An enzymatic deconjugation method for the analysis of small molecule active drugs on antibody-drug conjugates

Antibody-drug conjugates (ADCs) are complex therapeutic agents that use the specific targeting properties of antibodies and the highly potent cytotoxicity of small molecule drugs to selectively eliminate tumor cells while limiting the toxicity to normal healthy tissues. Two critical quality attributes of ADCs are the purity and stability of the active small molecule drug linked to the ADC, but these are difficult to assess once the drug is conjugated to the antibody. In this study, we report a enzyme deconjugation approach to cleave small molecule drugs from ADCs, which allows the drugs to be subsequently characterized by reversed-phase high performance liquid chromatography. The model ADC we used in this study utilizes a valine-citrulline linker that is designed to be sensitive to endoproteases after internalization by tumor cells. We screened several proteases to determine the most effective enzyme. Among the 3 cysteine proteases evaluated, papain had the best efficiency in cleaving the small molecule drug from the model ADC. The deconjugation conditions were further optimized to achieve complete cleavage of the small molecule drug. This papain deconjugation approach demonstrated excellent specificity and precision. The purity and stability of the active drug on an ADC drug product was evaluated and the major degradation products of the active drug were identified. The papain deconjugation method was also applied to several other ADCs, with the results suggesting it could be applied generally to ADCs containing a valine-citrulline linker. Our results indicate that the papain deconjugation method is a powerful tool for characterizing the active small molecule drug conjugated to an ADC, and may be useful in ensuring the product quality, efficacy and the safety of ADCs.     

抗体偶联药物的研究进展与质量控制

抗体偶联药物（antibody-drug conjugates,ADC）因其良好的靶向性及抗癌活性目前已成为抗肿瘤抗体药物研发的新热点和重要趋势,受到越来越多的关注。ADC药物由单克隆抗体、高效应的细胞毒性物质以及连接臂三部分组成,它将抗体的靶向性与细胞毒性药物的抗肿瘤作用相结合,可以降低细胞毒性抗肿瘤药物的不良反应,提高肿瘤治疗的选择性,还能更好地应对靶向单抗的耐药性问题。与传统单抗药物相比,因其结构复杂,ADC药物质量属性分析方法的建立具有更大的难度和特殊性。对抗体偶联药物的研发现状、质量属性分析方法和挑战以及质量控制要点进行了简要介绍,为ADC药物的研究和质量控制提供参考。     

Insights from native mass spectrometry approaches for top- and middle- level characterization of site-specific antibody-drug conjugates

Antibody-drug conjugates (ADCs) have emerged as a family of compounds with promise as efficient immunotherapies. First-generation ADCs were generated mostly via reactions on either lysine side-chain amines or cysteine thiol groups after reduction of the interchain disulfide bonds, resulting in heterogeneous populations with a variable number of drug loads per antibody. To control the position and the number of drug loads, new conjugation strategies aiming at the generation of more homogeneous site-specific conjugates have been developed. We report here the first multi-level characterization of a site-specific ADC by state-of-the-art mass spectrometry (MS) methods, including native MS and its hyphenation to ion mobility (IM-MS). We demonstrate the versatility of native MS methodologies for site-specific ADC analysis, with the unique ability to provide several critical quality attributes within one single run, along with a direct snapshot of ADC homogeneity/heterogeneity without extensive data interpretation. The capabilities of native IM-MS to directly access site-specific ADC conformational information are also highlighted. Finally, the potential of these techniques for assessing an ADC's heterogeneity/homogeneity is illustrated by comparing the analytical characterization of a site-specific DAR4 ADC to that of first-generation ADCs. Altogether, our results highlight the compatibility, versatility, and benefits of native MS approaches for the analytical characterization of all types of ADCs, including site-specific conjugates. Thus, we envision integrating native MS and IM-MS approaches, even in their latest state-of-the-art forms, into workflows that benchmark bioconjugation strategies.     

