Comparative analysis of Herceptin N-Linked glycosylation by HILIC-FLD and LC-MS/MS methods.

Monoclonal antibodies like Herceptin play a pivotal role in modern therapeutics, with their glycosylation patterns significantly influencing their bioactivity. To characterize the N-glycan profile and their relative abundance in Herceptin, we employed two analytical methods: hydrophilic interaction chromatography with fluorescence detection (HILIC-FLD) for released glycans and liquid chromatography tandem mass spectrometry (LC-MS/MS) for glycopeptides. Our analysis included 21 European Union (EU)-Herceptin lots and 14 United States (US)-Herceptin lots. HILIC-FLD detected 25 glycan species, including positional isomers, revealing comparable chromatographic profiles for both EU and US lots. On the other hand, LC-MS/MS identified 26 glycoforms within the glycopeptide EEQYNSTYR. Both methods showed that a subset of glycans dominated the total abundance. Notably, EU-Herceptin lots with an expiration date of October 2022 exhibited increased levels of afucosylated and high mannose N-glycans. Our statistical comparisons showed that the difference in quantitative results between HILIC-FLD and LC-MS/MS is significant, indicating that the absolute quantitative values depend on the choice of the analytical method. However, despite these differences, both methods demonstrated a strong correlation in relative glycan proportions. This study contributes to the comprehensive analysis of Herceptin's glycosylation, offering insights into the influence of analytical methods on glycan quantification and providing valuable information for the biopharmaceutical industry.

Design of a Ratiometric Plasmonic Biosensor for Herceptin Detection in HER2-Positive Breast Cancer.

Breast cancer is the most common cause of cancer death in women; therefore, its early detection and treatment are crucial. To achieve this goal, we designed an optical sensor based on direct interaction of trastuzumab [Herceptin (HER)], a monoclonal antibody used to treat HER2-positive breast cancer, with plasmonic nanoparticles. Surface-modified gold nanoparticles (AuNPs) have gained considerable attention in biosensing techniques over the last years, which actuated these nanoparticles to the heart of various biosensing notions. We have exploited the localized surface plasmon resonance (LSPR) of gold nanoparticles to determine HER in human serum. AuNPs were decorated with negatively charged citrate ions, yielding enhanced direct-surface interaction with HER antibodies. The AuNPs are mixed with silver nanoparticles (AgNPs) in an optimized ratio to increase selectivity and sensitivity further. AuNPs detect the HER antibodies using LSPR, whereas AgNPs help monitor interferences' effect on the sensing media. The three effective factors in HER sensing, including the nanoparticle ratio, temperature, and pH were optimized via response surface methodology (RSM) based on the central composite design (CCD). The sensor's response toward HER was achieved in the linear range of 0.5 × 10-7 to 40 × 10-7 M with the detection limit of 3.7 × 10-9 M and relative standard deviation (RSD) less than 5%. The selectivity of the LSPR sensor was assessed by monitoring its response toward HER in the presence of other biological molecules with similar physicochemical properties. Rapid response time (less than 1 min), selectivity, and the simplicity of the developed LSPR-based sensor are the key advantages of the developed sensor.

Coupling of Trastuzumab chromatographic profiling with machine learning tools: A complementary approach for biosimilarity and stability assessment.

Biosimilar products present a growing opportunity to improve the global healthcare systems. The amount of accepted variability during the comparative assessments of biosimilar products introduces a significant challenge for both the biosimilar developers and the regulatory authorities. The aim of this study was to explore unsupervised machine learning tools as a mathematical aid for the interpretation and visualization of such comparability under control and stress conditions using data extracted from high throughput analytical techniques. For this purpose, a head-to-head analysis of the physicochemical characteristics of three Trastuzumab (TTZ) approved biosimilars and the originator product (Herceptin®) was performed. The studied quality attributes included the primary structure and identity by peptide mapping (PM) with reversed-phase chromatography-UV detection, size and charge profiles by stability-indicating size exclusion and cation exchange chromatography. Stress conditions involved pH and thermal stress. Principal component analysis (PCA) and two of the widely used cluster analysis tools, namely, K-means and Density-based Spatial Clustering of Applications with Noise (DBSCAN), were explored for clustering and feature representation of varied analytical datasets. It has been shown that the clustering patterns delineated by the used algorithms changed based on the included chromatographic profiles. The applied data analysis tools were found effective in revealing patterns of similarity and variability between i) intact and stressed as well as ii) originator and biosimilar samples.

