Expression and Characterization of Intein-Cyclized Trimer of Staphylococcus aureus Protein A Domain Z.

Staphylococcus aureus protein A (SpA) is an IgG Fc-binding virulence factor that is widely used in antibody purification and as a scaffold to develop affinity molecules. A cyclized SpA Z domain could offer exopeptidase resistance, reduced chromatographic ligand leaching after single-site endopeptidase cleavage, and enhanced IgG binding properties by preorganization, potentially reducing conformational entropy loss upon binding. In this work, a Z domain trimer (Z3) was cyclized using protein intein splicing. Interactions of cyclic and linear Z3 with human IgG1 were characterized by differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). DSF showed a 5 ℃ increase in IgG1 melting temperature when bound by each Z3 variant. SPR showed the dissociation constants of linear and cyclized Z3 with IgG1 to be 2.9 nM and 3.3 nM, respectively. ITC gave association enthalpies for linear and cyclic Z3 with IgG1 of -33.0 kcal/mol and -32.7 kcal/mol, and -T∆S of association 21.2 kcal/mol and 21.6 kcal/mol, respectively. The compact cyclic Z3 protein contains 2 functional binding sites and exhibits carboxypeptidase Y-resistance. The results suggest cyclization as a potential approach toward more stable SpA-based affinity ligands, and this analysis may advance our understanding of protein engineering for ligand and drug development.

Continuous monitoring of IgG using immobilized fluorescent reporters.

In the manufacture of therapeutic monoclonal antibodies, the clarified cell culture fluid (CCF) is typically loaded onto an initial protein A affinity capture column. Imperfect mass transfer and loading to maximum capacity can risk antibody breakthrough and loss of valuable product, but conservative underloading wastes expensive protein A resin. In addition, the effects of column fouling and ligand degradation require the frequent optimization of immunoglobulin G (IgG) loading to avoid wastage. Continuous real-time monitoring of IgG flowthrough is of great interest, therefore. We previously developed a fluorescence-based monitoring technology that allows batch mix-and-read mAb detection in the CCF. Here, we report the use of reporters immobilized on cyanogenbromide-activated Sepharose 4B resin for continuous detection of IgG in column breakthrough. The column effluent is continuously contacted with immobilized fluorescein-labeled Fc-binding ligands in a small monitoring column to produce an immediately-detectable change in fluorescence intensity. The technology allows rapid and reliable monitoring of IgG in a flowing stream of clarified CCF emerging from a protein A column, without prior sample preparation. We observed a significant change in fluorescence intensity at 0.5 g/L human IgG, sufficient to detect a 5% breakthrough of a 10 g/L load, within 18 s at a flow rate of 0.5 ml/min. The current small-scale technology is suitable for use in process development, but the chemistry should be readily adaptable to larger scale applications using fiber-optic sensors, and continuous IgG monitoring could be applicable in a variety of upstream and downstream process settings.

Antibody mix-and-read assays based on fluorescence intensity probes.

Antibodies and Fc fusion proteins are a rapidly growing class of pharmaceuticals. Cell culture and purification process development and operation require frequent measurement of product concentrations, commonly by complex enzyme-linked immunosorbent assay and high-performance liquid chromatography methods. Here we report a fast (<30 s), and simple antibody Fc assay based on mix-and-read reporting by fluorescence emission. A soluble fluorescein-labeled Fc-affinity reporter produced by standard peptide synthesis is mixed with an Fc-containing sample to produce an immediate shift in both fluorescence polarization and intensity, compatible with on- and at-line measurements and microbioreactor monitoring. We observed significant shifts in fluorescence intensity in Chinese hamster ovary cell culture fluid spiked with IgG and detected an adalimumab biosimilar down to 100 ng/mL (10-4 g/L), despite the interferents in the complex sample matrix. Neither the fluorescence polarization nor the fluorescence intensity assay is significantly affected by the addition of clarified lysate of 2 million CHO-k1 cells/mL, suggesting applicability even to cultures of low viability. Biochemical and molecular docking approaches suggest that the fluorescence intensity enhancement is caused by changes in the fluorophore's local microenvironment upon binding to IgG Fc, especially by interactions with Fc His433.Abbreviations: CCF: Cell Culture Fluid; CHO: Chinese Hamster Ovary cells; ELISA: Enzyme Linked Immunosorbent Assay; Fc: Fragment Crystallizable of antibody; HPLC: High-Performance Liquid Chromatography; HPßCD: hydroxypropyl-ß-cyclodextrin; IgG: ImmunoglobulinG; mAb: Monoclonal Antibody; PBS: Phosphate-Buffered Saline; PDB: Protein Data Bank; SpA: Staphylococcal protein A; SpG: Staphylococcal protein G.

