Rapid and low-cost sampling for detection of airborne SARS-CoV-2 in dehumidifier condensate.

Airborne spread of coronavirus disease 2019 (COVID-19) by infectious aerosol is all but certain. However, easily implemented approaches to assess the actual environmental threat are currently unavailable. We present a simple approach with the potential to rapidly provide information about the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the atmosphere at any location. We used a portable dehumidifier as a readily available and affordable tool to collect airborne virus in the condensate. The dehumidifiers were deployed in selected locations of a hospital ward with patients reporting flu-like symptoms which could possibly be due to COVID-19 over three separate periods of one week. Samples were analyzed frequently for both virus envelope protein and SARS-CoV-2 RNA. In several samples across separate deployments, condensate from dehumidifiers tested positive for the presence of SARS-CoV-2 antigens as confirmed using two independent assays. RNA was detected, but not attributable to SARS-CoV-2. We verified the ability of the dehumidifier to rapidly collect aerosolized sodium chloride. Our results point to a facile pool testing method to sample air in any location in the world and assess the presence and concentration of an infectious agent to obtain quantitative risk assessment of exposure, designate zones as "hot spots" and minimize the need for individual testing which may often be time consuming, expensive, and laborious.

Low-cost customizable microscale toolkit for rapid screening and purification of therapeutic proteins.

Biopharmaceutical separations require tremendous amounts of optimization to achieve acceptable product purity. Typically, large volumes of reagents and biological materials are needed for testing different parameters, thus adding to the expense of biopharmaceutical process development. This study demonstrates a versatile and customizable microscale column (µCol) for biopharmaceutical separations using immobilized metal affinity chromatography (IMAC) as an example application to identify key parameters. µCols have excellent precision, efficiency, and reproducibility, can accommodate any affinity, ion-exchange or size-exclusion-based resin and are compatible with any high-performance liquid chromatography (HPLC) system. µCols reduce reagent amounts, provide comparable purification performance and high-throughput, and are easy to automate compared with current conventional resin columns. We provide a detailed description of the fabrication methods, resin packing methods, and µCol validation experiments using a conventional HPLC system. Finite element modeling using COMSOL Multiphysics was used to validate the experimental performance of the µCols. In this study, µCols were used for improving the purification achieved for granulocyte colony stimulating factor (G-CSF) expressed using a cell-free CHO in vitro translation (IVT) system and were compared to a conventional 1 ml IMAC column. Experimental data revealed comparable purity with a 10-fold reduction in the amount of buffer, resin, and purification time for the µCols compared with conventional columns for similar protein yields.

Cell-free production of a therapeutic protein: Expression, purification, and characterization of recombinant streptokinase using a CHO lysate.

The use of cell-free systems to produce recombinant proteins has grown rapidly over the past decade. In particular, cell-free protein synthesis (CFPS) systems based on mammalian cells provide alternative methods for the production of many proteins, including those that contain disulfide bonds, glycosylation, and complex structures such as monoclonal antibodies. In the present study, we show robust production of turbo green fluorescent protein (tGFP) and streptokinase in a cell-free system using instrumented mini-bioreactors for highly reproducible protein production. We achieved recombinant protein production (∼600 µg/ml of tGFP and 500 µg/ml streptokinase) in 2.5 hr of expression time, comparable to previously reported yields for cell-free protein expression. Also, we demonstrate the use of two different affinity tags for product capture and compare those to a tag-free self-cleaving intein capture technology. The intein purification method provided a product recovery of 86%, compared with 52% for conventionally tagged proteins, while resulting in a 30% increase in total units of activity of purified recombinant streptokinase compared with conventionally tagged proteins. These promising beneficial features combined with the intein technology makes feasible the development of dose-level production of therapeutic proteins at the point-of-care.

Improving the recombinant human erythropoietin glycosylation using microsome supplementation in CHO cell-free system.

