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.

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.

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.

Rapid evolution of virus sequences in intrinsically disordered protein regions.

Nodamura Virus (NoV) is a nodavirus originally isolated from insects that can replicate in a wide variety of hosts, including mammals. Because of their simplicity and ability to replicate in many diverse hosts, NoV, and the Nodaviridae in general, provide a unique window into the evolution of viruses and host-virus interactions. Here we show that the C-terminus of the viral polymerase exhibits extreme structural and evolutionary flexibility. Indeed, fewer than 10 positively charged residues from the 110 amino acid-long C-terminal region of protein A are required to support RNA1 replication. Strikingly, this region can be replaced by completely unrelated protein sequences, yet still produce a functional replicase. Structure predictions, as well as evolutionary and mutational analyses, indicate that the C-terminal region is structurally disordered and evolves faster than the rest of the viral proteome. Thus, the function of an intrinsically unstructured protein region can be independent of most of its primary sequence, conferring both functional robustness and sequence plasticity on the protein. Our results provide an experimental explanation for rapid evolution of unstructured regions, which enables an effective exploration of the sequence space, and likely function space, available to the virus.

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.

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.

A novel 'pipeline' system for downstream preparation of therapeutic monoclonal antibodies.

A novel 'pipeline' system for the preparation of therapeutic monoclonal antibodies (mAb) in a non-GMP compliant environment has been developed. We used sterile silica-gel pipes to connect individual process units, in order to form a fully-enclosed and seamlessly connected system. This 'pipeline' system was used to implement downstream preparation processes for a humanized anti-CD146 mAb, huAA98, which is a therapeutic mAb generated to inhibit cancer-related angiogenesis. The quality assessment of the huAA98 end-product indicated that endotoxin levels were 0.016 EU/ml, protein A levels were 1.08 ng/ml and host cell protein (HCP) was undetectable. Thus, all measures were below the clinical criteria set by the Chinese Pharmacopoeia (Edition 2010). Having passed our proof-of-concept test, this 'pipeline' system can be used as a universal platform for the preparation of mAbs suitable for pre-clinical studies, in a non-GMP compliant laboratory environment.

Enhancement of immunoassay's fluorescence and detection sensitivity using three-dimensional plasmonic nano-antenna-dots array.

Protein detection is universal and vital in biological study and medical diagnosis (e.g., cancer detection). Fluorescent immunoassay is one of the most widely used and most sensitive methods in protein detection (Giljohann, D. A.; Mirkin, C. A. Nature2009, 462, 461-464; Yager, P.; et al. Nature2006, 442, 412-418). Improvements of such assays have many significant implications. Here, we report the use of a new plasmonic structure and a molecular spacer to enhance the average fluorescence of an immunoassay of Protein A and human immunoglobulin G (IgG) by over 7400-fold and the immunoassay's detection sensitivity by 3,000,000-fold (the limit of detection is reduced from 0.9 × 10(-9) to 0.3 × 10(-15) molar (i.e., from 0.9 nM to 300 aM), compared to identical assays performed on glass plates). Furthermore, the average fluorescence enhancement has a dynamic range of 8 orders of magnitude and is uniform over the entire large sample area with a spatial variation ±9%. Additionally, we observed that, when a single molecule fluorophore is placed at a "hot spot" of the plasmonic structure, its fluorescence is enhanced by 4 × 10(6)-fold, thus indicating the potential to further significantly increase the average fluorescence enhancement and the detection sensitivity. Together with good spatial uniformity, wide dynamic range, and ease to manufacture, the giant enhancement in immunoassay's fluorescence and detection sensitivity (orders of magnitude higher than previously reported) should open up broad applications in biology study, medical diagnosis, and others.

3D porous sol-gel matrix incorporated microdevice for effective large volume cell sample pretreatment.

In this study, we demonstrated an effective sample pretreatment microdevice that could perform the capture, purification, and release of pathogenic bacteria with a large-volume sample and at a high speed and high-capture yield. We integrated a sol-gel matrix into the microdevice which forms three-dimensional (3D) micropores for the cell solution to pass through and provides a large surface area for the immobilization of antibodies to capture the target Staphylococcus aureus (S. aureus) cells. The antibody was linked to the surface of the sol-gel via a photocleavable linker, allowing the cell-captured antibody moiety to be released by UV irradiation. In addition to the optimization of the antibody immobilization and UV cleavage processes, the cell-capture efficiency was maximized by controlling the sample flow rate with a pumping scheme (3 steps, 5 steps: 3 steps with one flutter step, 7 steps: 3 steps with two flutter steps) and the pumping time (100, 200, and 300 ms). A quantitative capture analysis was performed by targeting a specific gene site of protein A of S. aureus in real-time PCR (RT-PCR). While the 3-step process with an actuation time of 100 ms showed the fastest flow rate (1 mL sample processing time in 10 min), the pumping scheme with the 7-step process and the 300 ms actuation time revealed the highest cell-capture efficiency. A limit of detection study with the 7-step and the 300 ms pumping scheme demonstrated that 100 cells per 100 µL were detected with a 70% yield, and even a single cell could be analyzed via on-chip sample preparation. Thus, our novel sol-gel based microdevice was proven more cost-effective, simple, and efficient in terms of its sample pretreatment ability compared to the use of a conventional 2D flat microdevice. This proposed sample pretreatment device can be further incorporated to an analytical functional unit to realize a micrototal analysis system (µTAS) with sample-in-answer-out capability in the fields of biomedical diagnostics, food safety testing, and environmental pollutant screening.

