Pioneering Just-in-Time (JIT) Strategy for Accelerating Raman Method Development and Implementation for Biologic Continuous Manufacturing.

Raman spectroscopy is a popular process analytical technology (PAT) tool that has been increasingly used to monitor and control the monoclonal antibody (mAb) manufacturing process. Although it allows the characterization of a variety of quality attributes by developing chemometric models, a large quantity of representative data is required, and hence, the model development process can be time-consuming. In recent years, the pharmaceutical industry has been expediting new drug development in order to achieve faster delivery of life-changing drugs to patients. The shortened development timelines have impacted the Raman application, as less time is allowed for data collection. To address this problem, an innovative Just-in-Time (JIT) strategy is proposed with the goal of reducing the time needed for Raman model development and ensuring its implementation. To demonstrate its capabilities, a proof-of-concept study was performed by applying the JIT strategy to a biologic continuous process for producing monoclonal antibody products. Raman spectroscopy and online two-dimensional liquid chromatography (2D-LC) were integrated as a PAT analyzer system. Raman models of antibody titer and aggregate percentage were calibrated by chemometric modeling in real-time. The models were also updated in real-time using new data collected during process monitoring. Initial Raman models with adequate performance were established using data collected from two lab-scale cell culture batches and subsequently updated using one scale-up batch. The JIT strategy is capable of accelerating Raman method development to monitor and guide the expedited biologics process development.

Automated Online-Sampling Multidimensional Liquid Chromatography with Feedback-Control Capability as a Framework for Real-Time Monitoring of mAb Critical Quality Attributes in Multiple Bioreactors.

Real-time monitoring of biopharmaceutical reactors is becoming increasingly important as the processes become more complex. During the continuous manufacturing of monoclonal antibodies (mAbs), the desired mAb product is continually created and collected over a 30 day process, where there can be changes in quality over that time. Liquid chromatography (LC) is the workhorse instrumentation capable of measuring mAb concentration as well as quality attributes such as aggregation, charge variants, oxidation, etc. However, traditional offline sampling is too infrequent to fully characterize bioprocesses, and the typical time from sample generation to data analysis and reporting can take weeks. To circumvent these limitations, an automated online sampling multidimensional workflow was developed to enable streamlined measurements of mAb concentration, aggregation, and charge variants. This analytical framework also facilitates automated data export for real-time analysis of up to six bioreactors, including feedback-controlling capability using readily available LC technology. This workflow increases the data points per bioreactor, improving the understanding of each experiment while also reducing the data turnaround time from weeks to hours. Examples of effective real-time analyses of mAb critical quality attributes are illustrated, showing substantial throughput improvements and accurate results while minimizing labor and manual intervention.

ESSENCE - A rapid, shear-enhanced, flow-through, capacitive electrochemical platform for rapid detection of biomolecules.

The rapid, sensitive, and selective detection of target analytes using electrochemical sensors is challenging. ESSENCE, a new Electrochemical Sensor that uses a Shear-Enhanced, flow-through Nanoporous Capacitive Electrode, overcomes current electrochemical sensors' response limitations, selectivity, and sensitivity limitations. ESSENCE is a microfluidic channel packed with transducer material sandwiched by a top and bottom microelectrode. The room-temperature instrument less integration process allows the switch of the transducer materials to make up the porous electrode without modifying the electrode architecture or device protocol. ESSENCE can be used to detect both biomolecules and small molecules by simply changing the packed transducer material. Electron microscopy results confirm the high porosity. In conjunction with the non-planar interdigitated electrode, the packed transducer material results in a flow-through porous electrode. Electron microscopy results confirm the high porosity. The enhanced shear forces and increased convective fluxes disrupt the electric double layer's (EDL) diffusive process in ESSENCE. This disruption migrates the EDL to high MHz frequency allowing the capture signal to be measured at around 100 kHz, significantly improving device timing (rapid detection) with a low signal-to-noise ratio. The device's unique architecture allows us multiple configuration modes for measuring the impedance signal. This allows us to use highly conductive materials like carbon nanotubes. We show that by combining single-walled carbon nanotubes as transducer material with appropriate capture probes, NP-µIDE has high selectivity and sensitivity for DNA (fM sensitivity, selective against non-target DNA), breast cancer biomarker proteins (p53, pg/L sensitivity, selective against non-target HER2).

Direct-from-sputum rapid phenotypic drug susceptibility test for mycobacteria.