Impact of linker-drug on ion exchange chromatography separation of antibody-drug conjugates

ABSTRACT

Charge variants are important attributes of monoclonal antibodies, including antibody-drug conjugates (ADCs), because charge variants can potentially influence the stability and biological activity of these molecules. Ion exchange chromatography (IEX) is widely used for charge variants analysis of mAbs and offers the feasibility of fractionation for in-depth characterization. However, the conjugated linker-drug on ADCs could potentially affect the separation performance of IEX, considering IEX separation relies on surface charge distribution of analyte and involves the interaction between analyte surface and IEX stationary phase. Here, we investigated weak cation exchange chromatography (WCX) for its application in analyzing three ADCs (two broad distribution ADCs and an ADC with controlled conjugation sites) and the 2-drug/4-drug loaded species isolated from the two broad distribution ADCs using hydrophobic interaction chromatography. The major peaks in WCX profile were characterized via fraction collection followed by capillary electrophoresis-sodium dodecyl sulfate or peptide mapping. Results suggested that both the number of drug loads and conjugation sites could impact WCX separation of an ADC. The hypothesis was that the linker drugs could interfere with the ionic interaction between its surrounding amino acids on the mAb surface and column resin, which reduced the retention of ADCs on WCX column in this study. Our results further revealed that WCX brings good selectivity towards positional isomers, but limited resolution for different drug load, which causes the peak compositions of the two broad-distribution ADCs to be highly complex. We also compared results from WCX and imaged capillary isoelectric focusing (icIEF). Results showed that separation in icIEF was less influenced by conjugated linker drugs for the ADCs studied in this work, and better alignment was found between the two techniques for the ADC with controlled conjugate sites. Overall, this work provides insights into the complexity of WCX analysis of ADCs, which should be considered during method development and sample characterization.     

In-depth structural characterization of Kadcyla® (ado-trastuzumab emtansine) and its biosimilar candidate

The biopharmaceutical industry has become increasingly focused on developing biosimilars as less expensive therapeutic products. As a consequence, the regulatory approval of 2 antibody-drug conjugates (ADCs), Kadcyla® and Adcetris® has led to the development of biosimilar versions by companies located worldwide. Because of the increased complexity of ADC samples that results from the heterogeneity of conjugation, it is imperative that close attention be paid to the critical quality attributes (CQAs) that stem from the conjugation process during ADC biosimilar development process. A combination of physicochemical, immunological, and biological methods are warranted in order to demonstrate the identity, purity, concentration, and activity (potency or strength) of ADC samples. As described here, we performed extensive characterization of a lysine conjugated ADC, ado-trastuzumab emtansine, and compared its CQAs between the reference product (Kadcyla®) and a candidate biosimilar. Primary amino acid sequences, drug-to-antibody ratios (DARs), conjugation sites and site occupancy data were acquired and compared by LC/MS methods. Furthermore, thermal stability, free drug content, and impurities were analyzed to further determine the comparability of the 2 ADCs. Finally, biological activities were compared between Kadcyla® and biosimilar ADCs using a cytotoxic activity assay and a HER2 binding assay. The in-depth characterization helps to establish product CQAs, and is vital for ADC biosimilars development to ensure their comparability with the reference product, as well as product safety.     

A rapid on-line method for mass spectrometric confirmation of a cysteine-conjugated antibody-drug-conjugate structure using multidimensional chromatography

Cysteine-conjugated antibody-drug conjugates (ADCs) are manufactured using controlled partial reduction and conjugation chemistry with drug payloads that typically occur in intervals of 0, 2, 4, 6, and 8. Control of heterogeneity is of particular importance to the quality of ADC product because drug loading and distribution can affect the safety and efficacy of the ADC. Liquid chromatography ultra-violet (LC-UV)-based methods can be used to acquire the drug distribution profiles of cysteine-conjugated ADCs when analyzed using hydrophobic interaction chromatography (HIC). However, alternative analysis techniques are often required for structural identification when conjugated drugs do not possess discrete ultra-violet absorbance properties for precise assessment of the drug-to-antibody ratio (DAR). In this study, multidimensional chromatography was used as an efficient method for combining non-compatible techniques, such as HIC, with analysis by mass spectrometry (LC/LC/QTOF-MS) for rapid on-line structural elucidation of species observed in HIC distribution profiles of cysteine-conjugated ADCs. The methodology was tested using an IgG1 mAb modified by cysteine conjugation with a non-toxic drug mimic. Structural elucidation of peaks observed in the HIC analysis (1^st dimension) were successfully identified based on their unique sub-unit masses via mass spectrometry techniques once dissociation occurred under denaturing reversed phase conditions (2^nd dimension). Upon identification, the DAR values were determined to be 2.83, 4.44, and 5.97 for 3 drug load levels (low-, medium-, and high-loaded ADC batches), respectively, based on relative abundance from the LC-UV data. This work demonstrates that multidimensional chromatography coupled with MS, provides an efficient approach for on-line biotherapeutic characterization to ensure ADC product quality.     