Isocratic reporter-exclusion immunoassay using restricted-access adsorbents.

We introduce analyte-dependent exclusion of reporter reagents from restricted-access adsorbents as the basis of an isocratic reporter-exclusion immunoassay for viruses, proteins, and other analytes. Capto™ Core 700 and related resins possess a noninteracting size-selective outer layer surrounding a high-capacity nonspecific mixed-mode capture adsorbent core. In the absence of analyte, antibody-enzyme reporter conjugates can enter the adsorbent and be captured, and their signal is lost. In the presence of large or artificially-expanded analytes, reporter reagents bind to analyte species to form complexes large enough to be excluded from the adsorbent core, allowing their signal to be observed. This assay principle is demonstrated using M13 bacteriophage virus and human chorionic gonadotropin as model analytes. The simple isocratic detection approach described here allows a rapid implementation of immunoassay for detection of a wide range of analytes and uses inexpensive, generally-applicable, and stable column materials instead of costly analyte-specific immunoaffinity adsorbents.

The design basis for the integrated and continuous biomanufacturing framework.

An 8 ton per year manufacturing facility is described based on the framework for integrated and continuous bioprocessing (ICB) common to all known biopharmaceutical implementations. While the output of this plant rivals some of the largest fed-batch plants in the world, the equipment inside the plant is relatively small: the plant consists of four 2000 L single-use bioreactors and has a maximum flow rate of 13 L/min. The equipment and facility for the ICB framework is described in sufficient detail to allow biopharmaceutical companies, vendors, contract manufacturers to build or buy their own systems. The design will allow the creation of a global ICB ecosystem that will transform biopharmaceutical manufacturing. The design is fully backward compatible with legacy fed-batch processes. A clinical production scale is described that can produce smaller batch sizes with the same equipment as that used at the commercial scale. The design described allows the production of as little as 10 g to nearly 35 kg of drug substance per day.

A common framework for integrated and continuous biomanufacturing.

There is a growing application of integrated and continuous bioprocessing (ICB) for manufacturing recombinant protein therapeutics produced from mammalian cells. At first glance, the newly evolved ICB has created a vast diversity of platforms. A closer inspection reveals convergent evolution: nearly all of the major ICB methods have a common framework that could allow manufacturing across a global ecosystem of manufacturers using simple, yet effective, equipment designs. The framework is capable of supporting the manufacturing of most major biopharmaceutical ICB and legacy processes without major changes in the regulatory license. This article reviews the ICB that are being used, or are soon to be used, in a GMP manufacturing setting for recombinant protein production from mammalian cells. The adaptation of the various ICB modes to the common ICB framework will be discussed, along with the pros and cons of such adaptation. The equipment used in the common framework is generally described. This review is presented in sufficient detail to enable discussions of IBC implementation strategy in biopharmaceutical companies and contract manufacturers, and to provide a road map for vendors equipment design. An example plant built on the common framework will be discussed. The flexibility of the plant is demonstrated with batches as small as 0.5 kg or as large as 500 kg. The yearly output of the plant is as much as 8 tons.