BACKGROUND: The spread of multi-drug resistant tuberculosis (MDR-TB) is a leading global public-health challenge. Because not all biological mechanisms of resistance are known, culture-based (phenotypic) drug-susceptibility testing (DST) provides important information that influences clinical decision-making. Current phenotypic tests typically require pre-culture to ensure bacterial loads are at a testable level (taking 2-4 weeks) followed by 10-14 days to confirm growth or lack thereof. METHODS AND FINDINGS: We present a 2-step method to obtain DST results within 3 days of sample collection. The first involves selectively concentrating live mycobacterial cells present in relatively large volumes of sputum (~2-10mL) using commercially available magnetic-nanoparticles (MNPs) into smaller volumes, thereby bypassing the need for pre-culture. The second involves using microchannel Electrical Impedance Spectroscopy (m-EIS) to monitor multiple aliquots of small volumes (~10µL) of suspension containing mycobacterial cells, MNPs, and candidate-drugs to determine whether cells grow, die, or remain static under the conditions tested. m-EIS yields an estimate for the solution "bulk capacitance" (Cb), a parameter that is proportional to the number of live bacteria in suspension. We are thus able to detect cell death (bactericidal action of the drug) in addition to cell-growth. We demonstrate proof-of-principle using M. bovis BCG and M. smegmatis suspended in artificial sputum. Loads of ~ 2000-10,000 CFU of mycobacteria were extracted from ~5mL of artificial sputum during the decontamination process with efficiencies of 84% -100%. Subsequently, suspensions containing ~105 CFU/mL of mycobacteria with 10 mg/mL of MNPs were monitored in the presence of bacteriostatic and bactericidal drugs at concentrations below, at, and above known MIC (Minimum Inhibitory Concentration) values. m-EIS data (ΔCb) showed data consistent with growth, death or stasis as expected and/or recorded using plate counts. Electrical signals of death were visible as early as 3 hours, and growth was seen in < 3 days for all samples, allowing us to perform DST in < 3 days. CONCLUSION: We demonstrated "proof of principle" that (a) live mycobacteria can be isolated from sputum using MNPs with high efficiency (almost all the bacteria that survive decontamination) and (b) that the efficacy of candidate drugs on the mycobacteria thus isolated (in suspensions containing MNPs) could be tested in real-time using m-EIS.

Metal-Organic Framework-Based Microfluidic Impedance Sensor Platform for Ultrasensitive Detection of Perfluorooctanesulfonate.

The growing global concerns to public health from human exposure to perfluorooctanesulfonate (PFOS) require rapid, sensitive, in situ detection where current, state-of-the-art techniques are yet to adequately meet sensitivity standards of the real world. This work presents, for the first time, a synergistic approach for the targeted affinity-based capture of PFOS using a porous sorbent probe that enhances detection sensitivity by embedding it on a microfluidic platform. This novel sorbent-containing platform functions as an electrochemical sensor to directly measure PFOS concentration through a proportional change in electrical current (increase in impedance). The extremely high surface area and pore volume of mesoporous metal-organic framework (MOF) Cr-MIL-101 is used as the probe for targeted PFOS capture based on the affinity of the chromium center toward both the fluorine tail groups as well as the sulfonate functionalities as demonstrated by spectroscopic (NMR and XPS) and microscopic (TEM) studies. Answering the need for an ultrasensitive PFOS detection technique, we are embedding the MOF capture probes inside a microfluidic channel, sandwiched between interdigitated microelectrodes (IDµE). The nanoporous geometry, along with interdigitated microelectrodes, increases the signal-to-noise ratio tremendously. Further, the ability of the capture probes to interact with the PFOS at the molecular level and effectively transduce that response electrochemically has allowed us achieve a significant increase in sensitivity. The PFOS detection limit of 0.5 ng/L is unprecedented for in situ analytical PFOS sensors and comparable to quantification limits achieved using state-of-the-art ex situ techniques.

Rapid culture-based detection of living mycobacteria using microchannel electrical impedance spectroscopy (m-EIS).

BACKGROUND: Multiple techniques exist for detecting Mycobacteria, each having its own advantages and drawbacks. Among them, automated culture-based systems like the BACTEC-MGIT™ are popular because they are inexpensive, reliable and highly accurate. However, they have a relatively long "time-to-detection" (TTD). Hence, a method that retains the reliability and low-cost of the MGIT system, while reducing TTD would be highly desirable. METHODS: Living bacterial cells possess a membrane potential, on account of which they store charge when subjected to an AC-field. This charge storage (bulk capacitance) can be estimated using impedance measurements at multiple frequencies. An increase in the number of living cells during culture is reflected in an increase in bulk capacitance, and this forms the basis of our detection. M. bovis BCG and M. smegmatis suspensions with differing initial loads are cultured in MGIT media supplemented with OADC and Middlebrook 7H9 media respectively, electrical "scans" taken at regular intervals and the bulk capacitance estimated from the scans. Bulk capacitance estimates at later time-points are statistically compared to the suspension's baseline value. A statistically significant increase is assumed to indicate the presence of proliferating mycobacteria. RESULTS: Our TTDs were 60 and 36 h for M. bovis BCG and 20 and 9 h for M. smegmatis with initial loads of 1000 CFU/ml and 100,000 CFU/ml respectively. The corresponding TTDs for the commercial BACTEC MGIT 960 system were 131 and 84.6 h for M. bovis BCG and 41.7 and 12 h for M smegmatis, respectively. CONCLUSION: Our culture-based detection method using multi-frequency impedance measurements is capable of detecting mycobacteria faster than current commercial systems.