Cell-Free Protein Synthesis (CFPS) offers many advantages for the production of recombinant therapeutic proteins using the CHO cell-free system. However, many complex proteins are still difficult to express using this method. To investigate the current bottlenecks in cell-free glycoprotein production, we chose erythropoietin (40% glycosylated), an essential endogenous hormone which stimulates the development of red blood cells. Here, we report the production of recombinant erythropoietin (EPO) using CHO cell-free system. Using this method, EPO was expressed and purified with a twofold increase in yield when the cell-free reaction was supplemented with CHO microsomes. The protein was purified to near homogeneity using an ion-metal affinity column. We were able to analyze the expressed and purified products (glycosylated cell-free EPO runs at 25-28 kDa, and unglycosylated protein runs at 20 kDa on an SDS-PAGE), identifying the presence of glycan moieties by PNGase shift assay. The purified protein was predicted to have ∼2,300 IU in vitro activity. Additionally, we tested the presence and absence of sugars on the cell-free EPO using a lectin-based assay system. The results obtained in this study indicate that microsomes augmented in vitro production of the glycoprotein is useful for the rapid production of single doses of a therapeutic glycoprotein drug and to rapidly screen glycoprotein constructs in the development of these types of drugs. CFPS is useful for implementing a lectin-based method for rapid screening and detection of glycan moieties, which is a critical quality attribute in the industrial production of therapeutic glycoproteins.

Immunoglobulin G elution in protein A chromatography employing the method of chromatofocusing for reducing the co-elution of impurities.

Purification processes for monoclonal Immunoglobulin G (IgG) typically employ protein A chromatography as a capture step to remove most of the impurities. One major concern of the post-protein A chromatography processes is the co-elution of some of the host cell proteins (HCPs) with IgG in the capture step. In this work, a novel method for IgG elution in protein A chromatography that reduces the co-elution of HCPs is presented where a two-step pH gradient is self-formed inside a protein A chromatography column. The complexities involved in using an internally produced pH gradient in a protein A chromatography column employing adsorbed buffering species are discussed though equation-based modeling. Under the conditions employed, ELISA assays show a 60% reduction in the HCPs co-eluting with the IgG fraction when using the method as compared to conventional protein A elution without affecting the IgG yield. Evidence is also obtained which indicates that the amount of leached protein A present in free solution in the purified product is reduced by the new method. Biotechnol. Bioeng. 2017;114: 154-162. © 2016 Wiley Periodicals, Inc.

Optimizing cell-free protein expression in CHO: Assessing small molecule mass transfer effects in various reactor configurations.

Cell-free protein synthesis (CFPS) is an ideal platform for rapid and convenient protein production. However, bioreactor design remains a critical consideration in optimizing protein expression. Using turbo green fluorescent protein (tGFP) as a model, we tracked small molecule components in a Chinese Hamster Ovary (CHO) CFPS system to optimize protein production. Here, three bioreactors in continuous-exchange cell-free (CECF) format were characterized. A GFP optical sensor was built to monitor the product in real-time. Mass transfer of important substrate and by-product components such as nucleoside triphosphates (NTPs), creatine, and inorganic phosphate (Pi) across a 10-kDa MWCO cellulose membrane was calculated. The highest efficiency measured by tGFP yields were found in a microdialysis device configuration; while a negative effect on yield was observed due to limited mass transfer of NTPs in a dialysis cup configuration. In 24-well plate high-throughput CECF format, addition of up to 40 mM creatine phosphate in the system increased yields by up to ∼60% relative to controls. Direct ATP addition, as opposed to creatine phosphate addition, negatively affected the expression. Pi addition of up to 30 mM to the expression significantly reduced yields by over ∼40% relative to controls. Overall, data presented in this report serves as a valuable reference to optimize the CHO CFPS system for next-generation bioprocessing. Biotechnol. Bioeng. 2017;114: 1478-1486. © 2017 Wiley Periodicals, Inc.

Understanding the mechanism of virus removal by Q sepharose fast flow chromatography during the purification of CHO-cell derived biotherapeutics.

During production of therapeutic monoclonal antibodies (mAbs) in mammalian cell culture, it is important to ensure that viral impurities and potential viral contaminants will be removed during downstream purification. Anion exchange chromatography provides a high degree of virus removal from mAb feedstocks, but the mechanism by which this is achieved has not been characterized. In this work, we have investigated the binding of three viruses to Q sepharose fast flow (QSFF) resin to determine the degree to which electrostatic interactions are responsible for viral clearance by this process. We first used a chromatofocusing technique to determine the isoelectric points of the viruses and established that they are negatively charged under standard QSFF conditions. We then determined that virus removal by this chromatography resin is strongly disrupted by the presence of high salt concentrations or by the absence of the positively charged Q ligand, indicating that binding of the virus to the resin is primarily due to electrostatic forces, and that any non-electrostatic interactions which may be present are not sufficient to provide virus removal. Finally, we determined the binding profile of a virus in a QSFF column after a viral clearance process. These data indicate that virus particles generally behave similarly to proteins, but they also illustrate the high degree of performance necessary to achieve several logs of virus reduction. Overall, this mechanistic understanding of an important viral clearance process provides the foundation for the development of science-based process validation strategies to ensure viral safety of biotechnology products.

