Evaluating a new mixed-mode resin Diamond MMC Mustang using Capto MMC ImpRes as a benchmark.

Diamond MMC Mustang is a relatively new mixed-mode resin, which mediates both cation exchange and hydrophobic interactions. In this work, we evaluated this resin using Cytiva's Capto MMC ImpRes, a well-established mixed-mode resin with similar properties, as a benchmark. The data suggest that in comparison with Capto MMC ImpRes, Diamond MMC Mustang exhibits comparable binding capacity and resolution. In addition, the resin under evaluation shows good lot-to-lot consistency. The information provided in this study allows users to have additional options when selecting mixed-mode resin for intermediate purification or final polishing, which is favourable especially at the present time when the supply chains of many manufacturers are negatively impacted by the coronavirus pandemic.

cDNA Display-Mediated Immuno-PCR (cD-IPCR): An Ultrasensitive Immunoassay for Biomolecular Detection.

Immuno-PCR (IPCR) is a sensitive antigen detection by means of specific antibody-DNA conjugates. To ensure the successful conjugation of a protein (an antibody) with a reporter DNA, immuno-PCR method based on cDNA display (cD-IPCR) has been introduced. The cDNA display molecule is a 1:1 covalent complex of a polypeptide and its encoding cDNA at the single molecule level, which is directly used for antigen detection and subsequent qPCR. This method can be applied to detect various antigens in biological samples, if sequences of their single-domain antibodies (VHHs) or peptide aptamers are known.

Integrated system for temperature-controlled fast protein liquid chromatography. III. Continuous downstream processing of monoclonal antibodies.

Three different applications of travelling heating zone reactor (THZR) chromatography for the downstream processing of monoclonal antibodies (mAbs) are described. mAb containing feedstocks were applied to a fixed bed of the thermoresponsive rProtein A matrix, Byzen Pro™, contained in a bespoke column (held at 15 °C) fitted with a travelling heating (42 °C) device encircling a narrow section of the column. For the demonstration of continuous concentration, uninterrupted loading of 1.0 g/L mAb in a pH 8 binding buffer was synchronized with 5 repeated movements of the heating zone along the column's full length at a velocity of 0.1 mm/s. Elution of mAbs was induced solely by the travelling heating zone's action, each full movement generating a sharp concentrated elution peak accompanied by a small transient mAb concentration-dependent dip in conductivity. Quasi-steady-state operation occurred from the third elution onwards, delivering a mean mAb concentration of 4.9 g/L and process yield >93%. Quasi-continuous separation of the target mAb (1.41 g/L) from bovine serum albumin, BSA (1.0 g/L), was achieved by cyclically alternating the feeding of the mAb + BSA feedstock, with that of the binding buffer alone; supply of the latter was timed to coincide with movement of the heating zone. Accurate coordination of the heating zone's travel and switching from feed to buffer permitted quasi-steady-state collection (elutions 3-6) of sharp peaks of mAb in high purity (98.7%) and yield (88.7%) in 4.5-fold concentrated form, with BSA exiting in the flow through fractions between successive mAb elution peaks. Fully automated THZR-mediated quasi-continuous buffer exchange of 1.34 g/L mAb from a phosphate buffer pH 8 into a HEPES buffer pH 8 of slightly lower conductivity was performed over a 19 h period by carefully timed switching from one feed solution to the other and back again, whilst synchronising movement of the heating zone with feeding of the exchange buffer. Quasi-steady-state operation (elutions 2-9) resulted in an average eluted mAb yield of 94.5% and concentration of 4.8 g/L. Triggering movement of the heating zone slightly ahead of the switch from mAb feed to exchange buffer permitted the positioning of mAb elution peaks in 9 mL volume segments with the lowest recorded conductivity. Measurements of buffer exchange performance conducted with two 'protein-free' systems demonstrated that compared to tangential flow filtration in diafiltration mode, which represents the 'state-of-the-art' technology for buffer exchange, the THZR chromatography based approach affords a >60% saving in minimum volume of exchange buffer required to remove 99.9% of the original buffer. Combined far and near UV circular dichroism, intrinsic fluorescence and thermal melting experiments showed that, unlike conventional Protein A/G affinity chromatography, the conditions for THZR Protein A chromatography respect maintenance of a favourable structural profile for mAbs.