The characterization and quantitation of glycomic changes in CHO cells during a bioreactor campaign.

Within the biotechnology industry there is a continuous drive to better and more fully understand the biopharmaceutical process in order to achieve better process control. A method to monitor and quantitate glycomic changes that occur in CHO cells during a bioreactor campaign could help to address this. The goal of the method presented here is to provide data that may help to understand the changes in glycosylation that are occurring, within the cell, to proteins other than the expressed biotherapeutic. The method involves the lysing of cells to gain access to intracellular proteins. The expressed biotherapeutic is specifically removed by affinity chromatography, while the remaining proteins are subjected to deglycosylation by treatment with PNGase F. The released glycans are derivatized with isotopic tags, and quantitative analysis by MALDI-TOF MS is performed. The MALDI-TOF MS method allows for the simultaneous analysis of both neutral and sialylated glycans, displays a linear dynamic range over two orders of magnitude for both neutral and sialylated glycans and achieves sub-picomolar sensitivity. This method may yield valuable information that gives further insight into the inner-workings of CHO cells, potentially taking another step towards fully understanding and controlling the biopharmaceutical process.

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.

Improved biomolecule microarrays by printing on nanoporous aluminum oxide using a continuous-flow microspotter.

Biomolecules, including protein A, albumin, and immunoglobulin G, are spotted on top of a nanoporous substrate by using a continuous-flow microspotter (CFM) system, which normally produces spots 3 to 4 orders of magnitude more sensitive than conventional biomolecule printing methods. The spots are observed with a fluorescence scanner. By using the CFM to print spots on nanoporous substrates, an additional order of magnitude increase in signal is observed, which leads to high signal-to-background ratios, highly saturated spots, and a measurable signal at printing concentrations as low as 1.6 ng mL(-1). This technique produces highly concentrated biomolecular spots from dilute samples and significantly increases the sensitivity of sensing platforms.

Rapid detection of mecA and spa by the loop-mediated isothermal amplification (LAMP) method.

AIM: To develop a detection assay for staphylococcal mecA and spa by using loop-mediated isothermal amplification (LAMP) method. METHODS AND RESULTS: Staphylococcus aureus and other related species were subjected to the detection of mecA and spa by both PCR and LAMP methods. The LAMP successfully amplified the genes under isothermal conditions at 64 degrees C within 60 min, and demonstrated identical results with the conventional PCR methods. The detection limits of the LAMP for mecA and spa, by gel electrophoresis, were 10(2) and 10 cells per tube, respectively. The naked-eye inspections were possible with 10(3) and 10 cells for detection of mecA and spa, respectively. The LAMP method was then applied to sputum and dental plaque samples. The LAMP and PCR demonstrated identical results for the plaque samples, although frequency in detection of mecA and spa by the LAMP was relatively lower for the sputum samples when compared to the PCR methods. CONCLUSION: Application of the LAMP enabled a rapid detection assay for mecA and spa. The assay may be applicable to clinical plaque samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The LAMP offers an alternative detection assay for mecA and spa with a great advantage of the rapidity.

The use of spa and phage typing for characterization of a MRSA population in a Belgian hospital: comparison between 2002 and 2007.

UNLABELLED: TARGET OF THE STUDY: Strain typing of pathogens is essential to pinpoint the sources and routes of transmission and to forecast future trends. In a general hospital, we studied possible changes in the MRSA population. PATIENTS AND METHODS: MRSA isolates received from a Belgian general hospital, during 2002 (n=150) and the second half of 2007 (n=105), were compared by phage and spa typing. RESULTS: In 2002, [J]* phage types characterized 45% of the MRSA isolates, 13% belonged to the [O]* phage types, 12% to a local phage type 29/42E/54/D11* and 28% were not assigned to a defined group. Thirteen different spa types were found among the isolates: 39% belonged to t038, 27% to t121, 14% to t041, 5% to t740, and 4% to t002 and t024 each. Two spa types were found respectively in two and three isolates, five were unique. In 2007, 35% belonged to [J]*, 23% to [O]* and 39% could not be put in a defined group. Eighteen different spa types were found: 30% belonged to t740, 29% to t121, 13% to t038 and 10% to t002. Three spa types were represented in two isolates, eleven were unique. The t041 spa type was specific for the 29/42E/54/D11* and the majority of the t121 isolates were related to [J]*. CONCLUSION: [J]* remained the dominant phage types group but decreased whereas [O]*, the second phage types group, increased. As to the spa types, t740 became dominant while t121 remained second. Phage and spa typing point to some quantitative changes among the Belgian MRSA population.