A rapid on-line method for mass spectrometric confirmation of a cysteine-conjugated antibody-drug-conjugate structure using multidimensional chromatography

Cysteine-conjugated antibody-drug conjugates (ADCs) are manufactured using controlled partial reduction and conjugation chemistry with drug payloads that typically occur in intervals of 0, 2, 4, 6, and 8. Control of heterogeneity is of particular importance to the quality of ADC product because drug loading and distribution can affect the safety and efficacy of the ADC. Liquid chromatography ultra-violet (LC-UV)-based methods can be used to acquire the drug distribution profiles of cysteine-conjugated ADCs when analyzed using hydrophobic interaction chromatography (HIC). However, alternative analysis techniques are often required for structural identification when conjugated drugs do not possess discrete ultra-violet absorbance properties for precise assessment of the drug-to-antibody ratio (DAR). In this study, multidimensional chromatography was used as an efficient method for combining non-compatible techniques, such as HIC, with analysis by mass spectrometry (LC/LC/QTOF-MS) for rapid on-line structural elucidation of species observed in HIC distribution profiles of cysteine-conjugated ADCs. The methodology was tested using an IgG1 mAb modified by cysteine conjugation with a non-toxic drug mimic. Structural elucidation of peaks observed in the HIC analysis (1^st dimension) were successfully identified based on their unique sub-unit masses via mass spectrometry techniques once dissociation occurred under denaturing reversed phase conditions (2^nd dimension). Upon identification, the DAR values were determined to be 2.83, 4.44, and 5.97 for 3 drug load levels (low-, medium-, and high-loaded ADC batches), respectively, based on relative abundance from the LC-UV data. This work demonstrates that multidimensional chromatography coupled with MS, provides an efficient approach for on-line biotherapeutic characterization to ensure ADC product quality.     

Potential drugs used in the antibody–drug conjugate (ADC) architecture for cancer therapy

Cytotoxic small-molecule drugs have a major influence on the fate of antibody–drug conjugates (ADCs). An ideal cytotoxic agent should be highly potent, remain stable while linked to ADCs, kill the targeted tumor cell upon internalization and release from the ADCs, and maintain its activity in multidrug-resistant tumor cells. Lessons learned from successful and failed experiences in ADC development resulted in remarkable progress in the discovery and development of novel highly potent small molecules. A better understanding of such small-molecule drugs is important for development of effective ADCs. The present review discusses requirements making a payload appropriate for antitumor ADCs and focuses on the main characteristics of commonly-used cytotoxic payloads that showed acceptable results in clinical trials. In addition, the present study represents emerging trends and recent advances of payloads used in ADCs currently under clinical trials.     

Cutting-edge multi-level analytical and structural characterization of antibody-drug conjugates: present and future

Introduction: The development and optimization of antibody drug conjugates (ADCs) rely on improving their analytical and bioanalytical characterization, by assessing critical quality attributes (CQAs). Among the CQAs, the glycoprofile, drug load distribution (DLD), the amount of unconjugated antibody (D0), the average drug-to-antibody ratio (DAR), the drug conjugation sites and the residual drug-linker and related product proportions (SMDs) in addition to high and low molecular weight species (H/LMWS), and charge variants are the most important ones.

Areas covered: The analytical and structural toolbox for the characterization of 1^st, 2^d and 3^d generation ADCs was significantly extended in the last 3 years. Here, we reviewed state-of-the-art techniques, such as liquid chromatography, high resolution native and ion mobility mass spectrometry, multidimensional liquid chromatography and capillary electrophoresis hyphenated to mass spectrometry, reported mainly since 2016.