A frontal analysis combined with a simultaneous chromatographic analysis of macromolecules using a single chromatographic system.

A chromatographic system was adapted to allow monitoring of eluent of preparative column via absorbance and with the chromatographic analysis of the target macromolecule on the same chromatographic system. The proposed approach was tested on important macromolecules, such as monoclonal antibodies, monoclonal antibody aggregates and plasmid DNA (pDNA). A frontal analysis was made on the preparative column, while a chromatographic on-line analysis was performed by sequentially injecting the preparative column outlet on a convection-based analytical column, operating on the same chromatographic system. Cation and/or anion exchangers were used as the chromatographic media (along with a protein A), depending on the sample to be purified. The method was found to be robust and reproducible. To adjust the limit of detection, an algorithm varying the number of injections was used, enabling accurate monitoring of an early breakthrough for concentrations below 1% of the feed concentration. The accuracy varies according to the applied flow rate, but it is typically in the range of few percent, or even below. Due to its simplicity and flexibility, the proposed method can be easily adapted to a pharmaceutical environment.

Whole-bacterium ribosome display selection for isolation of highly specific anti-Staphyloccocus aureus Affitins for detection- and capture-based biomedical applications.

Detection and capture methods using antibodies have been developed to ensure identification of pathogens in biological samples. Though antibodies have many attractive properties, they also have limitations and there are needs to expand the panel of available affinity proteins with different properties. Affitins, that we developed from the Sul7d proteins, are a solid class of affinity proteins, which can be used as substitutes to antibodies or to complement them. We report the generation and characterization of antibacterial Affitins with high specificity for Staphylococcus aureus. For the first time, ribosome display selections were carried out using whole-living-cell and naïve combinatorial libraries, which avoid production of protein targets and immunization of animals. We showed that Affitin C5 exclusively recognizes S. aureus among dozens of strains, including clinical ones. C5 binds staphylococcal Protein A (SpA) with a K D of 108 ± 2 nM and has a high thermostability (T m = 77.0°C). Anti-S. aureus C5 binds SpA or bacteria in various detection and capture applications, including ELISA, western blot analysis, bead-fishing, and fluorescence imaging. Thus, novel anti-bacteria Affitins which are cost-effective, stable, and small can be rapidly and fully designed in vitro with high affinity and specificity for a surface-exposed marker. This class of reagents can be useful in diagnostic and biomedical applications.

Three-dimensional origami paper-based device for portable immunoassay applications.

In this study, we demonstrate a three-dimensional surface-modified origami-paper-based analytical device (3D-soPAD) for immunoassay applications. The platform enables the sequential steps of immunoassays to be easily performed using a folded, sliding paper design featuring multiple pre-stored reagents, allowing us to take advantage of the vertical diffusion of the analyte through the different paper layers. The cellulose substrate is composed of carboxymethyl cellulose modified with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, which provide covalent bonding sites for bio-recognition molecules. After the optimization of the operation parameters, we determined the detection limit of the 3D-soPAD for human immunoglobulin G (HIgG) which can be as low as 0.01 ng mL-1, with a total turnaround time of 7 min. In order to study the long-term storage of the platform, anti-HIgG horseradish peroxidase (aHIgG-HRP) conjugates were stored by freeze-drying in sugar matrices composed of 10% sucrose/10% trehalose (w/w%) on the paper device, retaining 80% of their activity after 75 days of storage at 4 °C. To evaluate the performance of the paper device using real samples, we demonstrated the detection of protein A (a biomarker for Staphylococcus aureus infection) in highly viscous human synovial fluid. These results show that the proposed 3D-soPAD platform can provide sensitive, high-throughput, and on-site prognosis of infection in resource-limited settings.

Bio-barcode technology for detection of Staphylococcus aureus protein A based on gold and iron nanoparticles.

