring to this article, please cite it as A. Siew, “40 Years of PharmTech,” 

Pharmaceutical Technology marks 40 years of publishing.