Expert commentary: These emerging techniques allow a deep insight into important CQAs that are related to ADC Chemistry Manufacturing and Control (CMC) as well as an improved understanding of in vitro and in vivo ADC biotransformations. This knowledge and the development of quantitative bioanalytical assays will continue to contribute to early-developability assessment for the optimization of all the ADC components (i.e. antibody, drug, and linker) and help to bring next-generation ADCs into late clinical development and to the market.     

抗体偶联药物的技术现状和展望

抗体偶联药物（antibody drug conjugate,ADC）通常由抗体通过链接体与毒素小分子偶联而成,同时具备抗体的高靶向性和小分子药物的高活性,使之作为一种新兴的靶向治疗手段,在肿瘤治疗领域展现出了优秀的疗效和潜力,成为药物研发领域的新热点。目前全球已有14款ADC药物获批上市,处于临床研究阶段的ADC候选药物分子超过140个。为了进一步提高ADC药物的安全性和有效性,近年来涌现出了各种新颖的技术。本文对ADC药物分子的关键元素,包括抗体、链接体、毒素小分子以及偶联技术等方面的最新研究进展进行总结,并讨论其优缺点。期望这些讨论能够帮助增加对ADC药物研究和开发更加系统的理解,为研发出更加高效和安全的ADC药物带来一些思考。     

Antibody–drug conjugates as novel anti-cancer chemotherapeutics

Over the past couple of decades, antibody–drug conjugates (ADCs) have revolutionized the field of cancer chemotherapy. Unlike conventional treatments that damage healthy tissues upon dose escalation, ADCs utilize monoclonal antibodies (mAbs) to specifically bind tumour-associated target antigens and deliver a highly potent cytotoxic agent. The synergistic combination of mAbs conjugated to small-molecule chemotherapeutics, via a stable linker, has given rise to an extremely efficacious class of anti-cancer drugs with an already large and rapidly growing clinical pipeline. The primary objective of this paper is to review current knowledge and latest developments in the field of ADCs. Upon intravenous administration, ADCs bind to their target antigens and are internalized through receptor-mediated endocytosis. This facilitates the subsequent release of the cytotoxin, which eventually leads to apoptotic cell death of the cancer cell. The three components of ADCs (mAb, linker and cytotoxin) affect the efficacy and toxicity of the conjugate. Optimizing each one, while enhancing the functionality of the ADC as a whole, has been one of the major considerations of ADC design and development. In addition to these, the choice of clinically relevant targets and the position and number of linkages have also been the key determinants of ADC efficacy. The only marketed ADCs, brentuximab vedotin and trastuzumab emtansine (T-DM1), have demonstrated their use against both haematological and solid malignancies respectively. The success of future ADCs relies on improving target selection, increasing cytotoxin potency, developing innovative linkers and overcoming drug resistance. As more research is conducted to tackle these issues, ADCs are likely to become part of the future of targeted cancer therapeutics.     

The impact of trisulfide modification of antibodies on the properties of antibody-drug conjugates manufactured using thiol chemistry

Antibody-drug conjugates (ADCs) are promising biotherapeutic agents for the treatment of cancer. The careful monitoring of critical quality attributes is important for ADCs' development, manufacturing and production. In this work, the effect of the presence of a trisulfide bond in the monoclonal antibody (mAb) conjugated to DM4 cytotoxic payload through a disulfide-bond linker sulfo-SPDB (sSPDB) was investigated. Three lots of antibody containing variable levels of trisulfide bonds were used. The identity and levels of trisulfide bonds were determined by liquid chromatography/ mass spectrometry (MS)/MS analysis. The antibodies were conjugated to sSPDB-DM4 to generate ADCs. Further analysis indicated that the drug-to-antibody ratio (DAR) value, a critical quality attribute, slightly increased for the conjugates made from antibody containing higher levels of trisulfide bond. Also, higher fragmentation levels were observed in the conjugates with more trisulfide bond. Detailed characterization by MS revealed that a small amount of DM4 payload was directly attached to inter-chain cysteine residues by disulfide or trisulfide bonds. Overall, our investigation indicated that the trisulfide bond present in the mAb could react with DM4 during the conjugation process. Therefore, the presence of trisulfide bonds in the antibody moiety should be carefully monitored and well controlled during the development of a maytansinoid ADC.     

