Automated online deconjugation of antibody-drug conjugate for small molecule drug profiling.

Antibody-drug conjugates (ADCs) are designed by chemically linking highly potent cytotoxic small molecule drugs to monoclonal antibodies of unique specificity for targeted destruction of cancer cells. This innovative class of molecules incurs unique developmental challenges due to its structural complexity of having both small molecule and protein components. The stability of the small molecule payload on the ADC is a critical attribute as it directly relates to product efficacy and patient safety. This study describes the use of an end-to-end automated workflow for effective and robust characterization of the small molecule drug while it is conjugated to the antibody. In this approach, online deconjugation was accomplished by an autosampler user defined program and 1D size exclusion chromatography was utilized to provide separation between small molecule and protein species. The small molecule portion was then trapped and sent to the 2D for separation and quantification by reversed-phase liquid chromatography with identification of impurities and degradants by mass spectrometry. The feasibility of this system was demonstrated on an ADC with a disulfide-based linker. This fully automated approach avoids tedious sample preparation that may lead to sample loss and large assay variability. Under optimized conditions, the method was shown to have excellent specificity, sensitivity (LOD of 0.036 µg/mL and LOQ of 0.144 µg/mL), linearity (0.04-72.1 µg/mL), precision (system precision %RSD of 1.7 and method precision %RSD of 3.4), accuracy (97.4 % recovery), stability-indicating nature, and was successfully exploited to analyze the small molecule drug on a panel of stressed ADC samples. Overall, the workflow established here offers a powerful analytical tool for profiling the in-situ properties of small molecule drugs conjugated to antibodies and the obtained information could be of great significance for guiding process/formulation development and understanding pharmacokinetic/pharmacodynamic behavior of ADCs.

Turning on lanthanide luminescence via nanoencapsulation.

Encapsulation of macrocyclic europium(III) chelates by discrete, monodisperse SiO2 nanoparticles (NPs) has been carried out, and the resulting significant enhancement of metal-derived luminescence has been studied to rationalize this dramatic effect. The tetraiminodiphenolate motif chosen for this study is easily synthesized and incorporated into the NP matrix under ambient conditions. The free complex exhibits primarily weak ligand-derived emission at room temperature, typical for these compounds, and displays intense metal-centered luminescence from the europium only when cooled to 77 K. Upon encapsulation by the NPs, however, europium-derived luminescence is visibly "turned on" at room temperature, yielding strong emission peaks characteristic of europium(III) with a corresponding enhancement factor of 6 × 10(6). The similar ligand singlet and triplet excited-state energies determined for the free complex (20820 and 17670 cm(-1), respectively) versus the encapsulated complex (20620 and 17730 cm(-1)) indicate that encapsulation does not affect the energy levels of the ligand appreciably. Instead, a detailed analysis of the metal-centered emission and ligand singlet and triplet emission bands for the free and encapsulated complexes reveals that the enhanced metal emission is due to the rigid environment afforded by the silica NP matrix affecting vibrationally mediated energy transfer. Further, the metal-centered emission lifetimes in methanol versus deuterated methanol indicate a decrease in the number of coordinated solvent molecules upon encapsulation, changing from an average of 3.3 to 2.1 bound methanol molecules and reducing the known quenching effect on europium-centered luminescence due to nearby OH vibrations.

A DNA-conjugated magnetic nanoparticle assay for assessing genotoxicity.

The genotoxicity of a molecule refers to its ability to interact with DNA in a way that inhibits normal DNA replication and transcription possibly leading to mutagenesis or carcinogenesis. Assessing the genotoxicity of a compound is critical in the development of pharmaceuticals and other products designed for human consumption or use. Typically genotoxicity is established using expensive and time consuming methods using animals or bacteria like the Ames test, mouse lymphoma assay, or mouse and rat carcinogenicity tests. We have developed a magnetic nanoparticle-based assay that uses conjugated double-stranded DNA to serve as a substrate for interaction with genotoxic molecules. After application of a magnetic field, the genotoxic molecules are extracted with the DNA-conjugated magnetic nanoparticles. The genotoxic molecules can then be released and detected. To evaluate the potential of this assay, we have screened several genotoxic and non-genotoxic compounds and have demonstrated the ability to extract a genotoxic compound in the presence of a non-genotoxic molecule. The assay demonstrates suitable analytical performance and the ability to differentiate between genotoxic and non-genotoxic molecules providing a rapid and inexpensive alternative to more traditional methods of evaluating genotoxicity.

Aptamer-conjugated nanoparticles for cancer cell detection.

