Current approaches for the purification of antibody-drug conjugates.

In the past two decades, antibody-drug conjugates have gained increasing attention because they expand the therapeutic index when compared with that of traditional chemotherapies. Antibody-drug conjugates are highly complex structures consisting of antibodies covalently conjugated with small-molecule cytotoxic drugs. The complex structure of antibody-drug conjugates makes chemistry, manufacturing, and control difficult. In contrast to antibody production, distinct purification methods following conjugation of antibodies with drug-linkers are required for the manufacturing. For process development of antibody drug conjugates, the drug-to-antibody ratio, free drug-linkers, and aggregates are critical quality attributes that must be strictly controlled and removed by appropriate purification techniques. In this review, features of various purification methods used to purify antibody drug conjugates are described and evaluated. The future landscape of the antibody-conjugates field is also discussed briefly.

Using multimodal chromatography for post-conjugation antibody-drug conjugate purification: A methodology from high throughput screening to in-silico process development.

In this paper, a methodology for the development of a multimodal chromatography process is presented that is aimed at removal of under-conjugated antibody-drug conjugate (ADC) species. Two ADCs are used as case studies: One ADC results from site-directed conjugation to inserted cysteine residues and has a drug-to-antibody ratio (DAR) of two, the other is the product of conjugation to interchain disulfide bonds with a DAR of eight. First, filter plate screening studies are designed for the unconjugated antibody and the ADCs. Different metrics for the analysis of these data sets are presented and discussed. From this analysis, the selected process conditions are then carried out using a benchtop chromatography system to confirm the separations observed in the filter plate studies while simultaneously generating data to estimate steric mass-action isotherm and mass transport parameters for process simulation. This column model is then employed to develop separation processes in-silico for the removal of the unconjugated parent antibody and under-conjugated product variants. The optimized process conditions identified using the model are then verified experimentally. The methodology presented in this work utilizes multimodal chromatography for ADC purification and provides the framework for a streamlined systematic approach to process development.

RGD4C peptide mediates anti-p21Ras scFv entry into tumor cells and produces an inhibitory effect on the human colon cancer cell line SW480.

BACKGROUND: We prepared an anti-p21Ras scFv which could specifically bind with mutant and wild-type p21Ras. However, it cannot penetrate the cell membrane, which prevents it from binding to p21Ras in the cytoplasm. Here, the RGD4C peptide was used to mediate the scFv penetration into tumor cells and produce antitumor effects. METHODS: RGD4C-EGFP and RGD4C-p21Ras-scFv recombinant expression plasmids were constructed to express fusion proteins in E. coli, then the fusion proteins were purified with HisPur Ni-NTA. RGD4C-EGFP was used as reporter to test the factors affecting RGD4C penetration into tumor cell. The immunoreactivity of RGD4C-p21Ras-scFv toward p21Ras was identified by ELISA and western blotting. The ability of RGD4C-p21Ras-scFv to penetrate SW480 cells and colocalization with Ras protein was detected by immunocytochemistry and immunofluorescence. The antitumor activity of the RGD4C-p21Ras-scFv was assessed with the MTT, TUNEL, colony formation and cell migration assays. Chloroquine (CQ) was used an endosomal escape enhancing agent to enhance endosomal escape of RGD4C-scFv. RESULTS: RGD4C-p21Ras-scFv fusion protein were successfully expressed and purified. We found that the RGD4C fusion protein could penetrate into tumor cells, but the tumor cell entry of was time and concentration dependent. Endocytosis inhibitors and a low temperature inhibited RGD4C fusion protein endocytosis into cells. The change of the cell membrane potential did not affect penetrability. RGD4C-p21Ras-scFv could penetrate SW480 cells, effectively inhibit the growth, proliferation and migration of SW480 cells and promote this cells apoptosis. In addition, chloroquine (CQ) could increase endosomal escape and improve antitumor activity of RGD4C-scFv in SW480 cells. CONCLUSION: The RGD4C peptide can mediate anti-p21Ras scFv entry into SW480 cells and produce an inhibitory effect, which indicates that RGD4C-p21Ras-scFv may be a potential therapeutic antibody for the treatment of ras-driven cancers.

Use of Ultrashort Columns for Therapeutic Protein Separations. Part 1: Theoretical Considerations and Proof of Concept.