Quantification of HER2 in COS7 cells using quantum weak measurement.

A Mach-Zehnder interferometer system based on weak measurement was set up to determinate the concentration variation of molecule by measuring the phase difference change between the two optical paths. The spectrum of the light was recorded to monitor the concentration of trastuzumab (Herceptin), which is a humanised monoclonal antibody, targeted to human epidermal growth factor receptor 2 (HER2). The trastuzumab targeting to HER2 was real-time detected and continuously monitored, the HER2 numbers of COS7 cells on a coverslip was determined at pico-molar level. Our weak measurement enabled method proposes an alternative approach for the concentration detection of molecules, providing a promising functional tool for the quantification of HER2 in cancer cells, possibly promoting fields such as the diagnosis and treatment of cancer.

Collecting antibodies and large molecule biomarkers in mouse interstitial brain fluid: a comparison of microdialysis and cerebral open flow microperfusion.

The determination of concentrations of large therapeutic molecules, like monoclonal antibodies (mAbs), in the interstitial brain fluid (ISF) is one of the cornerstones for the translation from preclinical species to humans of treatments for neurodegenerative diseases. Microdialysis (MD) and cerebral open flow microperfusion (cOFM) are the only currently available methods for extracting ISF, and their use and characterization for the collection of large molecules in rodents have barely started. For the first time, we compared both methods at a technical and performance level for measuring ISF concentrations of a non-target-binding mAb, trastuzumab, in awake and freely moving mice. Without correction of the data for recovery, concentrations of samples are over 10-fold higher through cOFM compared to MD. The overall similar pharmacokinetic profile and ISF exposure between MD (corrected for recovery) and cOFM indicate an underestimation of the absolute concentrations calculated with in vitro recovery. In vivo recovery (zero-flow rate method) revealed an increased extraction of trastuzumab at low flow rates and a 6-fold higher absolute concentration at steady state than initially calculated with the in vitro recovery. Technical optimizations have significantly increased the performance of both systems, resulting in the possibility of sampling up to 12 mice simultaneously. Moreover, strict aseptic conditions have played an important role in improving data quality. The standardization of these complex methods makes the unraveling of ISF concentrations attainable for various diseases and modalities, starting in this study with mAbs, but extending further in the future to RNA therapeutics, antibody-drug conjugates, and even cell therapies.

Characterization of Glycosylated Proteins at Subunit Level by HILIC/MS.

Hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry (MS) is considered as the reference analytical technique for glycans profiling, especially for the characterization of glycosylated protein therapeutics such as monoclonal antibodies (mAbs) and mAbs-related products. Although HILIC/MS is mainly known to profile enzymatically released and fluorescently labeled N-glycans, the recent commercialization of new widepore HILIC amide bonded stationary phases packed with sub-2 µm particles has allowed for remarkable separations also at the subunit level. Here, we describe a simple protocol to perform the mAb glycans profiling at subunit level by HILIC/MS.

Platform Methods to Characterize the Charge Heterogeneity of Three Common Protein Therapeutics by Imaged Capillary Isoelectric Focusing.

