Incorporation of automated buffer exchange empowers high-throughput protein and plasmid purification for downstream uses.

The continued acceleration of time-to-market product development and rising demand for biotherapeutics have hastened the need for higher throughput within the biopharmaceutical industry. Automated liquid handlers (ALH) are increasingly popular due to flexible programming that enables processing of multiple samples with an array of functions. This flexibility is useful in streamlining research that requires chromatographic procedures to achieve product purity for downstream analysis. However, purification of biologics often requires additional off-deck buffer exchange steps due to undesirable elution conditions such as high acid or high salt content. Expanding the capability of ALHs to perform purification in sequence with buffer exchange would, therefore, increase workflow efficiency by eliminating the need for manual intervention, thus expediting sample preparation. Here we demonstrate two different automated purifications using pipet-based dispersive solid-phase extraction (dSPE). The first is an affinity purification of His-tagged proteins from bacterial lysate. The second is an anion-exchange purification of plasmid DNA. Both methods are followed by buffer exchange performed by an ALH. Percent recoveries for the three purified recombinant proteins ranged from 51 ± 1.2 to 86 ± 10%. The yields were inversely correlated to starting sample load and protein molecular weight. Yields for plasmid purification ranged between 11.4 ± 0.8 and 13.7 ± 0.9 µg, with the largest plasmid providing the highest yield. Both programs were rapid, with protein purification taking <80 min and plasmid purification <60 min. Our results demonstrate that high-quality, ready-to-use biologics can be obtained rapidly from a crude sample after two separate chromatographic processes without manual intervention.

Factors Compromising Glucuronidase Performance in Urine Drug Testing Potentially Resulting in False Negatives.

Next generation ß-glucuronidases can effectively cleave glucuronides in urine at room temperature. However, during the discovery studies, additional challenges were identified for urine drug testing across biologically relevant pH extremes and patient urine specimens. Different enzymes were evaluated across clinical urine specimens and commercially available urine control matrices. Each enzyme shows distinct substrate preferences, pH optima, and variability across clinical specimens. These results demonstrate how reliance on a single glucuronidated substrate as the internal hydrolysis control cannot ensure performance across a broader panel of analytes. Moreover, sample specific urine properties compromise ß-glucuronidases to varying levels, more pronounced for some enzymes, and thereby lower the recovery of some drug analytes in an enzyme-specific manner. A minimum of 3-fold dilution of urine with buffer yields measurable improvements in achieving target pH and reducing the impact of endogenous compounds on enzyme performance. After subjecting the enzymes to pH extremes and compromising chemicals, one particular ß-glucuronidase was identified that addressed many of these challenges and greatly lower the risk of failed hydrolyses. In summary, we present strategies to evaluate glucuronidases that aid in higher accuracy urine drug tests with lower potential for false negatives.

Automated multi-attribute method sample preparation using high-throughput buffer exchange tips.

RATIONALE: The multi-attribute method (MAM) has become a valuable mass spectrometry (MS)-based tool that can identify and quantify the site-specific product attributes and purity information for biotherapeutics such as monoclonal antibodies (mAbs) and fusion molecules in recent years. As we expand the use of the MAM at various stages of drug development, it is critical to enhance the sample preparation throughput without additional chemical modifications and variability. METHODS: In this study, a fully automated MAM sample preparation protocol is presented, where rapid desalting in less than 15 minutes is achieved using miniaturized size-exclusion chromatography columns in pipette tips on an automated liquid handler. The peptide samples were analyzed using an electrospray ionization (ESI) orbitrap mass spectrometer coupled to an ultra-high-performance liquid chromatography (UHPLC) system with a dual column switching system. RESULTS: No significant change was observed in product attributes and their quantities compared with manual, low-artifact sample preparation. The sample recovery using the buffer exchange tips was greatly enhanced over the manual spin cartridges while still demonstrating excellent reproducibility for a wide variety of starting sample concentrations. Unlike a plate desalting system, the individual columns provide flexibility in the number of samples prepared at a time and sample locations within plates. CONCLUSIONS: This automated protocol enables the preparation of up to 96 samples with less "at-bench" time than the manual preparation of a smaller batch of samples, thereby greatly facilitating support of process development and the use of the MAM in quality control.