Due to the particular elution mechanism observed with large solutes (e.g., proteins) in liquid chromatography, column length has less impact in controlling their retention compared to small solutes. Moreover, long columns-in theory-just broaden the peaks of large solutes since a great part of the column only acts as void (extra) volume. Such a theory suggests that using very short columns should result in comparable separation quality versus using long columns and make it possible to perform faster (high-throughput) analyses. Therefore, the elution behavior of various therapeutic monoclonal antibodies and their fragments (25-150 kDa) has been investigated using modern instrumentation and column formats. The possibilities offered by narrow-bore columns packed with state-of-the-art 2.7 µm superficially porous particles with 5, 50, 100, and 150 mm lengths have been compared. In particular, the impact of gradient steepness and column length on separation efficiency was evaluated. Using 5 mm × 2.1 mm columns, it has become possible to separate antibody fragments and antibody-drug conjugate species in less than 30 s. Such fast methods can be very useful for high-throughput screening purposes in biopharmaceutical industries.

Preclinical Development of MGC018, a Duocarmycin-based Antibody-drug Conjugate Targeting B7-H3 for Solid Cancer.

B7-H3, also referred to as CD276, is a member of the B7 family of immune regulatory proteins. B7-H3 is overexpressed on many solid cancers, including prostate cancer, renal cell carcinoma, melanoma, squamous cell carcinoma of the head and neck, non-small cell lung cancer, and breast cancer. Overexpression of B7-H3 is associated with disease severity, risk of recurrence and reduced survival. In this article, we report the preclinical development of MGC018, an antibody-drug conjugate targeted against B7-H3. MGC018 is comprised of the cleavable linker-duocarmycin payload, valine-citrulline-seco duocarmycin hydroxybenzamide azaindole (vc-seco-DUBA), conjugated to an anti-B7-H3 humanized IgG1/kappa mAb through reduced interchain disulfides, with an average drug-to-antibody ratio of approximately 2.7. MGC018 exhibited cytotoxicity toward B7-H3-positive human tumor cell lines, and exhibited bystander killing of target-negative tumor cells when cocultured with B7-H3-positive tumor cells. MGC018 displayed potent antitumor activity in preclinical tumor models of breast, ovarian, and lung cancer, as well as melanoma. In addition, antitumor activity was observed toward patient-derived xenograft models of breast, prostate, and head and neck cancer displaying heterogeneous expression of B7-H3. Importantly, MGC018 exhibited a favorable pharmacokinetic and safety profile in cynomolgus monkeys following repeat-dose administration. The antitumor activity observed preclinically with MGC018, together with the positive safety profile, provides evidence of a potentially favorable therapeutic index and supports the continued development of MGC018 for the treatment of solid cancers. GRAPHICAL ABSTRACT: http://mct.aacrjournals.org/content/molcanther/19/11/2235/F1.large.jpg.

Modeling of hydrophobic interaction chromatography for the separation of antibody-drug conjugates and its application towards quality by design.

Antibody-drug conjugates (ADCs) are hybrid molecules based on monoclonal antibodies (mAbs) with covalently attached cytotoxic small-molecule drugs. Due to their potential for targeted cancer therapy, they form part of the diversifying pipeline of various biopharmaceutical companies, in addition to currently seven commercial ADCs. With other new modalities, ADCs contribute to the increasing complexity of biopharmaceutical development in times of growing costs and competition. Another challenge is the implementation of quality by design (QbD), which receives a lot of attention. In order to answer these challenges, mechanistic models are gaining interest as tools for enhanced process understanding and efficient process development. The drug-to-antibody ratio (DAR) is a critical quality attribute (CQA) of ADCs. After the conjugation reaction, the DAR can still be adjusted by including a hydrophobic interaction chromatography (HIC) step. In this work, we developed a mechanistic model for the preparative separation of cysteine-engineered mAbs with different degrees of conjugation with a non-toxic surrogate drug. The model was successfully validated for varying load compositions with linear and optimized step gradient runs, applying conditions differing from the calibration runs. In two in silico studies, we then present scenarios for how the model can be applied profitably to ensure a more robust achievement of the target DAR and for the efficient characterization of the design space. For this, we also used the model in a linkage study with a kinetic reaction model developed by us previously. The combination of the two models effectively widens system boundaries over two adjacent process steps. We believe this work has great potential to help advance the incorporation of digital tools based on mechanistic models in ADC process development by illustrating their capabilities for efficient process development and increased robustness. Mechanistic models can support the implementation of QbD and eventually might be the basis for digital process twins able to represent multiple unit operations.