Imaged capillary isoelectric focusing (icIEF) is a gold standard method for characterizing the charge heterogeneity of protein therapeutics. A broad range of protein therapeutics such as monoclonal antibodies, antibody-drug conjugates (ADCs), and fusion proteins are routinely analyzed by icIEF due to its high resolution and high reproducibility. Platform methods, which can be applied without modification to the analysis of different protein therapeutics, save valuable time and resources in method development and quality control. Here, we provide platform methods for icIEF analysis of three classes of protein therapeutics, a biosimilar to the monoclonal antibody trastuzumab, recombinant human erythropoietin (rhEPO), and a fusion protein. The details of sample preparation and separation conditions for each molecule are described in this chapter.

Optimisation of the use of sliding window deconvolution for comprehensive characterisation of trastuzumab and adalimumab charge variants by native high resolution mass spectrometry.

The biopharmaceutical industry continues to develop mAb-based biotherapeutics in increasing numbers. Due to their complexity, there are several critical quality attributes (CQAs) that need to be measured and controlled to guarantee product safety and efficacy. Charge variant analysis is a widely used method to monitor changes in product quality during the manufacturing process of monoclonal antibodies (mAbs) and, together with a bottom-up peptide centred approach, acts as a key analytical platform to fulfil regulatory requirements. Native MS measures biomolecules under conditions that preserve most aspects of protein tertiary and quaternary structure, enabling direct characterization of large intact proteins such as mAbs. The resulting native mass spectrum of a mAb is characterized by a narrower charge-state envelope that simplifies the spectra and also condenses the ion signals into fewer peaks, increasing the signal-to-noise ratio. Algorithmic spectral deconvolution is needed for routine accurate and rapid molecular weight determination, and consequently, multiple deconvolution algorithms have evolved over the past decade. Here, we demonstrate the utility of the sliding window algorithm as a robust and powerful deconvolution tool for comprehensive characterisation of charge variant analysis data for mAbs. Optimum performance is evaluated by studying the impact of critical software parameters on detection, identification and relative quantitation of protein isoforms. By combining molecular mass and retention time information, it was possible to identify multiple modifications on adalimumab and trastuzumab, both IgG1 mAbs, including lysine truncation, deamidation and succinimide formation, along with the N-glycan distribution of each of the identified charge variants. Sliding window deconvolution also provides a key benefit of low abundant variant detection in a single analysis and the ability to detect co-eluting components with different relative abundances. The studied mAbs demonstrate the algoritms applicability for efficient data processing of both simple and complex mAbs analysed using pH gradient cation exchange chromatography coupled to native mass spectrometry.

Using differential scanning calorimetry for the development of non-reduced capillary electrophoresis sodium dodecyl sulfate methods for monoclonal antibodies.

Analysis of non-reduced and reduced monoclonal antibodies (mAbs) by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is routinely used to detect product size variants and process-related impurities. Levels of high molecular weight (HMW) forms obtained from this method usually trend comparably to those obtained by orthogonal methods such as size-exclusion ultra-high performance liquid chromatography (SE-UHPLC). However, in the presented case study, comparison of CE-SDS data for three IgG1 mAbs (trastuzumab, mAb1, and mAb2) showed a discrepancy between amounts of observed HMW forms in mAb2 compared with its native forms determined by SE-UHPLC (~17% vs. ~0.5%, respectively). SDS chemical denaturation, as measured by differential scanning calorimetry, demonstrated that the high thermal stability of mAb2 caused an unidentified HMW peak observed by non-reduced (NR)-CE-SDS, which was the result of improper denaturing, resulting in a partially folded species. More so, this strategy enabled the rapid identification of optimal SDS concentration and temperature conditions needed for suitable denaturation for mAb2. This case study presents an alternative option for quick optimization of NR-CE-SDS methods when characterizing mAbs or other thermally stable proteins. Also, this strategy can be used to understand basic biophysical mechanisms of protein unfolding and investigate the higher-order structure imparted by specific sequences and understand how these sequences might affect the results of an analytical method such as CE-SDS.