In Vitro and In Vivo Evaluation of Cysteine and Site Specific Conjugated Herceptin Antibody-Drug Conjugates

Antibody drug conjugates (ADCs) are monoclonal antibodies designed to deliver a cytotoxic drug selectively to antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are critical to the activity and development of the ADC.The cytotoxic drugs or payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous number of drugs per antibody. The number of drugs per antibody commonly referred to as the drug to antibody ratio (DAR), can vary between 0 and 8 drugs for a IgG₁ antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non target related toxicities.In these studies we incorporated a non-natural amino acid, para acetyl phenylalanine, at two unique sites within an antibody against Her2/neu. We covalently attached a cytotoxic drug to these sites to form an ADC which contains two drugs per antibody.We report the results from the first direct preclinical comparison of a site specific non-natural amino acid anti-Her2 ADC and a cysteine conjugated anti-Her2 ADC. We report that the site specific non-natural amino acid anti-Her2 ADCs have superior in vitro serum stability and preclinical toxicology profile in rats as compared to the cysteine conjugated anti-Her2 ADCs. We also demonstrate that the site specific non-natural amino acid anti-Her2 ADCs maintain their in vitro potency and in vivo efficacy against Her2 expressing human tumor cell lines. Our data suggests that site specific non-natural amino acid ADCs may have a superior therapeutic window than cysteine conjugated ADCs.     

Antibody drug conjugates of cleavable amino-alkyl and aryl maytansinoids

Natural products have been used for many medicinal purposes for centuries. Antibody drug conjugates (ADCs) have utilized this rich source of small molecule therapeutics to produce several clinically useful treatments. ADCs based on the natural product maytansine have been successful clinically. The authors further the utility of the anti-cancer natural product maytansine by developing efficacious payloads and linker-payloads for conjugating to antibodies. The success of our approach was realized in the EGFRvIII targeting ADC EGFRvIII-16. The ADC was able to regress tumors in 2 tumor models (U251/EGFRvIII and MMT/EGFRvIII). When compared to a positive control ADC, the efficacy observed was similar or improved while the isotype control ADCs had no effect.     

Microscale screening of antibody libraries as maytansinoid antibody-drug conjugates

Antibody-drug conjugates (ADCs) are of great interest as targeted cancer therapeutics. Preparation of ADCs for early stage screening is constrained by purification and biochemical analysis techniques that necessitate burdensome quantities of antibody. Here we describe a method, developed for the maytansinoid class of ADCs, enabling parallel conjugation of antibodies in 96-well format. The method utilizes ～100 µg of antibody per well and requires <5 µg of ADC for characterization. We demonstrate the capabilities of this system using model antibodies. We also provide multiple examples applying this method to early-stage screening of maytansinoid ADCs. The method can greatly increase the throughput with which candidate ADCs can be screened in cell-based assays, and may be more generally applicable to high-throughput preparation and screening of different types of protein conjugates.     

Control Strategy for Small Molecule Impurities in Antibody-Drug Conjugates

Antibody-drug conjugates (ADCs) are an emerging class of biopharmaceuticals. As such, there are no specific guidelines addressing impurity limits and qualification requirements. The current ICH guidelines on impurities, Q3A (Impurities in New Drug Substances), Q3B (Impurities in New Drug Products), and Q6B (Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products) do not adequately address how to assess small molecule impurities in ADCs. The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) formed an impurities working group (IWG) to discuss this issue. This white paper presents a strategy for evaluating the impact of small molecule impurities in ADCs. This strategy suggests a science-based approach that can be applied to the design of control systems for ADC therapeutics. The key principles that form the basis for this strategy include the significant difference in molecular weights between small molecule impurities and the ADC, the conjugation potential of the small molecule impurities, and the typical dosing concentrations and dosing schedule. The result is that exposure to small impurities in ADCs is so low as to often pose little or no significant safety risk.     