Serial displacement chromatofocusing and its applications in multidimensional chromatography and gel electrophoresis: I. Theory and general considerations.

The technique of "serial displacement chromatofocusing" (SDC) is investigated both theoretically and experimentally with model mixtures of proteins and peptides. The method employs a multistep, retained pH gradient formed using adsorbed buffering species to produce a series of discrete effluent fractions. Each of these fractions may contain several displaced protein bands under conditions of sufficient mass overloading, so that several displacement trains of adjoined bands can be produced in a single chromatographic run. Numerical simulations and experimental results showed selective concentration effects for minor components in a fraction when the feed amount was sufficient large. A computer-aided design method was developed to facilitate the use of the method and was applied to both anion- and cation-exchange column packings. Good agreement was achieved between the designed pH gradients and experimental results. The characteristics of SDC were also explored in terms of its loading capacity, scalability, repeatability, recovery, and differentiation of proteins between their true and apparent isoelectric point values.

Serial displacement chromatofocusing and its applications in multidimensional chromatography and gel electrophoresis: II. Experimental results.

Part I of this study investigated the theory and basic characteristics of "serial displacement chromatofocusing" (SDC). In Part II of this study, SDC is applied to two prototype applications which have potential uses in proteomics and related areas involving the analysis of complex analyte mixtures. In the first application, SDC was used as a prefractionation method prior to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to separate a human prostate cancer cell lysate. It was observed that the resolution achieved in narrow-pI-range 2D-PAGE was improved when using SDC prefractionation, so that SDC may be useful as a low-cost, high-speed, and highly scalable alternative to electrophoretic prefractionation methods for 2D-PAGE. The second application involves the use of SDC as the first dimension, and reversed-phase chromatography as the second dimension, to produce a novel, fully automated, two-dimensional high-performance liquid chromatography technique. The method was shown to have performance advantages over one-dimensional reversed-phase chromatography for peptide separations.

Reducing sample complexity in proteomics by chromatofocusing with simple buffer mixtures.

Chromatofocusing has many potential applications in the field of proteomics, such as for the isolation and removal of major sample components to facilitate the analysis of low-abundance components, and for sample prefractionation prior to a subsequent separation using SDS-PAGE, narrow-pI-range 2D-PAGE, or additional chromatography steps. However, the chromatofocusing techniques that are most commonly used employ propriety polyampholyte elution buffers and highly specialized column packings, both of which limit the use of chromatofocusing in practice. To expand the range of application for this technique, this chapter considers chromatofocusing methods which employ common ion-exchange column packings and elution buffers which are simple mixtures of readily available buffering species. Of particular interest is the use of chromatofocusing with a multistep pH gradient for the fractionation of protein mixtures into narrow-pI-range fractions. The cross-contamination characteristics of these fractions using SDS-PAGE are also assessed.

Characterization and purification of bacteriophages using chromatofocusing.

The technique of chromatofocusing was applied to the characterization and purification of three bacteriophages that are routinely used for testing virus filters: phiX174, PR772, and PP7. Chemically well-defined eluent buffers were used, instead of the more commonly used chromatofocusing polyampholyte buffers. Chromatographic column packings were selected to minimize band broadening by confining bacteriophage adsorption solely to the exterior particle surface. Under the conditions used it was determined that bacteriophages could be made to focus into narrow bands in a retained pH gradient with recoveries of live phage that ranged from 15 to nearly 100% as determined by a plaque-forming assay. Retention times and apparent isoelectric point data were obtained for samples consisting either of purified bacteriophage, or samples consisting of crude preparations of bacteriophages containing host cell impurities. Isoelectric point estimates were obtained using modified, previously described models. The results obtained suggest that chromatofocusing is a simple and rapid method for obtaining approximate isoelectric points for bacteriophages and probably other types of viruses. It is also likely a useful method for purifying these materials.

Evaluation of chromatofocusing as a capture method for monoclonal antibody products.