Evaluation of hydrophobic-interaction chromatography resins for purification of antibody-drug conjugates using a mimetic model with adjustable hydrophobicity.

Antibody drug conjugates are cytotoxic pharmaceuticals, designed to destroy malignant cells. A cytotoxic molecule is attached to an antibody that binds specific to a cancer-cell surface. Given the high toxicity of the drugs, strict safety standards have to be kept. For this reason, an antibody drug conjugates model was developed with fluorescein 5-isothiocyanate as the nontoxic payload surrogate. Due to the similar hydrophobicity, this model is used to establish a suitable purification process and characterization method for antibody drug conjugates. Because of the pH dependent solubility of fluorescein, the hydrophobicity of conjugates can be modulated by the pH value. Based on the complex heterogeneity and hydrophobicity of the conjugates a chromatographic purification is challenging. Hydrophobic interaction chromatography is used for analytical as well as for preparative separation. Because of the increased hydrophobicity of the conjugates compared to native antibody, hydrophobic interaction chromatography often suffer from resolution and recovery problems. Conjugates were separated differing on the number of payloads attached to the antibody. For this matter, the drug-antibody ratio is determined and used as a quantitative term. The conjugates are purified at high recoveries and resolution by step gradients using suitable resins, allowing the separation of the target drug-antibody ratio.

Streamlined Expressed Protein Ligation: Site-Specific Antibody-Drug Conjugate.

Protein semisynthesis is a powerful tool for studying proteins and has contributed to a better understanding of protein structure and function and also driven innovations in protein science. Expressed protein ligation (EPL) is a widely used method to generate chemically modified proteins. However, EPL has some limitations, particularly relevant to modify challenging proteins such as antibodies. The method termed streamlined expressed protein ligation (SEPL) overcomes some of the problems of EPL, and other methods of protein semisynthesis, to generate challenging modified proteins such as antibody-drug conjugates (ADCs). ADCs targeting highly cytotoxic molecules to cancer cells, offer an attractive strategy to selectively eliminate tumor cells with improved therapeutic index than the antibodies or cytotoxic molecules themselves. Despite the potential of ADCs, the development of such complex molecules is challenging. We provide here protocols to prepare site-specifically modified ADCs by streamlined expressed protein ligation (SEPL), which does not require the incorporation of unnatural modifications into the antibody. Therefore, fully native antibodies, with only the desired cytotoxic molecules attached, can be generated.

Site-Specific Conjugation to Cys-Engineered THIOMAB™ Antibodies.

Antibodies bearing engineered cysteine residues (termed THIOMAB™ antibodies) enable the site-selective attachment of a drug, label or other payload for specific delivery to certain tissues (e.g., tumors). This Chapter describes detailed methods we have developed and optimized for the conjugation, purification and analysis of THIOMAB™ antibody drug conjugates (TDCs).

Conjugations to Endogenous Cysteine Residues.

Interchain disulfide bonds of antibodies can be reduced by agents such as TCEP or DTT to form reactive cysteine residues. These endogenous cysteines are used for conjugation to biologically active drugs either directly or via linkers to prepare antibody drug conjugates (ADCs). The anti-notch 3 ADC described here is being evaluated in the early clinical development program as a potential treatment for a variety of cancers. The ADC is composed of an IgG1 mAb that is conjugated by endogenous cysteines to a cytotoxic microtubulin inhibitor via a maleimide-containing linker. The endogenous cysteine residues are produced by partial reduction of the mAb with TCEP reducing agent. The conjugation results in the formation of a mixture of 2, 4, 6, and 8 loaded ADC species. In addition to the desired product, several product-related impurities such as aggregates are generated during the conjugation reaction. The product- and process-related impurities are separated from the monomeric ADC by column chromatography and ultrafiltration-diafiltration techniques. The temperature of TCEP reduction step has an impact on the level of aggregates produced in the reaction. The temperature also impacts the isomeric composition of the 4 loaded ADC species.

Utilizing Solid-Phase to Enable High-Throughput, Site-Specific Conjugation and Dual-Labeled Antibody and Fab Conjugates.

For therapeutic and diagnostic applications, site-specific antibody conjugates have proven superior for both the ease of characterization as well as for optimal biophysical and therapeutic properties. Screening multiple antibodies on multiple sites with multiple linker-drugs can become very tedious and time-consuming. As solid-phase reactions are best suited to simplify multistep reactions, readily available protein A/L agarose beads can be utilized to generate site-specific, antibody -drug conjugates on engineered cysteines. Multiple site-specific labels on an antibody with either fluorophore or other-linker drugs is highly desired to evaluate antibody trafficking or payload-synergy for therapeutics. Utilizing solid-phase conjugation, a simple method to generate dual-labeled, site-specific antibody and Fab conjugates from antibody with engineered cysteine is also been described.