Enhancing the multi-attribute method through an automated and high-throughput sample preparation.

The multi-attribute method (MAM), a recent advance in the application of liquid chromatography-mass spectrometry within the pharmaceutical industry, enables the simultaneous monitoring of multiple product quality attributes in a single analytical method. While MAM is coupled with automated data processing and reporting, the sample preparation, based on proteolytic peptide mapping, remains cumbersome and low throughput. The standard sample preparation for MAM relies on protein denaturation, reduction, and alkylation prior to proteolytic digestion, but often a desalting step is required to maintain enzymatic activity. While most of the sample preparation can be automated on a standard robotic liquid handling system, a streamlined approach for protein desalting and temperature modulation is required for a viable, fully automated digestion. In this work, for the first time, a complete tip-based MAM sample preparation is automated on a single robotic liquid handling system, leveraging a deck layout that integrates both heating and cooling functionalities. The fully automated method documented herein achieves a high-throughput sample preparation for MAM, while maintaining superior method performance.Abbreviations: MAM: multi-attribute method; PQAs: product quality attributes; CE: capillary electrophoresis; IEX: ion-exchange chromatography; HILIC-FLR: hydrophilic interaction liquid chromatography coupled to a fluorescence detector; RP-LC/UV: reversed-phase liquid chromatography coupled to a UV detector; MS: mass spectrometry; NPD: new peak detection; GdnHCl: guanidine hydrochloride; TIC: total ion current; pAb: polyclonal antibody; IgG: immunoglobulin G; DTT: dithiothreitol; IAA: iodoacetic acid; TFA: trifluoroacetic acid; A280: absorbance at 280 nm wavelength; 96MPH: 96-channel multi-probe head; CPAC: Cold Plate Air Cooled; HHS: Hamilton Heater Shaker; DWP: Deep-Well Plate; PCR: Polymerase Chain Reaction; NTR: Nested Tip Rack; Met: methionine; Trp: tryptophan; N-term pQ: N-terminal glutamine cyclization; Lys: lysine; PAM: peptidylglycine α-amidating monooxygenase; G0F: asialo-, agalacto-, bi-antennary, core substituted with fucose; G1F: asialo-, mono-galactosylated bi-antennary, core substituted with fucose; G2F: asialo-, bi-galactosylated bi-antennary, core substituted with fucose; G0: asialo-, agalacto-, bi-antennary; Man5: oligomannose 5; Man8: oligomannose 8; TriF: asialo-, tri-galactosylated tri-antennary, core substituted with fucose.

Variants of glycosyl hydrolase family 2 ß-glucuronidases have increased activity on recalcitrant substrates.

Glucuronidated drug metabolites can be quantified from urine samples by first hydrolyzing conjugates with ß-glucuronidase (ß-GUS) and then separating free drug molecules by liquid chromatography and mass spectrometry detection (LC-MS). To improve the activity and specificity of various ß-GUS, we designed enzyme chimeras and generated site-saturation variants based on structural analyses, then screened them for improved activity on drug metabolites important to clinical and forensic drug-testing laboratories. Often, an increase of activity on one substrate of interest was countered by loss of activity against another, and there was no strong correlation of activity on standard ß-glucuronidase substrates to activity on recalcitrant drug glucuronides. However, we discovered a chimera of two enzymes from different species of Aspergillus that displays a 27 % increase in activity on morphine-3-glucuronide than the parent proteins. Furthermore, mutations in the M-loop, which is a loop near the active site, resulted in numerous variants with dramatically increased rates of hydrolysis on drug glucuronides. Specifically, the M-loop variant Q451D/A452E of a ß-GUS from Brachyspira pilosicoli has a 50-fold and 25-fold increase in activity on the recalcitrant substrates codeine-6-glucuronide and dihydrocodeine-6-glucuronide, respectively, compared to the parent enzyme.