Infected wound model development of an in vitro biomaterial-protected wound infection model to study microbial activity and antimicrobial treatment through microdialysis.

OBJECTIVE: Skin injuries provide a favorable environment for microbial infection if left untreated. This is problematic especially in nosocomial situations having a high prevalence of Staphylococcus aureus that can cause suppuration of wounds, systemic disease, and toxic shock. The objective of this investigation was to use a wound model system to study the interactions between microbial activity, host tissue, therapeutic treatments, and wound biomaterials. DESIGN: An in vitro wound model was developed using Sykes-Moore chambers filled with 1 of 2 biomaterials used for wound treatment (1% alginate and dialyzed HyFil hydrogel (B. Braun Medical, Inc, Bethlehem, Pennsylvania) and seeded with fibroblasts. The chambers were inoculated with S aureus, and half were later treated with antibiotics through in situ microdialysis tubing. MAIN OUTCOME MEASURES: The chambers were monitored by obtaining fluid samples and biomaterial samples at specific time intervals (0, 2, 8, and 24 hours) and were analyzed for (1) S aureus protein A (SPA) concentration, (2) viable S aureus numbers, and (3) fibroblast numbers and viability. Chambers containing each biomaterial with and without antibiotics were compared to controls. MAIN RESULTS: There was an inverse relationship between postinfection S aureus numbers and fibroblast viability. S aureus numbers were usually consistent with SPA concentration, which may have been underestimated because of SPA interactions with the biomaterials. CONCLUSION: This wound model may be useful to gain an understanding about the interactions between microbial activity, host tissue, therapeutic treatments, and wound biomaterials. Hypotheses about wound treatments derived by means of this model may direct future in vivo studies.

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.

Ultrasensitive detection of trace protein by Western blot based on POLY-quantum dot probes.

In this study, we describe an ultrasensitive quantum dots (QDs)-based Western blot. With the high affinity of avidin-functionalized POLY-QDs and simplification of the detection process, this enabled the quantitative analysis of protein by Western blotting. To prepare the POLY-QDs, CdTe quantum dots were first coated with biotinylated denatured bovine serum albumin and then, via the effect of the biotin-avidin system, the biotinylated denatured bovine serum albumin-coated QDs, which had strong fluorescence, were linked together. With this series of modifications, the fluorescence intensity of CdTe QDs was significantly increased. Using the POLY-QDs as labels, the signal of Western blotting was more sensitive in tracing the protein than traditional dyeing. In the present study, trace protein A was applied to POLY-QDs-based Western blotting as a model. The linearity of this method was from 30 pg to 1.5 ng, and the sensitivity was up to low pictogram values. The final fluorescence signal on the polyvinylidenedifluoride (PVDF) membrane was retained for at least 40 min. The results of this study indicate that the POLY-QDs-based Western blot is an excellent quantitative analytical method for trace protein analysis.

Affinity capture mass spectrometry of biomarker proteins using peptide ligands from biopanning.

Affinity capture mass spectrometry was used to isolate and ionize protein A from Staphylococcus aureus from both a commercial source and cell culture lysate using matrix assisted laser desorption/ionization (MALDI) mass spectrometry. Two surfaces are compared: gold surfaces with immunoglobulin G covalently immobilized and silica surfaces with a covalently bound small peptide discovered via biopanning. A detection limit of 2.22 bacterial cells/mL of culture fluid was determined for the immobilized peptide surfaces. This study emphasizes the ability to use peptide ligands to effectively capture a biomarker protein out of a complex mixture. This demonstrates the potential to use biopanning to generate capture ligands for a large variety of target proteins and subsequently detect the captured protein using MALDI mass spectrometry.

Trace level analysis of leached Protein A in bioprocess samples without interference from the large excess of rhMAb IgG.

Resins containing immobilized Staphylococcal Protein A (PA) are widely used in the commercial purification of recombinant human monoclonal antibody (rhuMAb IgG) biotherapeutics. Therefore, a sensitive assay for leached PA is needed to ensure that PA is not present at unacceptable levels as an impurity in the final product. PA impurities are measured by an ELISA using chicken anti-PA antibodies. However, PA in the presence of IgG product forms a PA/IgG complex that interferes in the assay. In this report a multi-product PA ELISA is described, wherein the PA/IgG complex is dissociated by heating in the presence of detergents and chelators prior to the ELISA. The dissociation facilitates the accessibility of the anti-PA antibodies to bind to PA in the immunoassay. Heat is provided by a novel microwave technology which allows brief heating time and high sample throughput using a microtiter plate for sample heating. Thus, broadly applicable dissociation conditions, suitable for all 21 rhMab IgGs tested to date were identified. This approach streamlines the measurement of leached PA, allows higher sample testing throughput, facilitates application across multiple products, and facilitates assay automation. Data comparing in-process samples tested with both the former product-specific ELISA and this new multi-product assay are shown.