Bridged Cysteine Conjugations.

Preparation of antibody-drug conjugates (ADCs) with a highly homogeneous drug loading in general requires site-selective conjugation of a cytotoxic payload. Typically, functionality utilized for attachment of the payload is achieved through engineering of suitable chemical handles or by enzymatic modification of the antibody. Relatively few methods to produce ADCs with homogeneous drug loading via endogenous amino acid conjugation have been developed. Herein we describe a robust method for the conjugation of antibodies using a cysteine rebridging approach to produce ADCs with highly homogeneous drug-to-antibody ratios (DAR) at the native interchain disulfides, called ThioBridge®. The process described relies upon an elegant cascade of addition-elimination reactions carried out under mild aqueous conditions that can be readily applied to wild-type antibodies without the need for prior modification via recombinant or enzymatic means. Using this method, conversions to a conserved DAR ADC are typically in the range of 70-95% and overall process yields of >70% are readily achieved.

ADC Analysis by Hydrophobic Interaction Chromatography.

Hydrophobic interaction chromatography (HIC) is a traditional technique used for the separation, purification, and characterization of proteins. As the number of antibody-drug conjugates (ADCs) continues to increase in clinical trials, HIC and other orthogonal methods utilizing changes in hydrophobicity are being used for ADC characterization and analysis. Unlike other techniques, HIC uniquely allows for protein analysis under mild nondenaturing conditions that preserve the native structure and activity of the molecules. Analysis of the ADC in its native form is advantageous. Herein, we describe a generic HIC protocol for the screening, analysis, and characterization of ADCs using an ammonium sulfate buffer and a high-pressure liquid chromatography system. Parameters affecting data quality and interpretation are addressed. In addition, several recommendations are included for method optimization and troubleshooting.

Two-Dimensional Liquid Chromatography Coupled to High-Resolution Mass Spectrometry for the Analysis of ADCs.

From a structural point of view, the complete characterization of ADCs is a challenging task due to their high complexity. ADCs combine the heterogeneity of the initial antibody to the variability associated with the conjugation strategy, the manufacturing process, and the storage. Given the inherent complexity of these biomolecules, online comprehensive two-dimensional liquid chromatography (LC × LC) is an attractive technique to address the challenges associated with ADC characterization. Compared to conventional one-dimensional liquid chromatography techniques (1D-LC), LC × LC combines two different and complementary separation systems. In the context of ADC analysis, LC × LC has been proven to be a rapid and efficient analytical tool: (1) to provide a higher resolving power by increasing the overall peak capacity and thus allowing to gain more information within a single run and (2) to allow mass spectrometry (MS) coupling with some chromatographic techniques that are not MS-compatible and hence to facilitate the structural elucidation of ADCs. In this chapter, we present the coupling of different chromatographic techniques including hydrophobic interaction chromatography (HIC), reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC), ion exchange chromatography (IEX), and hydrophilic liquid chromatography (HILIC). The interest of HIC × SEC, SEC × SEC, HIC × RPLC, IEX × RPLC, RPLC × RPLC, and HILIC × RPLC, all hyphenated to high-resolution mass spectrometry (HRMS), is discussed in the context of the characterization of ADCs.

High-Resolution Characterization of ADCs by Orbitrap LCMS.

Antibody-drug conjugates (ADCs) have complex molecular structures as they are composed of both small and large molecules, and they often undergo biotransformation over time in circulation. Here we describe a high-resolution Orbitrap MS approach for the characterization of ADC biotransformation and stability. Compared with conventional approach by Q-TOF MS, the method described here significantly improved the mass resolution and enabled more comprehensive characterization of ADC catabolites. It is particularly beneficial for characterizing ADC biotransformations with small mass changes.

Characterization of ADCs by Capillary Electrophoresis.

Capillary electrophoresis (CE) is a highly efficient separation technique that resolves ions based on their electrophoretic mobility in the presence of an applied voltage. It has been broadly applied for characterizing biotherapeutics including ADCs. In this chapter, step-by-step procedures for characterizing ADCs using CE will be described with focus placed on reduced and non-reduced capillary electrophoresis sodium dodecyl sulfate (CE-SDS) for purity determination and imaged capillary isoelectric focusing (iCIEF) for charge heterogeneity analysis.