Purification, Characterization, and Structural Studies of a Sulfatase from Pedobacter yulinensis.

Sulfatases are ubiquitous enzymes that hydrolyze sulfate from sulfated organic substrates such as carbohydrates, steroids, and flavones. These enzymes can be exploited in the field of biotechnology to analyze sulfated metabolites in humans, such as steroids and drugs of abuse. Because genomic data far outstrip biochemical characterization, the analysis of sulfatases from published sequences can lead to the discovery of new and unique activities advantageous for biotechnological applications. We expressed and characterized a putative sulfatase (PyuS) from the bacterium Pedobacter yulinensis. PyuS contains the (C/S)XPXR sulfatase motif, where the Cys or Ser is post-translationally converted into a formylglycine residue (FGly). His-tagged PyuS was co-expressed in Escherichia coli with a formylglycine-generating enzyme (FGE) from Mycobacterium tuberculosis and purified. We obtained several crystal structures of PyuS, and the FGly modification was detected at the active site. The enzyme has sulfatase activity on aromatic sulfated substrates as well as phosphatase activity on some aromatic phosphates; however, PyuS did not have detectable activity on 17α-estradiol sulfate, cortisol 21-sulfate, or boldenone sulfate.

Parallel sample processing using dispersive INtip micro-purification on programmable multichannel pipettes.

Automation gives researchers the ability to process and screen orders of magnitude higher numbers of samples than manual experimentation. Current biomacromolecule separation methodologies suffer from necessary manual intervention, making their translation to high-throughput automation difficult. Herein, we present the first characterization of biomacromolecule affinity purification via dispersive solid-phase extraction in a pipette tip (INtip). We use commercially available resin and compare efficiency with batch and spin column methodologies. Moreover, we measure the kinetics of binding and evaluate resin binding capacities. INtip technology is effective on, and scalable for, an automated platform (INTEGRA ASSIST). The results suggest that high-throughput biomolecular workflows will benefit from the integration of INtip separations.

Automating Complex, Multistep Processes on a Single Robotic Platform to Generate Reproducible Phosphoproteomic Data.

Mass spectrometry-based phosphoproteomics holds promise for advancing drug treatment and disease diagnosis; however, its clinical translation has thus far been limited. This is in part due to an unstandardized and segmented sample preparation process that involves cell lysis, protein digestion, peptide desalting, and phosphopeptide enrichment. Automating this entire sample preparation process will be key in facilitating standardization and clinical translation of phosphoproteomics. While peptide desalting and phosphopeptide enrichment steps have been individually automated, integrating these two extractions and, further, the entire process requires more advanced robotic platforms as well as automation-friendly extraction tools. Here we describe a fully automated peptide desalting and phosphopeptide enrichment method using IMCStips on a Hamilton STAR. Using our established automated method, we identified more than 10,000 phosphopeptides from 200 µg of HCT116 cell lysate without fractionation with >85% phosphopeptide specificities. Compared with titania-based Spin Tip products, the automated IMCStips-based method gave 50% higher phosphopeptide identifications. The method reproducibility was further assessed using multiple reaction monitoring (MRM) to show >50% phosphopeptide recoveries after the automated phosphopeptide extraction with coefficients of variation (CVs) of <20% over a 3-week period. The established automated method is a step toward standardization of the sample preparation of phosphopeptide samples and could be further expanded upon to create a fully automated "cells to phosphopeptides" method.

Comparison of Purified ß-glucuronidases in Patient Urine Samples Indicates a Lack of Correlation Between Enzyme Activity and Drugs of Abuse Metabolite Hydrolysis Efficiencies Leading to Potential False Negatives.