Rapid culture-based detection of living mycobacteria using microchannel electrical impedance spectroscopy (m-EIS)

BACKGROUND: Multiple techniques exist for detecting Mycobacteria, each having its own advantages and drawbacks. Among them, automated culture-based systems like the BACTEC-MGIT™ are popular because they are inexpensive, reliable and highly accurate. However, they have a relatively long "time-to-detection" (TTD). Hence, a method that retains the reliability and low-cost of the MGIT system, while reducing TTD would be highly desirable. METHODS: Living bacterial cells possess a membrane potential, on account of which they store charge when subjected to an AC-field. This charge storage (bulk capacitance) can be estimated using impedance measurements at multiple frequencies. An increase in the number of living cells during culture is reflected in an increase in bulk capacitance, and this forms the basis of our detection. M. bovis BCG and M. smegmatis suspensions with differing initial loads are cultured in MGIT media supplemented with OADC and Middlebrook 7H9 media respectively, electrical "scans" taken at regular intervals and the bulk capacitance estimated from the scans. Bulk capacitance estimates at later time-points are statistically compared to the suspension's baseline value. A statistically significant increase is assumed to indicate the presence of proliferating mycobacteria. RESULTS: Our TTDs were 60 and 36 h for M. bovis BCG and 20 and 9 h for M. smegmatis with initial loads of 1000 CFU/ml and 100,000 CFU/ml respectively. The corresponding TTDs for the commercial BACTEC MGIT 960 system were 131 and 84.6 h for M. bovis BCG and 41.7 and 12 h for M smegmatis, respectively. CONCLUSION: Our culture-based detection method using multi-frequency impedance measurements is capable of detecting mycobacteria faster than current commercial systems.

Foaming Betadine Spray as a potential agent for non-labor-intensive preoperative surgical site preparation.

BACKGROUND: The Centers for Disease Control and Prevention's (CDC) National Healthcare Safety Network (NHSN) report published in 2009 shows that there were about 16,000 cases of surgical site infection (SSI) following ~ 850,000 operative procedures making SSI one of the most predominant infection amongst nosocomial infections. Preoperative skin preparation is a standard procedure utilized to prevent SSIs thereby improving patient outcomes and controlling associated healthcare costs. Multiple techniques/ products have been used for pre-operative skin preparation, like 2 step scrubbing and painting, 2 step scrubbing and drying, and 1 step painting with a drying time. However, currently used products require strict, time consuming and labor-intensive protocols that involve repeated mechanical scrubbing. It can be speculated that a product requiring a more facile protocol will increase compliance, thus promoting a reduction in SSIs. Hence, the antimicrobial efficacy of a spray-on foaming formulation containing Betadine (povidone-iodine aerosol foam) that can be administered with minimum effort is compared to that of an existing formulation/technique (Wet Skin Scrub). METHODS: In vitro antimicrobial activities of (a) 5% Betadine delivered in aerosolized foam, (b) Wet Skin Scrub Prep Tray and (c) liquid Betadine are tested against three clinically representative microorganisms (S. aureus, S. epidermidis and P. aeruginosa,) on two surfaces (agar-gel on petri-dish and porcine skin). The log reduction/growth of the bacteria in each case is noted and ANOVA statistical analysis is used to establish the effectiveness of the antimicrobial agents, and compare their relative efficacies. RESULTS: With agar gel as the substrate, no growth of bacteria is observed for all the three formulations. With porcine skin as the substrate, the spray-on foam's performance was not statistically different from that of the Wet Skin Scrub Prep technique for the microorganisms tested. CONCLUSIONS: The povidone-iodine aerosolized foam could potentially serve as a non-labor intensive antimicrobial agent for surgical site preparation.

Sunlight-assisted route to antimicrobial plasmonic aminoclay catalysts.

We present a straightforward, environmentally-benign, one-pot photochemical route to generate alloyed AgAu bimetallic nanoparticle decorated aminoclays in water at room temperature. The protocol uses no reducing agent (e.g., NaBH4) nor is photocatalyst required. These hybrid materials show excellent promise as dual catalysts/antibacterial agents.

Coatings and surface modifications imparting antimicrobial activity to orthopedic implants.

Bacterial colonization and biofilm formation on an orthopedic implant surface is one of the worst possible outcomes of orthopedic intervention in terms of both patient prognosis and healthcare costs. Making the problem even more vexing is the fact that infections are often caused by events beyond the control of the operating surgeon and may manifest weeks to months after the initial surgery. Herein, we review the costs and consequences of implant infection as well as the methods of prevention and management. In particular, we focus on coatings and other forms of implant surface modification in a manner that imparts some antimicrobial benefit to the implant device. Such coatings can be classified generally based on their mode of action: surface adhesion prevention, bactericidal, antimicrobial-eluting, osseointegration promotion, and combinations of the above. Despite several advances in the efficacy of these antimicrobial methods, a remaining major challenge is ensuring retention of the antimicrobial activity over a period of months to years postoperation, an issue that has so far been inadequately addressed. Finally, we provide an overview of additional figures of merit that will determine whether a given antimicrobial surface modification warrants adoption for clinical use.