S. aureus is one of important causes of disease, food poisoning in humans and animals. The generally methods for detection of S. aureus is time consuming. Therefore, a new method is necessary for rapid, sensitive and specific diagnosis of S. aureus. In the present study, two probes and a Bio-barcode DNA were designed for detection of S. aureus (Protein A). Firstly, magnetic nanoparticle (MNPs) and gold nanoparticle (AuNPs) were synthesized at 80 °C and 100 °C, respectively. The AuNPs and the MNPs were functionalized with probe1, Bio-barcode DNA and probe2, respectively. Target DNA was added into the nanomaterial's system containing bio-barcode DNA-AuNPs-probe1 and probe2-MNPs to formed bio-barcode DNA-AuNPs-probe1-target DNA-probe2-MNPs complex. The bio-barcode DNA-AuNPs-probe1-target DNA-probe2-MNPs complex was separated with magnetic field. Finally, the bio-barcode DNA was released from surface of complex using DTT (0.8 M) and there was isolated of nanoparticles by magnetic field and centrifuge. The fluorescence intensity of bio-barcode DNA was measured in different concentrations of S. aureus (101 to 108 CFU mL-1) by fluorescence spectrophotometry. The results showed that standard curve was linearly from 102 to 107 CFU mL-1. Limit of detection of bio-barcode assay for both PBS and real samples was 86 CFU mL-1.

Development a stacking pad design for enhancing the sensitivity of lateral flow immunoassay.

Lateral flow immunoassays (LFIAs) have wide application in point-of-care testing, particularly in resource-poor settings. To achieve signal amplification in a gold nanoparticle-based lateral flow assay without an additional procedure or the need for complex fabrication, a new and simple method was developed for using a "stacking pad" configuration that adds an additional membrane between the conjugation pad and test pad to the conventional AuNP-based LFIA format. This design helps to accumulate the antibody and antigen on the stacking pad, hence extending the antigen/antibody binding interactions to enhance the test's detection sensitivity. With the enhanced lateral flow assay, as low as 1 ng/mL of Protein A and 15.5 ng/mL of C-reactive protein can be visualized with the naked eye. We also successfully applied the stacking pad system in the analysis of C-reactive protein in human serum and synovial fluid samples. These results suggest that this stacking pad LFIA can provide sensitive and on-site prognosis for detection in synovial fluid and serum samples in resource-limited settings.

Specific Detection of Proteins Using Exceptionally Responsive Magnetic Particles.

Sensitivity and specificity are among the most important parameters for viable sensor technologies based on magnetic nanoparticles. In this work, we describe synthetic routes and analytical approaches to improve both aspects. Magnetic iron oxide particles having diameters of 120, 440, and 700 nm were synthesized, and their surfaces were specifically functionalized. The larger particles showed significantly stronger magnetic signals and responses when compared to commercially available magnetic particles (Dynabeads). A force-based detection method was used to distinguish specifically bound particles (via protein interactions) and nonspecifically bound ones (e.g., via physisorption). In addition, an exchange platform, denoted as exchange-induced remnant magnetization (EXIRM), was developed and utilized to detect label-free proteins specifically. Using EXIRM, the 700 nm magnetic particles showed a 7-fold increase in detection sensitivity when compared to the markedly larger commercially available Dynabeads; furthermore, EXIRM exhibited high specificity, even in a 100-fold increase of nontargeted protein. More generally, particle size effects, reaction times, and dynamic ranges are evaluated and discussed herein.

Quantification of immobilized protein in pharmaceutical production by bio-assisted potentiometric multisensor system.

Quantification of proteins is a key biochemical assay in molecular biology, biotechnology, medicine and pharmacology. Protein quantification protocols can be based on spectrophotometry, enzyme-linked immunosorbent assay, mass spectrometry or quantitative immunoblotting depending on analyte. In case of immobilized protein these methods require suitable sample preparation. Thus, sophisticated analysis becomes even more complex, expensive and time-consuming. Such drawbacks are highly undesirable in industry. In this study we propose a new approach for evaluation of immobilized protein concentration based on application of bio-assisted potentiometric multisensor system. Surface-immobilized recombinant protein A from Staphylococcus aureus (SpA, expressed in Escherichia coli), which is commonly used as affinity ligand immobilized to stationary phase (сhromatography media) for monoclonal antibody purification was employed as the model object. Chromatography media samples containing different amounts of immobilized SpA were analyzed. Proteinase K from Tritirachium album was employed as a bio-transducer. We demonstrated that the suggested approach provides information about immobilized SpA concentration with 0.8mg/ml accuracy in the range 1-6.7mg/ml and within just 16min. Moreover, the proposed procedure requires no expensive materials and equipment and no bio-transducer immobilization. This method has potential of application for fast monitoring of other immobilized proteins in different tasks.