Aptamer-conjugated nanoparticles (ACNPs) have been used for a variety of applications, particularly dual nanoparticles for magnetic extraction and fluorescent labeling. In this type of assay, silica-coated magnetic and fluorophore-doped silica nanoparticles are conjugated to highly selective aptamers to detect and extract targeted cells in a variety of matrixes. However, considerable improvements are required in order to increase the selectivity and sensitivity of this two-particle assay to be useful in a clinical setting. To accomplish this, several parameters were investigated, including nanoparticle size, conjugation chemistry, use of multiple aptamer sequences on the nanoparticles, and use of multiple nanoparticles with different aptamer sequences. After identifying the best-performing elements, the improvements made to this assay's conditional parameters were combined to illustrate the overall enhanced sensitivity and selectivity of the two-particle assay using an innovative multiple aptamer approach, signifying a critical feature in the advancement of this technique.

Quantitation of plasmid DNA deposited on gold particles for particle-mediated epidermal delivery using ICP-MS.

DNA-plasmid-based vaccines are a promising class of next generation therapeutics. Particle-mediated epidermal delivery is an attractive method for the administration of DNA plasmid vaccines. This technology utilizes minute quantities of DNA plasmid which have been deposited onto the surface of 2-3-microm gold particles, and so the development of this technology requires the use of analytical methods that can accurately quantitate the amount of the DNA on the particle. Spectroscopic methods are generally insufficient for this task due to interference from the gold particle. ICP-MS circumvents this issue while allowing for the sensitive, reproducible, and accurate determination of the quantity of DNA on the particle surface. This report will detail the development and application of such a method.

Locked nucleic acid based beacons for surface interaction studies and biosensor development.

DNA sensors and microarrays permit fast, simple, and real-time detection of nucleic acids through the design and use of increasingly sensitive, selective, and robust molecular probes. Specifically, molecular beacons (MBs) have been employed for this purpose; however, their potential in the development of solid-surface-based biosensors has not been fully realized. This is mainly a consequence of the beacon's poor stability because of the hairpin structure once immobilized onto a solid surface, commonly resulting in a low signal enhancement. Here, we report the design of a new MB that overcomes some of the limitations of MBs for surface immobilization. Essentially, this new design adds locked nucleic acid bases (LNAs) to the beacon structure, resulting in a LNA molecular beacon (LMB) with robust stability after surface immobilization. To test the efficacy of LMBs against that of regular molecular beacons (RMBs), the properties of selectivity, sensitivity, thermal stability, hybridization kinetics, and robustness for the detection of target sequences were compared and evaluated. A 25-fold enhancement was achieved for the LMB on surface with detection limits reaching the low nanomolar range. In addition, the LMB-based biosensor was shown to possess better stability, reproducibility, selectivity, and robustness when compared to the RMB. Therefore, as an alternative to conventional DNA and as a prospective tool for use in both DNA microarrays and biosensors, these results demonstrate the potential of the locked nucleic acid bases for nucleic acid design for surface immobilization.

Synthesis and investigation of deoxyribonucleic acid/locked nucleic acid chimeric molecular beacons.

To take full advantage of locked nucleic acid (LNA) based molecular beacons (LNA-MBs) for a variety of applications including analysis of complex samples and intracellular monitoring, we have systematically synthesized a series of DNA/LNA chimeric MBs and studied the effect of DNA/LNA ratio in MBs on their thermodynamics, hybridization kinetics, protein binding affinity and enzymatic resistance. It was found that the LNA bases in a MB stem sequence had a significant effect on the stability of the hair-pin structure. The hybridization rates of LNA-MBs were significantly improved by lowering the DNA/LNA ratio in the probe, and most significantly, by having a shared-stem design for the LNA-MB to prevent sticky-end pairing. It was found that only MB sequences with DNA/LNA alternating bases or all LNA bases were able to resist nonspecific protein binding and DNase I digestion. Additional results showed that a sequence consisting of a DNA stretch less than three bases between LNA bases was able to block RNase H function. This study suggested that a shared-stem MB with a 4 base-pair stem and alternating DNA/LNA bases is desirable for intracellular applications as it ensures reasonable hybridization rates, reduces protein binding and resists nuclease degradation for both target and probes. These findings have implications on the design of LNA molecular probes for intracellular monitoring application, disease diagnosis and basic biological studies.

Molecular engineering of DNA: molecular beacons.

