Mitigation of Oxidation in Therapeutic Antibody Formulations: a Biochemical Efficacy and Safety Evaluation of N-Acetyl-Tryptophan and L-Methionine.

PURPOSE: Biotherapeutics can be susceptible to oxidation during manufacturing and storage. Free L-methionine is known to protect methionine residues in proteins from oxidation. Similarly, free tryptophan and other indole derivatives have been shown to protect tryptophan residues from oxidation. N-acetyl-DL-tryptophan was previously identified as a potentially superior antioxidant to tryptophan as it has a lower oxidation potential and produces less peroxide upon light exposure. This study sought to confirm the antioxidant efficacy and safety of N-acetyl-DL-tryptophan and L-methionine as formulation components for biotherapeutic drugs. METHODS: Antibodies were subjected to AAPH and light exposure in the presence of N-acetyl-DL-tryptophan and L-methionine. Oxidation in relevant CDR and Fc residues was quantified by peptide map. In silico, in vitro, and in vivo studies were performed to evaluate the safety of N-acetyl-DL-tryptophan and L-methionine. RESULTS: Peptide mapping demonstrated that N-acetyl-DL-tryptophan was effective at protecting tryptophans from AAPH stress, and that the combination of N-acetyl-DL-tryptophan and L-methionine protected both tryptophan and methionine from AAPH stress. The safety assessment suggested an acceptable safety profile for both excipients. CONCLUSIONS: N-acetyl-tryptophan and L-methionine effectively reduce the oxidation of susceptible tryptophan and methionine residues in antibodies and are safe for use in parenteral biotherapeutic formulations.

The pandemic and resilience for the future: AccBio 2021.

The year 2020 brought the onslaught of a global crisis in the form of the COVID-19 pandemic. While nearly every facet of everyday life and work was impacted by the pandemic, the biopharmaceutical industry found silver linings in innovation, partnership, and resiliency, all of which contributed to unprecedented speed in developing and delivering vaccines and therapies. The 7th International Conference on Accelerating Biopharmaceutical Development (AccBio 2021) brought together industry leaders to share experiences from the past year and discuss how lessons learned from the pandemic can be carried forward into the future of biopharmaceutical development. Presenters highlighted examples such as introducing biotherapeutics derived from non-clonal cell pools into the clinic, developing modular or platform technologies, and taking novel risks, among others. These strategies for enabling speed to clinic and launch, as well as for sustaining a robust supply chain, are likely to be integrated into future programs to ensure biomanufacturing resiliency and get medicines to patients faster than pre-pandemic times.

A universal method for detection of amyloidogenic misfolded proteins.

Diseases associated with the misfolding of endogenous proteins, such as Alzheimer's disease and type II diabetes, are becoming increasingly prevalent. The pathophysiology of these diseases is not totally understood, but mounting evidence suggests that the misfolded protein aggregates themselves may be toxic to cells and serve as key mediators of cell death. As such, an assay that can detect aggregates in a sensitive and selective fashion could provide the basis for early detection of disease, before cellular damage occurs. Here we report the evolution of a reagent that can selectively capture diverse misfolded proteins by interacting with a common supramolecular feature of protein aggregates. By coupling this enrichment tool with protein specific immunoassays, diverse misfolded proteins and sub-femtomole amounts of oligomeric aggregates can be detected in complex biological matrices. We anticipate that this near-universal approach for quantitative misfolded protein detection will become a useful research tool for better understanding amyloidogenic protein pathology as well as serve as the basis for early detection of misfolded protein diseases.

Ranking the selectivity of PubChem screening hits by activity-based protein profiling: MMP13 as a case study.