Automated 2D-HPLC method for characterization of protein aggregation with in-line fraction collection device.

Monoclonal antibodies are mainly produced by mammalian cell culture, which due to its complexity, results in a wide range of product variants/isoforms. With the growing implementation of Quality by Design (QbD) and Process Analytical Technology (PAT) in drug manufacturing, monitoring and controlling quality attributes within a predefined range during manufacturing may provide added consistency to product quality. To implement these concepts, more robust analytical tools could reduce the time needed for monitoring quality attributes during upstream processing. The formation of protein aggregates is one such quality attribute that can lead to safety and efficacy issues in the final drug product. Described in this study is a fully automated two-dimensional high performance liquid chromatography (2D-HPLC) method for characterizing protein aggregation of crude in-process bioreactor samples. It combines protein A purification and separation by size exclusion into a single analytical module that has the potential to be employed at-line within a bioprocessing system. This method utilizes a novel in-line fraction collection device allowing for the collection of up to twelve fractions from a single sample or peak which facilitates the subsequent linked analysis of multiple protein peaks of interest in one chromatography module.

G-quadruplex aptamer targeting Protein A and its capability to detect Staphylococcus aureus demonstrated by ELONA.

Aptamers for whole cell detection are selected mostly by the Cell-SELEX procedure. Alternatively, the use of specific cell surface epitopes as target during aptamer selections allows the development of aptamers with ability to bind whole cells. In this study, we integrated a formerly selected Protein A-binding aptamer PA#2/8 in an assay format called ELONA (Enzyme-Linked OligoNucleotide Assay) and evaluated the ability of the aptamer to recognise and bind to Staphylococcus aureus presenting Protein A on the cell surface. The full-length aptamer and one of its truncated variants could be demonstrated to specifically bind to Protein A-expressing intact cells of S. aureus, and thus have the potential to expand the portfolio of aptamers that can act as an analytical agent for the specific recognition and rapid detection of the bacterial pathogen. The functionality of the aptamer was found to be based on a very complex, but also highly variable structure. Two structural key elements were identified. The aptamer sequence contains several G-clusters allowing folding into a G-quadruplex structure with the potential of dimeric and multimeric assembly. An inverted repeat able to form an imperfect stem-loop at the 5'-end also contributes essentially to the aptameric function.

Quantitative Resolution of Monomer-Dimer Populations by Inversion Modulated DEER EPR Spectroscopy.

A simple method, based on inversion modulated double electron-electron resonance electron paramagnetic resonance (DEER EPR) spectroscopy, is presented for determining populations of monomer and dimer in proteins (as well as any other biological macromolecules). The method is based on analysis of modulation depth versus electron double resonance (ELDOR) pulse flip angle. High accuracy is achieved by complete deuteration, extensive sampling of a large number of ELDOR pulse flip angle values, and combined analysis of differently labeled spin samples. We demonstrate the method using two different proteins: an obligate monomer exemplified by the small immunoglobulin binding B domain of protein A, and the p66 subunit of HIV-1 reverse transcriptase which exists as an equilibrium mixture of monomer and dimer species whose relative populations are affected by glycerol content. This information is crucial for quantitative analysis of distance distributions involving proteins that may exist as mixtures of monomer, dimer and high order multimers under the conditions of the DEER EPR experiment.

A high-sensitivity fiber-optic evanescent wave sensor with a three-layer structure composed of Canada balsam doped with GeO2.