Continuous Fc detection for protein A capture process control.

Purification of therapeutic monoclonal antibodies usually involves a protein A affinity capture step. Because column breakthrough of antibody in complex, UV-absorbing culture fluid cannot be readily detected in real time, processes are designed so conservatively that column capacity is usually underutilized, wasting adsorbent and reducing productivity. We have developed a fluorescence-based monitoring technology which allows real-time mAb monitoring and used it to detect IgG in column breakthrough. The column effluent was continuously contacted with soluble fluorescein-labeled Fc-binding ligands to produce an immediately-detectable shift in both fluorescence polarization and intensity. To extend the upper limit of inlet flow rate, a 14:1 split-ratio flow splitter was tested with an inlet flow of 15 mL/min (0.9 L/h), producing a sampling stream at 1 mL/min while still enabling continuous detection functionality. We observed significant shifts in fluorescence intensity in CHO cell culture fluid spiked with human IgG, and detected 0.02-0.1 g/L human IgG in protein A column breakthrough at a flow velocity of 80 cm/h. The increase in fluorescence intensity upon 0.1% breakthrough of an 1 g/L feed was used to trigger column switching using Python-enabled two-way communication with the standard Unicorn OPC process control protocol. The technology allows rapid, continuous and reliable monitoring of IgG in a flowing process stream, without elaborate sample preparation.

Highland games: A benchmarking exercise in predicting biophysical and drug properties of monoclonal antibodies from amino acid sequences.

Biopharmaceutical product and process development do not yet take advantage of predictive computational modeling to nearly the degree seen in industries based on smaller molecules. To assess and advance progress in this area, spirited coopetition (mutually beneficial collaboration between competitors) was successfully used to motivate industrial scientists to develop, share, and compare data and methods which would normally have remained confidential. The first "Highland Games" competition was held in conjunction with the October 2018 Recovery of Biological Products Conference in Ashville, NC, with the goal of benchmarking and assessment of the ability to predict development-related properties of six antibodies from their amino acid sequences alone. Predictions included purification-influencing properties such as isoelectric point and protein A elution pH, and biophysical properties such as stability and viscosity at very high concentrations. Essential contributions were made by a large variety of individuals, including companies which consented to provide antibody amino acid sequences and test materials, volunteers who undertook the preparation and experimental characterization of these materials, and prediction teams who attempted to predict antibody properties from sequence alone. Best practices were identified and shared, and areas in which the community excels at making predictions were identified, as well as areas presenting opportunities for considerable improvement. Predictions of isoelectric point and protein A elution pH were especially good with all-prediction average errors of 0.2 and 1.6 pH unit, respectively, while predictions of some other properties were notably less good. This manuscript presents the events, methods, and results of the competition, and can serve as a tutorial and as a reference for in-house benchmarking by others. Organizations vary in their policies concerning disclosure of methods, but most managements were very cooperative with the Highland Games exercise, and considerable insight into common and best practices is available from the contributed methods. The accumulated data set will serve as a benchmarking tool for further development of in silico prediction tools.

Recombinant expression, characterization, and quantification in human cancer cell lines of the Anaplastic Large-Cell Lymphoma-characteristic NPM-ALK fusion protein.

Systemic anaplastic large cell lymphoma (ALCL) is an aggressive T-cell lymphoma most commonly seen in children and young adults. The majority of pediatric ALCLs are associated with the t(2;5)(p23;q35) translocation which fuses the Anaplastic Lymphoma Kinase (ALK) gene with the Nucleophosmin (NPM) gene. The NPM-ALK fusion protein is a constitutively-active tyrosine kinase, and plays a major role in tumor pathogenesis. In an effort to advance novel diagnostic approaches and the understanding of the function of this fusion protein in cancer cells, we expressed in E. coli, purified and characterized human NPM-ALK fusion protein to be used as a standard for estimating expression levels in cultured human ALCL cells, a key tool in ALCL pathobiology research. We estimated that NPM-ALK fusion protein is expressed at substantial levels in both Karpas 299 and SU-DHL-1 cells (ca. 4-6 million molecules or 0.5-0.7 pg protein per cell; based on our in-house developed NPM-ALK ELISA; LOD of 40 pM) as compared to the ubiquitous ß-actin protein (ca. 64 million molecules or 4.5 pg per lymphocyte). We also compared NPM-ALK/ ß-actin ratios determined by ELISA to those independently determined by two-dimensional electrophoresis and showed that the two methods are in good agreement.