High-throughput screening (HTS) has become an integral part of academic and industrial efforts aimed at developing new chemical probes and drugs. These screens typically generate several 'hits', or lead active compounds, that must be prioritized for follow-up medicinal chemistry studies. Among primary considerations for ranking lead compounds is selectivity for the intended target, especially among mechanistically related proteins. Here, we show how the chemical proteomic technology activity-based protein profiling (ABPP) can serve as a universal assay to rank HTS hits based on their selectivity across many members of an enzyme superfamily. As a case study, four metalloproteinase-13 (MMP13) inhibitors of similar potency originating from a publically supported HTS and reported in PubChem were tested by ABPP for selectivity against a panel of 27 diverse metalloproteases. The inhibitors could be readily separated into two groups: (1) those that were active against several metalloproteases and (2) those that showed high selectivity for MMP13. The latter set of inhibitors was thereby designated as more suitable for future medicinal chemistry optimization. We anticipate that ABPP will find general utility as a platform to rank the selectivity of lead compounds emerging from HTS assays for a wide variety of enzymes.

Optimization of activity-based probes for proteomic profiling of histone deacetylase complexes.

Histone deacetylases (HDACs) are key enzymatic regulators of the epigenome and serve as promising targets for anticancer therapeutics. Recently, we developed a photoreactive "clickable" probe, SAHA-BPyne, to report on HDAC activity and complex formation in native biological systems. Here, we investigate the selectivity, sensitivity, and inhibitory properties of SAHA-BPyne and related potential activity-based probes for HDACs. While we identified several probes that are potent HDAC inhibitors and label HDAC complex components in native proteomic preparations, SAHA-BPyne was markedly superior for profiling HDAC activities in live cells. Interestingly, the enhanced performance of SAHA-BPyne as an in situ activity-based probe could not be solely ascribed to potency in HDAC binding, implying that other features of the molecule were key to efficient active site-directed labeling in living systems. Finally, we demonstrate the value of in situ profiling of HDACs by comparing the activity and expression of HDAC1 in cancer cells treated with the cytotoxic agent parthenolide. These results underscore the utility of activity-based protein profiling for studying HDAC function and may provide insight for the future development of click chemistry-based photoreactive probes for the in situ analysis of additional enzyme activities.

The Use of a 2,2'-Azobis (2-Amidinopropane) Dihydrochloride Stress Model as an Indicator of Oxidation Susceptibility for Monoclonal Antibodies.

Protein oxidation is a major pathway for degradation of biologic drug products. Past literature reports have suggested that 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH), a free radical generator that produces alkoxyl and alkyl peroxyl radicals, is a useful model reagent stress for assessing the oxidative susceptibility of proteins. Here, we expand the applications of the AAPH model by pairing it with a rapid peptide map method to enable site-specific studies of oxidative susceptibility of monoclonal antibodies and their derivatives for comparison between formats, the evaluation of formulation components, and comparisons across the stress models. Comparing the free radical-induced oxidation model by AAPH with a light-induced oxidation model suggests that light-sensitive residues represent a subset of AAPH-sensitive residues and therefore AAPH can be used as a preliminary screen to highlight molecules that need further assessment by light models. In sum, these studies demonstrate that AAPH stress can be used in multiple ways to evaluate labile residues and oxidation sensitivity as it pertains to developability and manufacturability.

Peptide-nanoparticle hybrid SERS probes for optical detection of protease activity.

Real-time in situ detection of active proteases is crucial for early-stage cancer screening and cell signaling pathway study; however, it is difficult to achieve using fluorescence or radioactive probes at volumes below 1 nL. Here we demonstrated a hybrid optical probe by incorporating nanocrescent particle and peptides with artificial tag molecules. We performed a proof-of-concept study using prostate specific antigen (PSA), one of the most prominent prostate cancer markers, and a serine protease present in patients' seminal fluid and serum. The Raman spectral signal from the tag molecules is enhanced by the nanocrescent and the signal is monitored as the indicator for peptide cleavage in a femtoliter reaction volume, at levels close to a single proteolytically active PSA molecule. The high reaction specificity of the peptides on individual nanoparticles minimizes the false detection of other serine proteases and background Raman signal, which results in a high-fidelity and high-signal-to-noise-ratio cancer nanoprobe that can be easily incorporated into nano/microfluidic devices.