Molecular beacons (MBs) are specifically designed DNA hairpin structures that are widely used as fluorescent probes. Applications of MBs range from genetic screening, biosensor development, biochip construction, and the detection of single-nucleotide polymorphisms to mRNA monitoring in living cells. The inherent signal-transduction mechanism of MBs enables the analysis of target oligonucleotides without the separation of unbound probes. The MB stem-loop structure holds the fluorescence-donor and fluorescence-acceptor moieties in close proximity to one another, which results in resonant energy transfer. A spontaneous conformation change occurs upon hybridization to separate the two moieties and restore the fluorescence of the donor. Recent research has focused on the improvement of probe composition, intracellular gene quantitation, protein-DNA interaction studies, and protein recognition.

Investigation of the hybrid molecular probe for intracellular studies.

Monitoring gene expression in vivo is essential to the advancement of biological studies, medical diagnostics, and drug discovery. Adding to major efforts in developing molecular probes for mRNA monitoring, we have recently developed an alternative tool, the hybrid molecular probe (HMP). To optimize the probe, a series of experiments were performed to study the properties of HMP hybridization kinetics and stability. The results demonstrated the potential of the HMP as a prospective tool for use in both hybridization studies and in vitro and in vivo analyses. The HMP has shown no tendency to produce false positive signals, which is a major concern for living cell studies. Moreover, HMP has shown the ability to detect the mRNA expression of different genes inside single cells from both basal and stimulated genes. As an effective alternative to conventional molecular probes, the proven sensitivity, simplicity, and stability of HMPs show promise for their use in monitoring mRNA expression in living cells.

Antibody-drug conjugate characterization by chromatographic and electrophoretic techniques.

Due to the inherent structure complexity and component heterogeneity of antibody drug conjugates (ADCs), separation technologies play a critical role in their characterization. In this review, we focus on chromatographic and electrophoretic approaches used to characterize ADCs with respect to drug-to-antibody ratio, drug distribution and conjugation sites, free small molecule drugs, charge variants, aggregates and fragments, etc. Chromatographic techniques including reversed-phase, ion exchange, size exclusion, hydrophobic interaction, two-dimensional liquid chromatography, and gas chromatography as well as capillary electrophoretic techniques including capillary electrophoresis sodium dodecyl sulfate, capillary zone electrophoresis and capillary isoelectric focusing are reviewed for their applications in the characterization of ADCs.

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.

Investigation of low recovery in the free drug assay for antibody drug conjugates by size exclusion-reversed phase two dimensional-liquid chromatography.

Antibody drug conjugates (ADCs) are complex therapeutic agents combining the selectivity of monoclonal antibodies and highly efficacious small molecule drugs to successfully eliminate tumor cells while limiting the general toxicity and side effects of the therapeutic to protect patient safety. One unique attribute critical to the safety of ADCs is the residual content of unconjugated small molecule drug present from either incomplete conjugation or degradation of the ADC. Typically for quality control assays, quantifying the amount of the free drug is performed through precipitation of the protein species using an organic solvent and then assaying the amount of free drug left in the supernatant. During the validation of an assay of this type for a maleimide based linker drug, issues were experienced with low and variable recovery in the spiked samples of the drug substance and drug product. A two-dimensional heart-cutting method coupling Size Exclusion Chromatography (SEC) with Reverse Phase (RP) chromatography was utilized to explore possible mechanisms leading to the low recovery of the free linker drug. The results of the investigation indicated that the spiked linker drug reacts with residual reactive groups on the ADC; a conclusion which was confirmed by the observed increase of average Drug to Antibody Ratio (DAR) determined by Hydrophobic Interaction Chromatography (HIC). Finally, several approaches were evaluated to minimize the recovery loss. Capping the residual reactive groups on the ADC with maleimide containing reagents effectively mitigated the low recovery issue.

Chemical de-conjugation for investigating the stability of small molecule drugs in antibody-drug conjugates.

Antibody-drug conjugates (ADCs) offer new therapeutic options for advanced cancer patients through precision killing with fewer side effects. The stability and efficacy of ADCs are closely related, emphasizing the urgency and importance of gaining a comprehensive understanding of ADC stability. In this work, a chemical de-conjugation approach was developed to investigate the in-situ stability of the small molecule drug while it is conjugated to the antibody. This method involves chemical-mediated release of the small molecule drug from the ADC and subsequent characterization of the released small molecule drug by HPLC. The feasibility of this technique was demonstrated utilizing a model ADC containing a disulfide linker that is sensitive to the reducing environment within cancer cells. Five reducing agents were screened for use in de-conjugation; tris(2-carboxyethyl) phosphine (TCEP) was selected for further optimization due to its high efficiency and clean impurity profile. The optimized de-conjugation assay was shown to have excellent specificity and precision. More importantly, it was shown to be stability indicating, enabling the identification and quantification of the small molecule drug and its degradation products under different formulation pHs and storage temperatures. In summary, the chemical de-conjugation strategy demonstrated here offers a powerful tool to assess the in-situ stability of small molecule drugs on ADCs and the resulting information will shed light on ADC formulation/process development and storage condition selection.