Engineered ChymotrypsiN for Mass Spectrometry-Based Detection of Protein Glycosylation.

We have engineered the substrate specificity of chymotrypsin to cleave after Asn by high-throughput screening of large libraries created by comprehensive remodeling of the substrate binding pocket. The engineered variant (chymotrypsiN, ChyB-Asn) demonstrated an altered substrate specificity with an expanded preference for Asn-containing substrates. We confirmed that protein engineering did not compromise the stability of the enzyme by biophysical characterization. Comparison of wild-type ChyB and ChyB-Asn in profiling lysates of HEK293 cells demonstrated both qualitative and quantitative differences in the nature of the peptides and proteins identified by liquid chromatography and tandem mass spectrometry. ChyB-Asn enabled the identification of partially glycosylated Asn sites within a model glycoprotein and in the extracellular proteome of Jurkat T cells. ChymotrypsiN is a valuable addition to the toolkit of proteases to aid the mapping of N-linked glycosylation sites within proteins and proteomes.

Identifying Challenges and Risks Associated with the Analysis of Major Mycotoxins in Feed and Botanicals.

Changing weather conditions have heightened the risk of growth of mycotoxigenic molds on crops and various agricultural commodities. Mycotoxins, which are linked to carcinogenic and nephrotoxic effects in animals and humans, have been traditionally analyzed by immunoassays, gas, and LC techniques with spectrophotometric detectors. This review discusses the current techniques and challenges in commercial settings associated with the analysis of mycotoxins in unique matrices such as animal feeds, herbal products, and dietary supplements containing botanicals. Because of the advantages and growing acceptance of LC-tandem MS (MS/MS) over traditional approaches, discussion is mainly based on LC-MS/MS-based approaches. Considering the impact of sample preparation on accuracy of quantitative results, discussion about pros and cons of recently introduced sample preparation techniques is integrated with analytical methods. A section of the review explains the importance and availability of reference materials for mycotoxins. The present discussion provides good insight into the current challenges and developments during mycotoxin analysis of feed and botanicals and addresses the need for researchers in terms of an official MS-based method.

Competitive multicomponent anion exchange adsorption of proteins at the single molecule level.

We use single molecule spectroscopy to study a multicomponent, competitive protein adsorption system. Fluorescently-labeled α-lactalbumin proteins are super-resolved adsorbing to cationic anion-exchange ligands in the presence of a competitor, insulin. We find that the competitor reduces the number of binding events by blocking ligands throughout the observed measurement time while the single-site adsorption kinetics are unchanged.

In Vitro/In Vivo Toxicity Evaluation and Quantification of Iron Oxide Nanoparticles.

Increasing biomedical applications of iron oxide nanoparticles (IONPs) in academic and commercial settings have alarmed the scientific community about the safety and assessment of toxicity profiles of IONPs. The great amount of diversity found in the cytotoxic measurements of IONPs points toward the necessity of careful characterization and quantification of IONPs. The present document discusses the major developments related to in vitro and in vivo toxicity assessment of IONPs and its relationship with the physicochemical parameters of IONPs. Major discussion is included on the current spectrophotometric and imaging based techniques used for quantifying, and studying the clearance and biodistribution of IONPs. Several invasive and non-invasive quantification techniques along with the pitfalls are discussed in detail. Finally, critical guidelines are provided to optimize the design of IONPs to minimize the toxicity.