Pain management laboratories analyze biological fluids (urine, saliva or blood) from patients treated for chronic pain to ensure compliance and to detect undisclosed drug use. The quantitation of multi-panel drugs in urine and tissues utilizes ß-glucuronidase to cleave the glucuronic acid and liberate the parent drug for mass spectrometry analysis. This work focuses on the comparison of three different, purified and commercially available ß-glucuronidases across 83 patient urine samples. One enzyme is genetically modified, expressed in bacteria and the other two enzymes are purified from abalone. The results indicate that the source of ß-glucuronidase plays an important role in substrate specificity which in turn dictates hydrolysis efficiency. Contaminants in the enzyme solutions also interfere with analyte detection. Altogether, these factors impact precision and accuracy of data interpretation, leading up to 13% positive/negative disagreement.

Increased activity of ß-glucuronidase variants produced by site-directed mutagenesis.

ß-glucuronidase (BGus) is an essential glycosyl hydrolase which has been widely used in biological and biomedical applications. In this paper, we report the construction and screening of nineteen Escherichia coli BGus (EBGus) mutants using site-directed mutagenesis. The mutants G559N, G559S and G559T showed a 3-5 fold increase in enzyme activity in comparison to wild type EBGus. In particular, G559S, with the highest activity, showed 2-6 fold enhanced activity compared to abalone and snail BGus extracts. Moreover, the glycine to serine mutagenesis for the same site in Staphylococcus sp. RLH1 BGus (StBGus) exhibited significantly enhanced activity, which indicated the importance of the G559→S mutation on BGus function. Based on this structural analysis, we postulate that the mutation at G559 plays an important role in the stabilization of the enzyme conformation, and thereby facilitates the effective binding of substrate.

Polymer-Protein Core-Shell Nanoparticles for Enhanced Antigen Immunogenicity.

Nanoengineered vaccine platforms can be modeled after viruses and other pathogens with highly organized and repetitive structures that trigger the host immune system. Here we demonstrated a pyridine-grafted poly(ε-caprolactone)-based polymer-protein core-shell nanoparticles (PPCS-NPs) platform can effectively trigger the host immune system and lead to significantly higher antibody titers.

Degradation of Opioids and Opiates During Acid Hydrolysis Leads to Reduced Recovery Compared to Enzymatic Hydrolysis.

Drug monitoring laboratories utilize a hydrolysis process to liberate the opiates from their glucuronide conjugates to facilitate their detection by tandem mass spectrometry (MS). Both acid and enzyme hydrolysis have been reported as viable methods, with the former as a more effective process for recovering codeine-6-glucuronide and morphine-6-glucuronide. Here, we report concerns with acid-catalyzed hydrolysis of opioids, including a significant loss of analytes and conversions of oxycodone to oxymorphone, hydrocodone to hydromorphone and codeine to morphine. The acid-catalyzed reaction was monitored in neat water and patient urine samples by liquid chromatography-time-of-flight and tandem MS. These side reactions with acid hydrolysis may limit accurate quantitation due to loss of analytes, possibly lead to false positives, and poorly correlate with pharmacogenetic profiles, as cytochrome P450 enzyme (CYP2D6) is often involved with oxycodone to oxymorphone, hydrocodone to hydromorphone and codeine to morphine conversions. Enzymatic hydrolysis process using the purified, genetically engineered ß-glucuronidase (IMCSzyme®) addresses many of these concerns and demonstrates accurate quantitation and high recoveries for oxycodone, hydrocodone, oxymorphone and hydromorphone.

Variations in enzymatic hydrolysis efficiencies for amitriptyline and cyclobenzaprine in urine.

A collaborative study was conducted to investigate discrepancies in recoveries of two commonly prescribed compounds, amitriptyline and cyclobenzaprine, in patient urine samples when hydrolyzed with different enzymes from different sources. A 2- to 10-fold increase in analyte recoveries was seen for patient samples hydrolyzed using a recombinant ß-glucuronidase (IMCSzyme™) over samples hydrolyzed with ß-glucuronidase from Haliotis rufescens We report outcomes from four commercially available ß-glucuronidase enzymes (IMCSzyme™, Patella vulgata, Helix pomatia and H. rufescens) on patient samples that tested positive for amitriptyline and cyclobenzaprine. Our results confirm reduced hydrolysis of glucuronides by ß-glucuronidases isolated from mollusks, but near complete conversion when using the recombinant enzyme. Our premise is that systematic differences in hydrolysis efficiencies due to varying substrate affinity among enzyme subtypes potentially impacts accuracy and reliability of measurements.