Characterization of N-Acetyl-Tryptophan Degradation in Protein Therapeutic Formulations.

N-Acetyl-tryptophan (NAT) is used as a stabilizer for preparations of human serum albumin and has more recently been demonstrated to provide oxidative protection for labile Trp residues in monoclonal antibodies. As a component in the formulations of protein therapeutics, NAT is sacrificially degraded; therefore, understanding the identity and quantity of NAT degradants potentially formed in these drug products is essential to understanding the potential patient impact of this additive. Here, we report a simple reversed-phase chromatography approach that allows systematic investigation of NAT degradation in relevant formulations under stressed conditions. Screening a panel of NAT-containing samples following a variety of forced stress conditions led to a range of NAT degradation from minimal (3%) to significant (83%). NAT degradants were observed to be largely conserved between oxidative and thermal stress conditions. Online mass spectrometry and standard compound synthesis allowed for identification of the major degradants in the stressed sample panel. NAT degradation was minimal under recommended storage conditions and in relevant thermal stress conditions for a representative protein therapeutic drug product, suggesting that NAT is stable under normal manufacturing, storage, and handling conditions. This work supports the use of NAT as an antioxidant in liquid drug product formulations.

Microfluidic barcode assay for antibody-based confirmatory diagnostics.

Confirmatory diagnostics offer high clinical sensitivity and specificity typically by assaying multiple disease biomarkers. Employed in clinical laboratory settings, such assays confirm a positive screening diagnostic result. These important multiplexed confirmatory assays require hours to complete. To address this performance gap, we introduce a simple 'single inlet, single outlet' microchannel architecture with multiplexed analyte detection capability. A streptavidin-functionalized, channel-filling polyacrylamide gel in a straight glass microchannel operates as a 3D scaffold for a purely electrophoretic yet heterogeneous immunoassay. Biotin and biotinylated capture reagents are patterned in discrete regions along the axis of the microchannel resulting in a barcode-like pattern of reagents and spacers. To characterize barcode fabrication, an empirical study of patterning behaviour was conducted across a range of electromigration and binding reaction timescales. We apply the heterogeneous barcode immunoassay to detection of human antibodies against hepatitis C virus and human immunodeficiency virus antigens. Serum was electrophoresed through the barcode patterned gel, allowing capture of antibody targets. We assess assay performance across a range of Damkohler numbers. Compared to clinical immunoblots that require 4-10 h long sample incubation steps with concomitant 8-20 h total assay durations; directed electromigration and reaction in the microfluidic barcode assay leads to a 10 min sample incubation step and a 30 min total assay duration. Further, the barcode assay reports clinically relevant sensitivity (25 ng ml(-1) in 2% human sera) comparable to standard HCV confirmatory diagnostics. Given the low voltage, low power and automated operation, we see the streamlined microfluidic barcode assay as a step towards rapid confirmatory diagnostics for a low-resource clinical laboratory setting.

Activity-based probes for proteomic profiling of histone deacetylase complexes.

Histone deacetylases (HDACs) are key regulators of gene expression that require assembly into larger protein complexes for activity. Efforts to understand how associated proteins modulate the function of HDACs would benefit from new technologies that evaluate HDAC activity in native biological systems. Here, we describe an active site-directed chemical probe for profiling HDACs in native proteomes and live cells. This probe, designated SAHA-BPyne, contains structural elements of the general HDAC inhibitor suberoylanilide hydroxamic acid (SAHA), as well as benzophenone and alkyne moieties to effect covalent modification and enrichment of HDACs, respectively. Both class I and II HDACs were identified as specific targets of SAHA-BPyne in proteomes. Interestingly, multiple HDAC-associated proteins were also enriched by SAHA-BPyne, even after denaturation of probe-labeled proteomes. These data indicate that certain HDAC-associated proteins are directly modified by SAHA-BPyne, placing them in close proximity to HDAC active sites where they would be primed to regulate substrate recognition and activity. We further show that SAHA-BPyne can be used to measure differences in HDAC content and complex assembly in human disease models. This chemical proteomics probe should thus prove valuable for profiling both the activity state of HDACs and the binding proteins that regulate their function.