Cleavable ester linked magnetic nanoparticles for labeling of solvent exposed primary amine groups of peptides/proteins.

Covalent labeling of solvent exposed amino acid residues using chemical reagents/crosslinkers followed by mass spectrometric analysis can be used to determine the solvent accessible amino acids of a protein. A variety of chemical reagents containing cleavable bonds were developed to label abundantly found lysine residues on the surface of protein. To achieve efficient separation of labeled peptides prior to mass spectrometric analysis, magnetic nanoparticles can be decorated with amino acid reactive functional groups and utilized for quick recovery of labeled peptides. [1] In this work, iron oxide magnetic nanoparticles (Fe3O4 MNPs) were synthesized by thermal decomposition method and coated with silica (SiO2@Fe3O4 MNPs) by reverse micro emulsion approach. The Fe3O4 MNPs and SiO2@Fe3O4 MNPs were characterized by TEM and XRD. The SiO2@Fe3O4 MNPs were further coated with amine groups and conjugated to N-hydroxysuccinimidyl (NHS) ester groups via a cleavable ester bond. Fluorescence based qualitative analysis of ester linked NHS ester modified SiO2@Fe3O4 MNPs was performed to confirm the presence of NHS ester group. The active NHS ester sites on the surface of SiO2@Fe3O4 MNPs were determined by depletion approach and found to be 694 active sites per 1 mg of SiO2@Fe3O4 MNPs. Free amine groups of a small peptide, ACTH (4-11) were labeled by ester linked, NHS ester modified SiO2@Fe3O4 MNPs under physiological conditions. Superparamagnetic nature of SiO2@Fe3O4 MNPs allowed quick and efficient magnetic separation of labeled peptides from the solution. The ester bond was further cleaved to separate labeled peptides followed by mass spectrometric analysis. The ester linked, NHS ester modified SiO2@Fe3O4 MNPs introduced a mass shift of 115.09 Da on amine groups of ACTH (4-11), which was confirmed by mass spectrometry.

Cleavable ester-linked magnetic nanoparticles for labeling of solvent-exposed primary amine groups of peptides/proteins.

To study the solvent-exposed lysine residues of peptides/proteins, we previously reported disulfide-linked N-hydroxysuccinimide ester-modified silica-coated iron oxide magnetic nanoparticles (NHS-SS-SiO2@Fe3O4 MNPs). The presence of a disulfide bond in the linker limits the use of disulfide reducing agent during protein digestion and allows unwanted disulfide formation between the MNPs and protein. In the current work, the disulfide bond was replaced with a cleavable ester group to synthesize NHS ester-modified SiO2@Fe3O4 MNPs. Use of the cleavable ester group provides an improved method for protein labeling and allows the use of disulfide reducing agents during protein digestion.

Labeling primary amine groups in peptides and proteins with N-hydroxysuccinimidyl ester modified Fe3O4@SiO2 nanoparticles containing cleavable disulfide-bond linkers.

The surface of superparamagnetic silica coated iron oxide (Fe3O4@SiO2) nanoparticles was functionalized with a disulfide bond linked N-hydroxysuccinimidyl (NHS) ester group in order to develop a method for labeling primary amines in peptides/proteins. The nanoparticle labeled proteins/peptides formed after NHS ester reaction with the primary amine groups were isolated using a magnet without any additional purification step. Nanoparticle moieties conjugated to peptides/proteins were then trimmed by cleavage at the disulfide linker with a reducing agent. The labeled peptides were analyzed by LC-MS/MS to determine their sequences and the sites of NHS ester labeling. This novel approach allowed characterization of lysine residues on the solvent accessible surface of native bovine serum albumin. Low cost, rapid magnetic separation, and specificity toward primary amine groups make NHS ester coated Fe3O4@SiO2 nanoparticles a potential labeling probe to study proteins on living cell surfaces.