Aptamers as Versatile Molecular Tools for Antibody Production Monitoring and Quality Control.

Antibody drugs have been used to treat many diseases, and to date, this has been the most rapidly growing drug class. However, the lack of suitable methods for real-time and high-throughput monitoring of antibody production and quality control has been a hindrance to the further advancement of antibody drugs or biosimilars. Therefore, we herein report a versatile tool for one-step fluorescence monitoring of antibody production by using aptamer probes selected through the in vitro SELEX method. In this case, DNA aptamers were selected against the humanized IgG1 antibody drug trastuzumab with high specificity and affinity with a Kd value of aptamer CH1S-3 of 10.3 nM. More importantly, the obtained aptamers were able to distinguish native from heat-treated, whereas antibodies failed this test. On the basis of the advantages of rapid detection for aptamers, we designed aptamer molecular beacons for direct and sensitive detection of trastuzumab in complex samples. Unlike traditional antibody-based ELISA, the signal was observed directly upon interaction with the target without the need for time-consuming binding and multiple washing steps. To further highlight biomedical applications, the use of aptamers as potential tools for quality control and traceless purification of antibody drugs was also demonstrated. Thus, aptamers are shown to be promising alternatives for antibody production monitoring, quality control, and purification, providing technical support to accelerate antibody drug development.

An application of Nano Differential Scanning Fluorimetry for Higher Order Structure assessment between mAb originator and biosimilars: Trastuzumab and Rituximab as case studies.

Differential scanning fluorimetry (DSF) or thermal shift has emerged in recent years as a high-throughput screening method in biotherapeutic formulation studies. The present article reports on a fast-track assessment platform for rapid investigation of therapeutic proteins such as monoclonal antibodies (mAb) with minimal sample concentration, volume, and preparation. The proposed nanoDSF platform has been demonstrated for rapid assessment of two commercial IgG 1 drug products (DP), trastuzumab and rituximab, and their biosimilars with respect to their conformational and colloidal stability. Domain specific differences for each of the IgGs have been elucidated with respect to onset of domain unfolding (Tonset) and melting temperatures. These thermal unfolding and transition midpoint (Tm) measurements are based on the intrinsic aromatic amino acid residue fluorescence of proteins. Moreover, to understand the possibility of nanoDSF as a predictive tool, data from nanoDSF has been correlated with accelerated stability studies. Melting temperatures across brands were found to be highly comparable to the rate of heating, thereby exhibiting a significant domain specific effect on melting temperatures for both trastuzumab and rituximab. Conservation of higher order structure (HOS) through reversible unfolding was also examined and both the mAbs were found to regain tertiary structure up till the first transition midpoint. No clear correlation was found between formation of higher molecular weight species (HMWS) and unfolding parameters (Tonset and Tagg) for accelerated stability studies. Finally, a discussion on the need for fast predictive assessment of conformation and colloidal stability as well as a comparison of advantages and limitations of the technique with routine/classical tools such as circular dichroism spectrophotometry and differential scanning calorimetry has been presented.

Improving the throughput of immunoaffinity purification and enzymatic digestion of therapeutic proteins using membrane-immobilized reagent technology.

Continued interest in protein therapeutics has motivated the development of improved bioanalytical tools to support development programs. LC-MS offers specificity, sensitivity, and multiplexing capabilities without the need for target-specific reagents, making it a valuable alternative to ligand binding assays. Immunoaffinity purification (IP) and enzymatic digestion are critical, yet extensive and time-consuming components of the "gold standard" bottom-up approach to LC-MS-based protein quantitation. In the present work, commercially available technology, based on membrane-immobilized reagents in spin column and plate format, is applied to reduce IP and digestion times from hours to minutes. For a standard monoclonal antibody, the lower limit of quantitation was 0.1 ng µL-1 compared to 0.05 ng µL-1 for the standard method. A pharmacokinetics (PK) study dosing Herceptin in rat was analyzed by both the membrane and the standard method with a total sample processing time of 4 h and 20 h, respectively. The calculated concentrations at each time point agreed within 8% between both methods, and PK values including area under the curve (AUC), half-life (T1/2), mean residence time (MRT), clearance (CL), and volume of distribution (Vdss) agreed within 6% underscoring the utility of the membrane methodology for quantitative bioanalysis workflows.