Functional phenotyping of human plasma using a 361-fluorogenic substrate biosensing microarray.

A microarray presenting glycerol nanodroplets of fluorogenic peptide substrates was used as a biosensor for the detection of multiple enzyme activities within human plasma. Using 10 different plasma proteases (kallikrein, factor XIIa, factor XIa, factor IXa, factor VIIa, factor Xa, thrombin, activated protein C, uPA and plasmin) and a 361-compound fluorogenic substrate library (Ac-Ala-P3-P2-Arg-coumarin for P = all amino acids except Cys), a database was created for deconvoluting the relative activity of each individual enzyme signal in human plasma treated with various activators (calcium, kaolin, or uPA). Three separate deconvolution protocols were tested: searching for "optimal" sensing substrate sequences for a set of 5 enzymes and using these substrates to detect protease signals in plasma; ranking the "optimal" sensing substrates for 10 proteases using local error minimization, resulting in a set of substrates which were bundled via weighted averaging into a super-pixel that had biosensing properties not obtainable by any individual fluorogenic substrate; and treating each 361-element map measured for each plasma preparation as a weighted sum of the 10 maps obtained for the 10 purified enzymes using a global error minimization. The similarity of the results from these latter two protocols indicated that a small subset of <90 substrates contained the majority of biochemical information. The results were consistent with the state of the coagulation cascade expected when treated with the given activators. This method may allow development of future biosensors using minimal and non-specific markers. These substrates can be applied to real-time diagnostic biosensing of complex protease mixtures.

High throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays.

Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized (X=all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXabeta, factor XIa and factor alpha XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates x 24 different proteases x 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P2 preference for Leu, Val, Phe, and Tyr in both the P1=Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage.

Profiling serine protease substrate specificity with solution phase fluorogenic peptide microarrays.

A novel microarray-based proteolytic profiling assay enabled the rapid determination of protease substrate specificities with minimal sample and enzyme usage. A 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized and microarrayed, along with fluorescent calibration standards, in glycerol nanodroplets on microscope slides. The arrays were then activated by deposition of an aerosolized enzyme solution, followed by incubation and fluorometric scanning. The specificities of human blood serine proteases (human thrombin, factor Xa, plasmin, and urokinase plasminogen activator) were examined. The arrays provided complete maps of protease specificity for all of the substrates tested and allowed for detection of cooperative interactions between substrate subsites. The arrays were further utilized to explore the conservation of thrombin specificity across species by comparing the proteolytic fingerprints of human, bovine, and salmon thrombin. These enzymes share nearly identical specificity profiles despite approximately 390 million years of divergent evolution. Fluorogenic substrate microarrays provide a rapid way to determine protease substrate specificity information that can be used for the design of selective inhibitors and substrates, the study of evolutionary divergence, and potentially, for diagnostic applications.

Peptide microarrays for the determination of protease substrate specificity.

A method is described for the preparation of substrate microarrays that allow for the rapid determination of protease substrate specificity. Peptidyl coumarin substrates, synthesized on solid support using standard techniques, are printed onto glass slides using DNA microarraying equipment. The linkage from the peptide to the slide is formed through a chemoselective reaction, resulting in an array of uniformly displayed fluorogenic substrates. The arrays can be treated with proteases to yield substrate specificity profiles. Standard instrumentation for visualization of microarrays can be used to obtain comparisons of the specificity constants for all of the prepared substrates. The utility of these arrays is demonstrated by the selective cleavage of preferred substrates with trypsin, thrombin, and granzyme B, and by assessing the extended substrate specificity of thrombin using a microarray of 361 different peptidyl coumarin substrates.