A Small-scale Model to Assess the Risk of Leachables from Single-use Bioprocess Containers through Protein Quality Characterization.

Leachables from single-use bioprocess containers (BPCs) are a source of process-related impurities that have the potential to alter product quality of biotherapeutics and affect patient health. Leachables often exist at very low concentrations, making it difficult to detect their presence and challenging to assess their impact on protein quality. A small-scale stress model based on assessing protein stability was developed to evaluate the potential risks associated with storing biotherapeutics in disposable bags caused by the presence of leachables. Small-scale BPCs were filled with protein solution at high surface area-to-volume ratios (≥3× the surface area-to-volume ratio of manufacturing-scale BPCs) and incubated at stress temperatures (e.g., 25 °C or 30 °C for up to 12 weeks) along with an appropriate storage vessel (e.g., glass vial or stainless steel) as a control for side-by-side comparison. Changes in protein size variants measured by size exclusion chromatography, capillary electrophoresis, and particle formation for two monoclonal antibodies using both the small-scale stress model and a control revealed a detrimental effect of gamma-irradiated BPCs on protein aggregation and significant BPC difference between earlier and later batches. It was found that preincubation of the empty BPCs prior to protein storage improved protein stability, suggesting the presence of volatile or heat-sensitive leachables (heat-labile or thermally degraded). In addition, increasing the polysorbate 20 concentration lowered, but did not completely mitigate, the leachable-protein interactions, indicating the presence of a hydrophobic leachable. Overall, this model can inform the risk of BPC leachables on biotherapeutics during routine manufacturing and assist in making decisions on the selection of a suitable BPC for the manufacturing process by assessing changes in product quality. LAY ABSTRACT: Leachables from single-use systems often exist in small quantities and are difficult to detect with existing analytical methods. The presence of relevant detrimental leachables from single-use bioprocess containers (BPCs) can be indirectly detected by studying the stability of monoclonal antibodies via changes by size exclusion chromatography, capillary electrophoresis sodium dodecyl sulfate, and visible/sub-visible particles using a small-scale stress model containing high surface area-to-volume ratio at elevated temperature alongside with an appropriate control (e.g., glass vials or stainless steel containers). These changes in protein quality attributes allowed the evaluation of potential risks associated with adopting single-use bioprocess containers for storage as well as bag quality and bag differences between earlier and later batches. These leachables appear to be generated during the bag sterilization process by gamma irradiation. Improvements in protein stability after storage in "preheated" bags indicated that these leachables may be thermally unstable or volatile. The effect of surfactant levels, storage temperatures, surface area-to-volume ratios, filtration, and buffer exchange on leachables and protein stability were also assessed.

Monitoring nucleic acids using molecular beacons.

The ability to observe the dynamic of RNA in single living cells offers many exciting opportunities in biology and medicine. In the last few years, molecular beacons (MBs) have shown great potential in monitoring RNA synthesis, transportation, and localization with good sensitivity and selectivity. A hairpin structure probe, MB is a dual-labeled single stranded oligonucleotide that only fluoresces in the presence of target sequences. In this paper, the basic principle and design of MB will be described. The application of MB for RNA imaging in living cells will be reviewed. The limitations of MB for in vivo application will be identified. In the last section of the article, the efforts on designing better MBs for highly sensitive and selective RNA imaging will be discussed.

A size exclusion-reversed phase two dimensional-liquid chromatography methodology for stability and small molecule related species in antibody drug conjugates.

Antibody drug conjugates (ADCs) are complex therapeutic agents combining the specific targeting properties of antibodies and highly potent cytotoxic small molecule drugs to selectively eliminate tumor cells while limiting the toxicity to normal healthy tissues. One unique critical quality attribute of ADCs is the content of unconjugated small molecule drug present from either incomplete conjugation or degradation of the ADC. In this work, size exclusion chromatography (SEC) was coupled with reversed-phase (RP) HPLC in an online 2-dimensional chromatography format for identification and quantitation of unconjugated small molecule drugs and related small molecule impurities in ADC samples directly without sample preparation. The SEC method in the 1st dimension not only separated the small molecule impurities from the intact ADC, but also provided information about the size variants (monomer, dimer, aggregates, etc.) of the ADC. The small molecule peak from the SEC was trapped and sent to a RP-HPLC in the 2nd dimension to further separate and quantify the different small molecule impurities present in the ADC sample. This SEC-RP 2D-LC method demonstrated excellent precision (%RSD<2.0), linearity (r(2)=0.9999), sensitivity (LOQ of 0.05µg/mL of free drug in ADC sample) and accuracy (95-105% recovery of spiked samples). The 2D-LC method was further utilized to study the stability of an ADC drug product at different temperatures and pHs. Both small molecule degradation products and aggregation of the conjugate were observed in the stability samples and the degradation pathways of the ADC were investigated. This 2D-LC method offers a powerful tool for ADC characterization and provides valuable information for conjugation and formulation development.