Conformation assessment of therapeutic monoclonal antibodies by SEC-MS: Unravelling analytical biases for application to quality control.

Immunogenicity related to the degradation of therapeutic monoclonal antibodies (mAbs) remains a major concern for their therapeutic efficacy and safety. Therefore, an analytical method allowing characterization and detection of mAbs degradation is mandatory. In this study, a simultaneous coupling of size exclusion chromatography (SEC) to native mass spectrometry (MS) and fluorescence detection (FLD) is proposed to detect degraded therapeutic mAbs and biases of structural changes (e.g. dimerization, denaturation) that may occur during native MS. A comprehensive study on infliximab behaviors have been performed under different mobile phase conditions (e.g. composition, pH, organic solvent, etc.) and MS parameters (e.g. gas temperatures, CID energies, etc.). Experimental conditions avoiding artificial denaturation and/ or dimerization have been defined. We have also demonstrated that under the developed conditions infliximab affinity towards its biological target TNFα is preserved. In addition, using this method dimers, denatured monomers and fragments could be detected in trastuzmab samples stressed by a long-term storage. These results were confirmed by using SEC coupled to ion mobility mass spectrometry as an orthogonal method for the detection of denatured monomer.

In-depth analysis of monoclonal antibodies using microfluidic capillary electrophoresis and native mass spectrometry.

Charge variant profiling of therapeutic proteins is required by the International Council for Harmonisation guidelines and is traditionally performed by capillary electrophoresis or ion exchange chromatography. Recently, improvements in the hyphenation of capillary electrophoresis with mass spectrometry and the introduction of mass spectrometry compatible background electrolytes has allowed the implementation of native mass spectrometric determination of the charge variant profile obtained from the electrophoretic separation. The low flow operation of the microfluidic electrophoretic platform significantly boosts mass spectrometric sensitivity and increases the dynamic range, even when using sample amounts as low as 1 ng in capillary. In the current study, rituximab, trastuzumab and bevacizumab drug products were analysed using the ZipChip microfluidic CE-ESI-MS platform that facilitated confident identification of proteoforms with an average mass accuracy of <15 ppm. Up to 52 proteoforms were identified for trastuzumab drug product, while rituximab sample revealed the presence of fragments and sialylated N-glycans. Overall, the CE-ESI-MS platform proved to be a fast and robust tool for therapeutic protein charge variant profiling and facilitated efficient coupling with native mass spectrometry for the generation of highly informative characterisation data.

Detection of doxorubicin, cisplatin and therapeutic antibodies in formalin-fixed paraffin-embedded human cancer cells.

A major limitation in the pharmacological treatment of clinically detectable primary cancers and their metastases is their limited accessibility to anti-cancer drugs (cytostatics, inhibitory antibodies, small-molecule inhibitors) critically impairing therapeutic efficacies. Investigations on the tissue distribution of such drugs are rare and have only been based on fresh frozen material or methanol-fixed cell culture cells so far. In this paper, we expand the detection of cisplatin-induced DNA adducts and anthracyclines as well as therapeutic antibodies to routinely prepared formalin-fixed, paraffin-embedded sections (FFPE). Using pre-treated cell lines prepared as FFPE samples comparable to tissues from routine analysis, we demonstrate that our method allows for the detection of chemotherapeutics (anthracyclines by autofluorescence, cisplatin by immune detection of DNA adducts) as well as therapeutic antibodies. This methodology thus allows for analyzing archival FFPE tissues, as demonstrated here for the detection of cisplatin, doxorubicin and trastuzumab in FFPE sections of tumor xenografts from drug-treated mice. Analyzing human tumor samples, this will lead to new insights into the tissue penetration of drugs.