Synthesis of self-assembled IL-1Ra-presenting nanoparticles for the treatment of osteoarthritis.

Osteoarthritis is a progressive joint disease that results in degradation of cartilage in load-bearing joints. Pain and inflammation in the joint are the hallmarks of this condition, which further exacerbate the cartilage destruction and health of the patient. It is hence imperative to treat the joint inflammation at the earliest. Interleukin 1 (IL-1) blockade by IL-1 receptor antagonist (IL-1Ra) has shown promise in the clinic but this therapy suffers from rapid clearance, high doses, and frequent intervention. Use of carrier particles that result in longer residence time has been proposed. Here we have synthesized a new class of nanoparticles presenting IL-1Ra on the surface and with tunable size from 300 to 700 nm. These IL-1Ra-poly(2-hydroxyethyl methacrylate)-pyridine nanoparticles are cytocompatible and stable in serum-containing solutions for several days. Our results further demonstrate that these nanoparticles are capable of blocking IL-1ß signaling in an NF-κB inducible reporter cell line. These engineered nanoparticles are promising for localized intra-articular delivery in joint space to reduce inflammation in osteoarthritis and other inflammatory diseases. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 595-599, 2016.

Plant virus incorporated hydrogels as scaffolds for tissue engineering possess low immunogenicity in vivo.

Viruses are no longer recognized purely for being ubiquitous pathogens, but have served as building blocks for material chemistry and nanotechnology. Thousands of coat protein subunits of a viral particle can be modified chemically and/or genetically. We have previously shown that the three-dimensional porous hydrogels can easily be functionalized by Tobacco mosaic virus (TMV), a rod-like plant virus, using its mutant, RGD-TMV. RGD-TMV hosted bioadhesive peptide (RGD) in the hydrogel, which was shown to enhance cell attachment and promote osteogenic differentiation of cultured stem cell. To translate this technology to potential clinical applications, we sought to study the biocompatibility of the hydrogel. In this paper, the hydrogels were implanted in vivo and assessed for their immunogenicity, toxicity, and biodegradability. Immune response for TMV substantially decreased when incorporated in the hydrogel implants. The implanted TMV hydrogels exhibited no apparent toxicity and were degradable in mice. The results highlighted the feasibility of using TMV incorporated hydrogels as scaffolding materials for regenerative medicine in terms of biocompatibility and biodegradability.

RGD-conjugated rod-like viral nanoparticles on 2D scaffold improve bone differentiation of mesenchymal stem cells.

Viral nanoparticles have uniform and well-defined nano-structures and can be produced in large quantities. Several plant viral nanoparticles have been tested in biomedical applications due to the lack of mammalian cell infectivity. We are particularly interested in using Tobacco mosaic virus (TMV), which has been demonstrated to enhance bone tissue regeneration, as a tunable nanoscale building block for biomaterials development. Unmodified TMV particles have been shown to accelerate osteogenic differentiation of adult stem cells by synergistically upregulating bone morphogenetic protein 2 (BMP2) and integrin-binding bone sialoprotein (IBSP) expression with dexamethasone. However, their lack of affinity to mammalian cell surface resulted in low initial cell adhesion. In this study, to increase cell binding capacity of TMV based material the chemical functionalization of TMV with arginine-glycine-aspartic acid (RGD) peptide was explored. An azide-derivatized RGD peptide was "clicked" to tyrosine residues on TMV outer surface via an efficient copper(I) catalyzed azide-alkyne cycloaddition (CuAAC) reaction. The ligand spacing is calculated to be 2-4 nm, which could offer a polyvalent ligand clustering effect for enhanced cell receptor signaling, further promoting the proliferation and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs).

Nanoengineered particles for enhanced intra-articular retention and delivery of proteins.