In this paper, we present a high-sensitivity polymer fiber-optic evanescent wave (FOEW) sensor with a three-layer structure that includes bottom, inter-, and surface layers in the sensing region. The bottom layer and inter-layer are POFs composed of standard cladding and the core of the plastic optical fiber, and the surface layer is made of dilute Canada balsam in xylene doped with GeO2. We examine the morphology of the doped GeO2, the refractive index and composition of the surface layer and the surface luminous properties of the sensing region. We investigate the effects of the content and morphology of the GeO2 particles on the sensitivity of the FOEW sensors by using glucose solutions. In addition, we examine the response of sensors incubated with staphylococcal protein A plus mouse IgG isotype to goat anti-mouse IgG solutions. Results indicate very good sensitivity of the three-layer FOEW sensor, which showed a 3.91-fold improvement in the detection of the target antibody relative to a conventional sensor with a core-cladding structure, and the novel sensor showed a lower limit of detection of 0.2ng/l and a response time around 320s. The application of this high-sensitivity FOEW sensor can be extended to biodefense, disease diagnosis, biomedical and biochemical analysis.

Bio-assisted potentiometric multisensor system for purity evaluation of recombinant protein A.

Recombinant proteins became essential components of drug manufacturing. Quality control of such proteins is routine task, which usually requires a lot of time, expensive reagents, specialized equipment and highly educated personnel. In this study we propose a new concept for protein purity evaluation that is based on application of bio-assisted potentiometric multisensor system. The model object for analysis was recombinant protein A from Staphylococcus aureus (SpA), which is commonly used for monoclonal antibody purification. SpA solutions with different amount of host cell related impurities (Escherichia coli, bacterial lysate) were analyzed. Two different bio-transducers were employed: proteinase K from Tritirachium album and baker's yeast Saccharomyces cerevisiae. It was shown that both bio-transducers are able to induce changes in pure and lysate-contaminated SpA samples. Different products of yeast digestion and proteolysis with proteinase of pure SpA and lysate were detected with size exclusion high-performance liquid chromatography (SE-HPLC). The induced changes of chemical composition are detectible with potentiometric multisensor system and can be related to SpA purity through projection on latent structures (PLS) regression technique. The proposed method allows for estimation of the impurity content with 12% accuracy using proteinase K and 16% accuracy using baker's yeast. The suggested approach could be useful for early contamination warning at initial protein purification steps. The analysis requires no expensive materials and equipment, no bio-material immobilization, and its duration time is comparable with other commonly used methods like chromatography or electrophoresis though the main part of this time is related to the sample preparation.

Quantitative definition and monitoring of the host cell protein proteome using iTRAQ - a study of an industrial mAb producing CHO-S cell line.

There are few studies defining CHO host cell proteins (HCPs) and the flux of these throughout a downstream purification process. Here we have applied quantitative iTRAQ proteomics to follow the HCP profile of an antibody (mAb) producing CHO-S cell line throughout a standard downstream purification procedure consisting of a Protein A, cation and anion exchange process. We used both 6 sample iTRAQ experiment to analyze technical replicates of three samples, which were culture harvest (HCCF), Protein A flow through and Protein A eluate and an 8 sample format to analyze technical replicates of four sample types; HCCF compared to Protein A eluate and subsequent cation and anion exchange purification. In the 6 sample iTRAQ experiment, 8781 spectra were confidently matched to peptides from 819 proteins (including the mAb chains). Across both the 6 and 8 sample experiments 936 proteins were identified. In the 8 sample comparison, 4187 spectra were confidently matched to peptides from 219 proteins. We then used the iTRAQ data to enable estimation of the relative change of individual proteins across the purification steps. These data provide the basis for application of iTRAQ for process development based upon knowledge of critical HCPs.

Electrokinetic effect for molecular recognition: A label-free approach for real-time biosensing.

We present a simple and inexpensive method for label-free detection of biomolecules. The method monitors the changes in streaming current in a fused silica capillary as target biomolecules bind to immobilized receptors on the inner surface of the capillary. To validate the concept, we show detection and time response of different protein-ligand and protein-protein systems: biotin-avidin and biotin-streptavidin, barstar-dibarnase and Z domain-immunoglobulin G (IgG). We show that specific binding of these biomolecules can be reliably monitored using a very simple setup. Using sequential injections of various proteins at a diverse concentration range and as well as diluted human serum we further investigate the capacity of the proposed technique to perform specific target detection from a complex sample. We also investigate the time for the signal to reach equilibrium and its dependence on analyte concentration and demonstrate that the current setup can be used to detect biomolecules at a concentration as low as 100pM without requiring any advanced device fabrication procedures. Finally, an analytical model based on diffusion theory has been presented to explain the dependence of the saturation time on the analyte concentration and capillary dimensions and how reducing length and inner diameter of the capillary is predicted to give faster detection and in practice also lower limit of detection.