Effects of antibody,drug and linker on the preclinical and clinical toxicities of antibody-drug conjugates

Antibody-drug conjugates (ADCs) represent a new class of cancer therapeutics. Their design involves a tumor-specific antibody, a linker and a cytotoxic payload. They were designed to allow specific targeting of highly potent cytotoxic agents to tumor cells whilst sparing normal cells. Frequent toxicities that may be driven by any of the components of an ADC have been reported. There are currently more than 50 ADCs in active clinical development, and a further ～20 that have been discontinued. For this review, the reported toxicities of ADCs were analysed, and the mechanisms for their effects are explored in detail. Methods to reduce toxicities, including dosing strategies and drug design, are discussed. The toxicities reported for active and discontinued drugs are important to drive the rational design and improve the therapeutic index of ADCs of the future.     

Antibody-drug conjugate model fast characterization by LC-MS following IdeS proteolytic digestion

Here we report the design and production of an antibody-fluorophore conjugate (AFC) as a non-toxic model of an antibody-drug conjugate (ADC). This AFC is based on the conjugation of dansyl sulfonamide ethyl amine (DSEA)-linker maleimide on interchain cysteines of trastuzumab used as a reference antibody. The resulting AFC was first characterized by routine analytical methods (SEC, SDS-PAGE, CE-SDS, HIC and native MS), resulting in similar chromatograms, electropherograms and mass spectra to those reported for hinge Cys-linked ADCs. IdeS digestion of the AFC was then performed, followed by reduction and analysis by liquid chromatography coupled to mass spectrometry analysis. Dye loading and distribution on light chain and Fd fragments were calculated, as well as the average dye to antibody ratio (DAR) for both monomeric and multimeric species. In addition, by analyzing the Fc fragment in the same run, full glyco-profiling and demonstration of the absence of additional conjugation was easily achieved.   As for naked antibodies and Fc-fusion proteins, IdeS proteolytic digestion may rapidly become a reference analytical method at all stages of ADC discovery, preclinical and clinical development. The method can be routinely used for comparability assays, formulation, process scale-up and transfer, and to define critical quality attributes in a quality-by-design approach.     

Antibody-drug conjugate model fast characterization by LC-MS following IdeS proteolytic digestion

Here we report the design and production of an antibody-fluorophore conjugate (AFC) as a non-toxic model of an antibody-drug conjugate (ADC). This AFC is based on the conjugation of dansyl sulfonamide ethyl amine (DSEA)-linker maleimide on interchain cysteines of trastuzumab used as a reference antibody. The resulting AFC was first characterized by routine analytical methods (SEC, SDS-PAGE, CE-SDS, HIC and native MS), resulting in similar chromatograms, electropherograms and mass spectra to those reported for hinge Cys-linked ADCs. IdeS digestion of the AFC was then performed, followed by reduction and analysis by liquid chromatography coupled to mass spectrometry analysis. Dye loading and distribution on light chain and Fd fragments were calculated, as well as the average dye to antibody ratio (DAR) for both monomeric and multimeric species. In addition, by analyzing the Fc fragment in the same run, full glyco-profiling and demonstration of the absence of additional conjugation was easily achieved.

As for naked antibodies and Fc-fusion proteins, IdeS proteolytic digestion may rapidly become a reference analytical method at all stages of ADC discovery, preclinical and clinical development. The method can be routinely used for comparability assays, formulation, process scale-up and transfer, and to define critical quality attributes in a quality-by-design approach.     

Methods for site-specific drug conjugation to antibodies

Antibody drug conjugates (ADCs) are an emerging class of targeted therapeutics with the potential to improve therapeutic index over traditional chemotherapy. Drugs and linkers have been the current focus of ADC development, in addition to antibody and target selection. Recently, however, the importance of conjugate homogeneity has been realized. The current methods for drug attachment lead to a heterogeneous mixture, and some populations of that mixture have poor in vivo performance. New methods for site-specific drug attachment lead to more homogeneous conjugates and allow control of the site of drug attachment. These subtle improvements can have profound effects on in vivo efficacy and therapeutic index. This review examines current methods for site-specific drug conjugation to antibodies, and compares in vivo results with their non-specifically conjugated counterparts. The apparent improvement in pharmacokinetics and the reduced off target toxicity warrant further development of this site-specific modification approach for future ADC development.