Demonstrating Analytical Similarity of Trastuzumab Biosimilar HLX02 to Herceptin® with a Panel of Sensitive and Orthogonal Methods Including a Novel FcγRIIIa Affinity Chromatography Technology.

BACKGROUND: A biosimilar needs to demonstrate its similarity to the originator reference product (RP) in terms of structural and functional properties as well as nonclinical and clinical outcomes. OBJECTIVES: The aim was to assess the analytical similarity between the trastuzumab biosimilar HLX02 and Europe-sourced Herceptin® (EU-Herceptin®) and China-sourced Herceptin® (CN-Herceptin®) following a quality-by-design (QbD) quality study and tier-based quality attribute evaluation. METHODS: A panel of highly sensitive and orthogonal methods, including a novel Fc gamma receptor IIIa (FcγRIIIa) affinity chromatography technique that enables quantitative comparison of glycan effects on effector function, was developed for the assessment. To ensure the full product variability was captured, ten batches of HLX02 were compared with 39 RP batches with expiry dates from August 2017 to March 2021. RESULTS: The extensive three-way similarity assessment demonstrated that HLX02 is highly similar to the RPs. Furthermore, the %afucose, %galactose, and FcγRIIIa affinity of the RPs were observed to first decrease and then return to the original level in relation to their expiry dates, and the RP batches can be subgrouped by their FcγRIIIa affinity chromatograms. HLX02 is demonstrated to be more similar to the RPs of the high FcγRIIIa affinity group. CONCLUSION: Besides having an overall high analytical similarity to both EU-Herceptin® and CN-Herceptin®, HLX02 is more similar to Herceptin® with high FcγRIIIa affinity, a result that demonstrates the power of the novel FcγRIIIa affinity chromatography technology in biosimilarity evaluation.

Evaluation of Quantitative Relationship Between Target Expression and Antibody-Drug Conjugate Exposure Inside Cancer Cells.

Antibody-drug conjugates (ADCs) employ overexpressed cell surface antigens to deliver cytotoxic payloads inside cancer cells. However, the relationship between target expression and ADC efficacy remains ambiguous. In this manuscript, we have addressed a part of this ambiguity by quantitatively investigating the effect of antigen expression levels on ADC exposure within cancer cells. Trastuzumab-valine-citrulline-monomethyl auristatin E was used as a model ADC, and four different cell lines with diverse levels of human epidermal growth factor receptor 2 (HER2) expression were used as model cells. The pharmacokinetics (PK) of total trastuzumab, released monomethyl auristatin E (MMAE), and total MMAE were measured inside the cells and in the cell culture media following incubation with two different concentrations of ADC. In addition, target expression levels, target internalization rate, and cathepsin B and MDR1 protein concentrations were determined for each cell line. All the PK data were mathematically characterized using a cell-level systems PK model for ADC. It was found that SKBR-3, MDA-MB-453, MCF-7, and MDA-MB-468 cells had ∼800,000, ∼250,000, ∼50,000, and ∼10,000 HER2 receptors per cell, respectively. A strong linear relationship (R 2 > 0.9) was observed between HER2 receptor count and released MMAE exposure inside the cancer cells. There was an inverse relationship found between HER2 expression level and internalization rate, and cathepsin B and multidrug resistance protein 1 (MDR1) expression level varied slightly among the cell lines. The PK model was able to simultaneously capture all the PK profiles reasonably well while estimating only two parameters. Our results demonstrate a strong quantitative relationship between antigen expression level and intracellular exposure of ADCs in cancer cells. SIGNIFICANCE STATEMENT: In this manuscript, we have demonstrated a strong linear relationship between target expression level and antibody-drug conjugate (ADC) exposure inside cancer cells. We have also shown that this relationship can be accurately captured using the cell-level systems pharmacokinetics model developed for ADCs. Our results indirectly suggest that the lack of relationship between target expression and efficacy of ADC may stem from differences in the pharmacodynamic properties of cancer cells.