Chromatofocusing is investigated as an alternative to protein A chromatography for the initial capture step in a purification process for several monoclonal antibodies and antibody fusion products. For comparison, this work also investigates the use of ion-exchange chromatography with either pH or salt gradient elution as additional alternatives to protein A chromatography. The specific conditions employed for the capture step for the case of chromatofocusing were selected on a rational basis using a computer-aided design method implemented in the form of a Microsoft Excel spreadsheet. Alternative operating conditions were compared experimentally with regard to the product yield achieved as well as the removal of total host cell proteins (HCPs) and of a specific HCP major component. Results from this study indicate that both chromatofocusing and ion-exchange chromatography are useful alternatives to a protein A chromatography capture step in many practical cases. This is especially true for the case of chromatofocusing when it is possible to exploit the ability of the method to create complex gradient shapes that are self-forming inside the column and to simultaneous focus and separate proteins inside the column.

Point-of-care production of therapeutic proteins of good-manufacturing-practice quality.

Manufacturing technologies for biologics rely on large, centralized, good-manufacturing-practice (GMP) production facilities and on a cumbersome product-distribution network. Here, we report the development of an automated and portable medicines-on-demand device that enables consistent, small-scale GMP manufacturing of therapeutic-grade biologics on a timescale of hours. The device couples the in vitro translation of target proteins from ribosomal DNA, using extracts from reconstituted lyophilized Chinese hamster ovary cells, with the continuous purification of the proteins. We used the device to reproducibly manufacture His-tagged granulocyte-colony stimulating factor, erythropoietin, glucose-binding protein and diphtheria toxoid DT5. Medicines-on-demand technology may enable the rapid manufacturing of biologics at the point of care.

Characterization of Non-Infectious Virus-Like Particle Surrogates for Viral Clearance Applications.

Viral clearance is a critical aspect of biopharmaceutical manufacturing process validation. To determine the viral clearance efficacy of downstream chromatography and filtration steps, live viral "spiking" studies are conducted with model mammalian viruses such as minute virus of mice (MVM). However, due to biosafety considerations, spiking studies are costly and typically conducted in specialized facilities. In this work, we introduce the concept of utilizing a non-infectious MVM virus-like particle (MVM-VLP) as an economical surrogate for live MVM during process development and characterization. Through transmission electron microscopy, size exclusion chromatography with multi-angle light scattering, chromatofocusing, and a novel solute surface hydrophobicity assay, we examined and compared the size, surface charge, and hydrophobic properties of MVM and MVM-VLP. The results revealed that MVM and MVM-VLP exhibited nearly identical physicochemical properties, indicating the potential utility of MVM-VLP as an accurate and economical surrogate to live MVM during chromatography and filtration process development and characterization studies.

Validation of an optical sensor-based high-throughput bioreactor system for mammalian cell culture.

Cell culture optimization is a labor-intensive process requiring a large number of experiments to be conducted under varying conditions. Here we describe a high-throughput bioreactor system that allows 12 mini stirred-tank bioreactors to be operated simultaneously. All bioreactors are monitored by low-cost minimally invasive optical sensors for pH and dissolved oxygen. The sensors consist of single-use patches affixed inside the bioreactors and monitored optically from the outside. Experimental results show that different sensing patches with the same composition respond consistently. The discrepancy between different pH sensors is less than 0.1 pH units over most of their responsive range. The discrepancy between different dissolved oxygen sensors is less than 10% over the whole range from 0% to 100% dissolved oxygen. The consistency of the sensing system ensures that only an initial one-time calibration is required for the sensing patches. After that, a calibration code is generated and sensing patches of the same composition can be used directly. This greatly reduces the time and cost required for monitored multi-bioreactor operations. We used SP2/0 myeloma/mouse hybridoma cell cultures to demonstrate reactor performance consistency. Transcriptional profiling, HPLC analysis, viable cell count, and viability inspection show that the presence of sensing patches and the use of optical monitoring have no apparent effect on the metabolism of the cells.

New concepts in the chromatography of peptides and proteins.

Although chromatography using a variety of novel bed configurations (e.g. fluidized beds, expanded beds, simulated moving beds, annular rotating beds, etc.) has been of recent interest, the majority of practical applications of analytical and preparative chromatography employ a stationary adsorbent bed into which a feed slug is charged periodically, similar to the technique first described by Mikhail Tswett over 100 years ago. However, new concepts in both the practice and theory of fixed-bed chromatography are continuing to expand the available range of applications for separating peptides and proteins.