Localized intra-articular delivery of anti-inflammatory proteins can reduce inflammation in osteoarthritis but poses a challenge because of raid clearance within few hours of injection. A new class of polymer is developed that forms self-assembled nanoparticles ranging from 300 to 900 nm and demonstrates particle size dependent prolonged retention in intra-articular joint spaces compared to bolus protein over a period of 14 d.

In vivo virus-based macrofluorogenic probes target azide-labeled surface glycans in MCF-7 breast cancer cells.

Chemical addressability of viral particles has played a pivotal role in adapting these biogenic macromolecules for various applications ranging from medicine to inorganic catalysis. Cowpea mosaic virus possesses multiple features that are advantageous for the next generation of virus-based nanotechnology: consistent multimeric assemblies dictated by its genetic code, facile large scale production, and lack of observable toxicity in humans. Herein, the chemistry of the viral particles is extended with the use of Cu-free strain-promoted azide-alkyne cycloaddition reaction, or SPAAC reaction. The elimination of Cu, its cocatalyst and reducing agent, simplifies the reaction scheme to a more straightforward approach, which can be directly applied to living systems. As a proof of concept, the viral particles modified with the azadibenzylcyclooctyne functional groups are utilized to trigger and amplify a weak fluorescent signal (azidocoumarin) in live cell cultures to visualize the non-natural sugars. Future adaptations of this platform may be developed to enhance biosensing applications.

Porous alginate hydrogel functionalized with virus as three-dimensional scaffolds for bone differentiation.

In regenerative medicine, a synthetic extracellular matrix is crucial for supporting stem cells during its differentiation process to integrate into surrounding tissues. Hydrogels are used extensively in biomaterials as synthetic matrices to support the cells. However, to mimic the biological niche of a functional tissue, various chemical functionalities are necessary. We present here, a method of functionalizing a highly porous hydrogel with functional groups by mixing the hydrogel with a plant virus, tobacco mosaic virus (TMV), and its mutant. The implication of this process resides with the three important features of TMV: its well-defined genetic/chemical modularity, its multivalency (TMV capsid is composed of 2130 copies of identical subunits), and its well-defined structural features. Previous studies utilizing the native TMV on two-dimensional supports accelerated mesenchymal stem cell differentiation, and surfaces modified with genetically modified viral particles further enhanced cell attachment and differentiation. Herein we demonstrate that functionalization of a porous alginate scaffold can be achieved by the addition of viral particles with minimal processing and downstream purifications, and the cell attachment and differentiation within the macroporous scaffold can be effectively manipulated by altering the peptide or small molecule displayed on the viral particles.

Thermally controlled release of anticancer drug from self-assembled γ-substituted amphiphilic poly(ε-caprolactone) micellar nanoparticles.

A thermo-responsive poly{γ-2-[2-(2-methoxyethoxy)ethoxy]ethoxy-ε-caprolactone}-b-poly(γ-octyloxy-ε-caprolactone) (PMEEECL-b-POCTCL) diblock copolymer was synthesized by ring-opening polymerization using tin octanoate (Sn(Oct)(2)) catalyst and a fluorescent dansyl initiator. The PMEEECL-b-POCTCL had a lower critical solution temperature (LCST) of 38 °C, and it was employed to prepare thermally responsive micelles. Nile Red and Doxorubicin (DOX) were loaded into the micelles, and the micellar stability and drug carrying ability were investigated. The size and the morphology of the cargo-loaded micelles were determined by DLS, AFM, and TEM. The Nile-Red-loaded polymeric micelles were found to be stable in the presence of both fetal bovine serum and bovine serum albumin over a 72 h period and displayed thermo-responsive in vitro drug release. The blank micelles showed a low cytotoxicity. As comparison, the micelles loaded with DOX showed a much higher in vitro cytotoxicity against MCF-7 human breast cancer cell line when the incubation temperature was elevated above the LCST. Confocal laser scanning microscopy was used to study the cellular uptake and showed that the DOX-loaded micelles were internalized into the cells via an endocytosis pathway.