Ultrafast enzymatic digestion of proteins by microdroplet mass spectrometry.

Enzymatic digestion for protein sequencing usually requires much time, and does not always result in high sequence coverage. Here we report the use of aqueous microdroplets to accelerate enzymatic reactions and, in particular, to improve protein sequencing. When a room temperature aqueous solution containing 10 µM myoglobin and 5 µg mL-1 trypsin is electrosonically sprayed (-3 kV) from a homemade setup to produce tiny (∼9 µm) microdroplets, we obtain 100% sequence coverage in less than 1 ms of digestion time, in sharp contrast to 60% coverage achieved by incubating the same solution at 37 °C for 14 h followed by analysis with a commercial electrospray ionization source that produces larger (∼60 µm) droplets. We also confirm the sequence of the therapeutic antibody trastuzumab (∼148 kDa), with a sequence coverage of 100% for light chains and 85% for heavy chains, demonstrating the practical utility of microdroplets in drug development.

Optimal Collision Energies and Bioinformatics Tools for Efficient Bottom-up Sequence Validation of Monoclonal Antibodies.

Rigorous validation of amino acid sequence is fundamental in the characterization of original and biosimilar protein biopharmaceuticals. Widely accepted workflows are based on bottom-up mass spectrometry, and they often require multiple techniques and significant manual work. Here, we demonstrate that optimization of a set of tandem mass spectroscopy (MS/MS) collision energies and automated combination of all available information in the measurements can increase the sequence validated by one technique close to the inherent limits. We created a software (called "Serac") that consumes results of the Mascot database search engine and identifies the amino acids validated by bottom-up MS/MS experiments using the most rigorous, industrially acceptable definition of sequence coverage (we term this "confirmed sequence coverage"). The software can combine spectra at the level of amino acids or fragment ions to exploit complementarity, provides full transparency to justify validation, and reduces manual effort. With its help, we investigated collision energy dependence of confirmed sequence coverage of individual peptides and full proteins on trypsin-digested monoclonal antibody samples (rituximab and trastuzumab). We found the energy dependence to be modest, but we demonstrated the benefit of using spectra taken at multiple energies. We describe a workflow based on 2-3 LC-MS/MS runs, carefully selected collision energies, and a fragment ion level combination, which yields ∼85% confirmed sequence coverage, 25%-30% above that from a basic proteomics protocol. Further increase can mainly be expected from alternative digestion enzymes or fragmentation techniques, which can be seamlessly integrated to the processing, thereby allowing effortless validation of full sequences.

Microfabricated calorimeters for thermometric enzyme linked immunosorbent assay in one-Nanoliter droplets.

Advances in microfabrication allow for highly sensitive calorimeters with dramatically reduced volume, decreased response time and increased energy resolution. These calorimeters hold the potential for designs of ELISA platforms competitive with fluorescent and chemiluminescent technologies. We have developed a new assay platform using conventional ELISA reagents to produce a thermal signal quantifiable using calorimetry. Our optimized micromachined calorimeters have nL reaction volumes and a minimum detectable power of 375 pW/Hz1/2. We demonstrate rapid quantification in a model system of trastuzumab, a humanized monoclonal antibody used in the treatment of HER2 overexpressing breast cancers, in human serum using a HER2 peptide mimetic. Trastuzumab concentration and reaction time constant correlated well (R2 = 0.954) and can be used to determine trastuzumab concentrations. The limit of detection for the ThermometricELISA (TELISA) was 10 µg/ml trastuzumab in human serum. TELISA allows for a simple readout, reduction in assay time, sample and reagent volumes and has the potential to become a point of care multiplexed platform technology.