Conformational plasticity of IgG during protein A affinity chromatography.

Single step elution of a protein A column with 100mM acetate pH 3.5 produced a curvilinear gradient with pH dropping steeply at first then more gradually as it approached endpoint. IgG with a native hydrodynamic diameter of 11.5 nm began to elute at pH 6.0 with a size of 9.4 nm. IgG size continued to decrease across the peak, reaching a minimum of 2.2 nm at pH 3.9. Secondary structure of early eluting IgG was only mildly affected but later eluting fractions became increasingly non-native with the 2.2 nm population exhibiting the highest proportion of ß-sheet and lowest random coil of all conformations. Size reduction and structural change of IgG through this portion of the elution peak were attributed dominantly to a pre-existing tendency of highly concentrated IgG to adopt reduced size conformations at low pH and conductivity, facilitated by the known conformational relaxation of IgG by its interaction with protein A. IgG size increased to 10.4 nm as elution pH approached 3.5 across the tailing fractions. Major loss of ß-sheet and increase of α-helix and random coil were observed in parallel. Late elution of this population was attributed to it being eluted from interactions with 2 distinct protein A domains, one bound to each side of the Fc region, creating a higher dissociation constant than single-site Fc-protein A interactions, and requiring more severely disruptive conditions for elution. The high degree of conformational disruption was attributed to simultaneous interaction of both heavy chains with protein A.

Sandwich fluorimetric method for specific detection of Staphylococcus aureus based on antibiotic-affinity strategy.

A novel antibiotic-affinity strategy was designed for fluorimetric detection of pathogenic bacteria based on the strong affinity of antibiotic agent to the cell wall of bacteria. In this proof-of-concept work, vancocin, a glycopeptide antibiotic for Gram-positive bacteria, was used as a molecular recognition agent to anchor Staphylococcus aureus (S. aureus) cell. To improve the specificity of this method for S. aureus detection, IgG was adopted as the second recognition agent utilizing the binding between Fc region of IgG and S. aureus protein A in the cell wall, to form a sandwich complex. By using fluorescein isothiocyanate as the signal probe, S. aureus whole cells could be directly assayed within a linear range of 1.0 × 10(3)-1.0 × 10(9) CFU mL(-1) with a detection limit of 2.9 × 10(2) CFU mL(-1). The whole assay process could be completed within 130 min when a ready-for-use microplate was adopted. This proposed strategy for pathogenic bacteria detection possessed some attractive characteristics such as high sensitivity, wide linear range, simple manipulation, short assay time, and low cost. Furthermore, this sandwich mode also showed ideal specificity because vancocin and IgG bound with S. aureus at two distinct sites. It opened up a new pathway for high-throughput screening of pathogenic bacteria in medical diagnosis, food safety, bioterrorism defense, and drug discovery.

Protein A chromatography increases monoclonal antibody aggregation rate during subsequent low pH virus inactivation hold.

Protein A chromatography is a near-ubiquitous method of mAb capture in bioprocesses. The use of low pH buffer for elution from protein A is known to contribute to product aggregation. Yet, a more limited set of evidence suggests that low pH may not be the sole cause of aggregation in protein A chromatography, rather, other facets of the process may contribute significantly. This paper presents a well-defined method for investigating this problem. An IgG4 was incubated in elution buffer after protein A chromatography (typical of the viral inactivation hold) and the quantity of monomer in neutralised samples was determined by size exclusion chromatography; elution buffers of different pH values predetermined to induce aggregation of the IgG4 were used. Rate constants for monomer decay over time were determined by fitting exponential decay functions to the data. Similar experiments were implemented in the absence of a chromatography step, i.e. IgG4 aggregation at low pH. Rate constants for aggregation after protein A chromatography were considerably higher than those from low pH exposure alone; a distinct shift in aggregation rates was apparent across the pH range tested.