Effect of charge regulation on steric mass-action equilibrium for the ion-exchange adsorption of proteins.

A thermodynamic formalism is developed for incorporating the effects of charge regulation on the ion-exchange adsorption of proteins under mass-overloaded conditions as described by the steric mass-action (SMA) isotherm. To accomplish this, the pH titration behavior of a protein and the associated adsorption equilibrium of the various charged forms of a protein are incorporated into a model which also accounts for the steric hindrance of salt counterions caused by protein adsorption. For the case where the protein is dilute, the new model reduces to the protein adsorption model described recently by the authors which accounts for charge regulation. Similarly, the new model reduces to the steric mass-action isotherm developed by Brooks and Cramer which applies to mass-overloaded conditions for the case where charge regulation is ignored so that the protein has a fixed charge. Calculations using the new model were found to agree with experimental data for the adsorption of bovine serum albumin (BSA) on an anion-exchange column packing when using reasonable physical properties. The new model was also used to develop an improved theoretical criterion for determining the conditions required for an adsorbed species to displace a protein in displacement chromatography when the pH is near the protein pI.

Behavior of the inadvertent pH transient formed by a salt gradient in the ion-exchange chromatography of proteins.

The inadvertent pH transient produced when a stepwise change in salt concentration is used as the eluent in ion-exchange chromatography was studied theoretically using a local-equilibrium theory and experimentally using both strong-base and weak-base anion-exchange column packings. The accuracy of the local-equilibrium theory was verified by comparing it to a full numerical solution of the governing partial differential equations obtained using the method of characteristics. The predictions from the local-equilibrium theory were observed to largely agree with experimental results. Detailed comparisons of experimental results and the local-equilibrium theory permitted the observed trends for the pH transients to be interpreted in terms of the physical properties of the column packing and mobile phase. The results of this study are useful for the design of ion-exchange processes using salt gradient elution where it is desired to limit the exposure of eluted proteins to the inadvertent pH transient caused by the salt gradient.

Displacement chromatography of proteins using a retained pH front in a hydrophobic charge induction chromatography column.

The chromatographic separation of two proteins into a displacement train of two adjoined rectangular bands was accomplished using a novel method for hydrophobic charge induction chromatography (HCIC) which employs a self-sharpening pH front as the displacer. This method exploits the fact that protein elution in HCIC is promoted by a pH change, but is relatively independent of salt effects, so that a retained pH front can be used in place of a traditional displacer in displacement chromatography. The retained pH front was produced using the two adsorbed buffering species tricine and acetic acid. The separation of lysozyme and α-chymotrypsinogen A into adjoined, rectangular bands was accomplished with overall recoveries based on the total mass injected greater than 90 and 70%, respectively. The addition of urea to the buffer system increased the sharpness of the pH front by 36% while the yields of lysozyme and α-chymotrypsinogen A based on the total mass eluted increased from 76% to 99% and from 37% to 85%, respectively, when the purities of both proteins in their product fractions were fixed at 85%. The results demonstrate that the method developed in this study is a useful variant of HCIC and is also a useful alternative to other displacement chromatography methods.

Charge regulation in protein ion-exchange chromatography: development and experimental evaluation of a theory based on hydrogen ion Donnan equilibrium.

An extension of the stoichiometric displacement (SD) model for the ion-exchange adsorption of dilute proteins is developed which accounts for the effects of hydrogen ion Donnan equilibrium on the protein charge. The ability of the new model to fit retention data when the fluid phase pH is near the protein pI and the effects of hydrogen ion Donnan equilibrium are important is examined using four different proteins and four different column packings. The results indicate that the model is able to fit retention data using values for the protein pI and the change in protein charge with pH at the pI, i.e., (dz/dpH)pI, that are significantly closer to the values of these parameters determined by isoelectric focusing and acid-base titrametry in free solution, respectively, as compared to the values obtained by determining the characteristic binding change as a function of pH using the traditional stoichiometric displacement model. This suggests that when the fluid phase pH is near the protein pI, charge regulation is an important cause of the discrepancy between the electrical charge of a protein in free solution and the characteristic binding charge from the stoichiometric displacement model. The results also indicate that for the case where the fluid phase pH is near the protein pI, the new model accounts for the effect of charge regulation during protein ion-exchange adsorption more accurately than previous models in the literature.