Limiting degradation of reactive antibody drug conjugate intermediates in HPLC method development.

The development of an accurate HPLC method for a reactive linker drug intermediate for antibody drug conjugates (ADCs) is challenging as the linker drug is designed to be reactive. This reactivity can lead to the generation of artifact peaks in the chromatograms and deliver inaccurate impurity content results. In this study, the linker drug contains an amine reactive tetrafluorophenyl (TFP) ester that readily undergoes hydrolysis and internal succinimide cyclization in aqueous solution. While complete elimination of the on-column degradation was nearly impossible, optimization of the critical chromatography conditions guided by chemical kinetics minimizes the degree of on-column degradation enabling an accurate assay and purity method for this reactive linker drug intermediate. Kinetics of the linker drug reactions were studied in different solution pHs and temperatures while the reaction rates were used to guide the selection of the optimum diluent, mobile phase pH, and column temperature to minimize the on-column degradation. An UHPLC column was used to achieve fast analysis to further reduce the degree of on-column degradation. The actual amount of on-column degradation of the final HPLC method was determined to be <0.1%, lower than the ICH reporting limit of, therefore demonstrate the effectiveness of the strategy.

Quantification of residual solvents in antibody drug conjugates using gas chromatography.

The detection and quantification of residual solvents present in clinical and commercial pharmaceutical products is necessary from both patient safety and regulatory perspectives. Head-space gas chromatography is routinely used for quantitation of residual solvents for small molecule APIs produced through synthetic processes; however residual solvent analysis is generally not needed for protein based pharmaceuticals produced through cultured cell lines where solvents are not introduced. In contrast, antibody drug conjugates and other protein conjugates where a drug or other molecule is covalently bound to a protein typically use solvents such as N,N-dimethylacetamide (DMA), N,N­dimethylformamide (DMF), dimethyl sulfoxide (DMSO), or propylene glycol (PG) to dissolve the hydrophobic small molecule drug for conjugation to the protein. The levels of the solvent remaining following the conjugation step are therefore important to patient safety as these parental drug products are introduced directly into the patients bloodstream. We have developed a rapid sample preparation followed by a gas chromatography separation for the detection and quantification of several solvents typically used in these conjugation reactions. This generic method has been validated and can be easily implemented for use in quality control testing for clinical or commercial bioconjugated products.

Gold nanoparticle-based colorimetric assay for the direct detection of cancerous cells.

Early and accurate detection of cancer often requires time-consuming techniques and expensive instrumentation. To address these limitations, we developed a colorimetric assay for the direct detection of diseased cells. The assay uses aptamer-conjugated gold nanoparticles to combine the selectivity and affinity of aptamers and the spectroscopic advantages of gold nanoparticles to allow for the sensitive detection of cancer cells. Samples with the target cells present exhibited a distinct color change while nontarget samples did not elicit any change in color. The assay also showed excellent sensitivity with both the naked eye and based on absorbance measurements. In addition, the assay was able to differentiate between different types of target and control cells based on the aptamer used in the assay indicating the wide applicability of the assay for diseased cell detection. On the basis of these qualities, aptamer-conjugated gold nanoparticles could become a powerful tool for point of care diagnostics.

Antibody-functionalized nano test tubes target breast cancer cells.

AIM: To develop nano test tubes that will deliver a biomedical payload to a specific cell type. METHODS: The template-synthesis method was used to prepare silica nano test tubes. An antibody that is specific for breast cancer cells was attached to the outer tube surfaces. A fluorophore was attached to the inner surfaces of the nano test tubes. The tubes were incubated with the breast cancer cells and the extent of attachment to the cell surfaces was investigated by fluorescence microscopy. RESULTS: Tubes modified on their outer surfaces with the target antibody showed enhanced attachment to breast-cancer cells, relative to tubes modified on their outer surfaces with a species and isotype-matched control antibody. CONCLUSIONS: This work is a first step toward demonstrating that nano test tubes can be used as cell-specific delivery vehicles.