Purification of ADCs by Hydrophobic Interaction Chromatography.

Antibody-drug conjugate (ADC) in vitro potency has been shown to be dependent on drug load, with higher drug load providing lower IC50 values. However, in vivo potency is affected by intrinsic biological effects as well, such as plasma clearance, dose-limiting toxicity, etc. Developing a preparative HIC process for ADC purification to isolate species with a specific drug loading involves several steps including conjugation optimization, resin selection, solubility studies gradient screening, and step gradient development (buffer selection). In this chapter, the rationale and general considerations for developing a preparative hydrophobic interaction chromatography (HIC) method are described for isolation of an example ADC with specific drug load, e.g., two monomethyl auristatin E (MMAE) payloads (E2).

Detection and Removal of Small Molecule and Endotoxin Contaminants in ADC Preparations.

Incomplete removal of free (unconjugated) drug or drug-linker species used to prepare ADCs results in contaminated ADC samples which may pose a risk for toxicity. Due to the extreme potency of typical small molecule toxins employed in ADCs, even relatively low levels of free drug contaminants in ADC samples have been hypothesized to result in nonspecific (i.e., off-target) activity in biological systems. It is possible for trace levels of certain free drug species to persist in final ADC samples despite the inclusion of common purification steps during the preparation processes. Therefore, methods for the detection, quantification, and removal of residual free drug present in ADC samples are ultimately required for the preparation of safe and efficacious final ADC drug products. Herein we report general methods for the detection and removal of such contaminants.

Proof of Concept To Achieve Infinite Selectivity for the Chromatographic Separation of Therapeutic Proteins.

Reversed phase liquid chromatography (RPLC) is a widely used technique for the analytical characterization of proteins biopharmaceuticals, due to its inherent compatibility with mass spectrometry (MS). However, this chromatographic mode suffers from limited selectivity when analyzing large molecules. Due to the on/off mechanism observed with large solutes in RPLC (S values were higher than 100 for intact proteins), we have developed a new analytical strategy based on the use of multi-isocratic elution mode, to achieve arbitrary selectivity for protein variants. In this work, it has been demonstrated that the combination of multi-isocratic steps and very short steep gradient segments at solute elution allows one to set the selectivity as desired, while maintaining sharp peaks due to significant band compression effects. The strategy was successfully applied to the analysis of intact and subunits of monoclonal antibodies (mAbs) as well as antibody-drug conjugates (ADCs), illustrating the possibility to achieve a uniform peak distribution (equidistant band spacing) and much higher resolution than in the case of common linear, multilinear, or nonlinear gradients.

Site-Selective Enzymatic Labeling of Designed Ankyrin Repeat Proteins Using Protein Farnesyltransferase.

Affinity agents coupled to a functional moiety play an ever-increasing role in modern medicine, ranging from radiolabeled selective binders in diagnosis to antibody-drug conjugates in targeted therapies. In biomedical research, protein coupling to fluorophores, surfaces and nanoparticles has become an integral part of many procedures. In addition to antibodies, small scaffold proteins with similar target binding properties are being widely explored as alternative targeting moieties. To label these binders of interest with different functional moieties, conventional chemical coupling methods can be employed, but often result in heterogeneously modified protein products. In contrast, enzymatic labeling methods are highly site-specific and efficient. Protein farnesyltransferase (PFTase) catalyzes the transfer of an isoprenoid moiety from farnesyl diphosphate (FPP) to a cysteine residue in a C-terminal CaaX motif at the C-terminus of a protein substrate. The addition of only four amino acid residues minimizes the influence on the native protein structure. In addition, a variety of isoprenoid analogs containing different bioorthogonal functional groups, including azides, alkynes, and aldehydes, have been developed to enable conjugation to various cargos after being incorporated onto the target protein by PFTase. In this protocol, we present a detailed procedure for labeling Designed Ankyrin Repeat Proteins (DARPins) engineered with a C-terminal CVIA sequence using an azide-containing FPP analog by yeast PFTase (yPFTase). In addition, procedures to subsequently conjugate the labeled DARPins to a TAMRA fluorophore using strained-promoted alkyne-azide cycloaddition (SPAAC) reactions as well as the sample preparation to evaluate the target binding ability of the conjugates by flow cytometry are described.