Femtosecond (fs) pulsed laser irradiation techniques have attracted interest as a photonic approach for the selective inactivation of virus contaminations in biological samples. Conventional pulsed laser approaches require, however, relatively long irradiation times to achieve a significant inactivation of virus. In this study, we investigate the enhancement of the photonic inactivation of Murine Leukemia Virus (MLV) via 805 nm femtosecond pulses through gold nanorods whose localized surface plasmon resonance overlaps with the excitation laser. We report a plasmonically enhanced virus inactivation, with greater than 3.7-log reduction measured by virus infectivity assays. Reliable virus inactivation was obtained for 10 s laser exposure with incident laser powers ≥0.3 W. Importantly, the fs-pulse induced inactivation was selective to the virus and did not induce any measurable damage to co-incubated antibodies. The loss in viral infection was associated with reduced viral fusion, linking the loss in infectivity with a perturbation of the viral envelope. Based on the observations that physical contact between nanorods and virus particles was not required for viral inactivation and that reactive oxygen species (ROS) did not participate in the detected viral inactivation, a model of virus inactivation based on plasmon enhanced shockwave generation is proposed.

Viral inactivation plays a critical role in assuring the safety of monoclonal antibody (mAb) therapeutics. Traditional viral inactivation involves large holding tanks in which product is maintained at a target low pH for a defined hold time, typically 30-60 min. The drive toward continuous processing and improved facility utilization has provided motivation for development of a continuous viral inactivation process. To this end, a lab-scale prototype viral inactivation system was designed, built, and characterized. Multiple incubation chamber designs are evaluated to identify the optimal design that enables narrow residence time distributions in continuous flow systems. Extensive analysis is conducted supporting rapid low pH viral inactivation and included evaluations with multiple viruses, a range of pH levels, buffer compositions, mAb concentrations, and temperatures. Multiple test conditions are evaluated using the in-line system and results compared to traditional batch-mode viral inactivation. Comparability in kinetics of virus inactivation suggests equivalency between the two approaches.

An integrated all flow-through technology platform for the purification of therapeutic monoclonal antibodies (mAb), consisting of activated carbon and flow-through cation and anion exchange chromatography steps, can replace a conventional chromatography platform. This new platform was observed to have excellent impurity clearance at high mAb loadings with overall mAb yield exceeding 80%. Robust removal of DNA and host cell protein was demonstrated by activated carbon and a new flow-through cation exchange resin exhibited excellent clearance of mAb aggregate with high monomer recoveries. A ten-fold improvement of mAb loading was achieved compared to a traditional cation exchange resin designed for bind and elute mode. High throughput 96-well plate screening was used for process optimization, focusing on mAb loading and solution conditions. Optimum operating windows for integrated flow-through purification are proposed based on performance characteristics. The combination of an all flow-through polishing process presents significant opportunities for improvements in facility utilization and process economics.

A quantitative study using laser confocal microscopy combined with differential interference microscopy on the kinetics and thermodynamics of the crystallization of glucose isomerase is presented. Fundamental crystallization parameters are determined from the kinetics of step advancement and rates of two-dimensional (2D) nucleation. The ruling mass transfer pathway and accompanying activation barriers are discussed. In brief, the solubility exhibits normal temperature dependence and the crystallization enthalpy is the thermodynamic driving force. The diminishing entropic cost for higher PEG concentrations is attributed to water structuring and a decrease in water activity. The prominent step generation mechanism is homogeneous 2D nucleation for high supersaturations. At low driving forces 2D nucleation occurs on anomalously hyperactive sites and the step edge free energies for homogeneous and heterogeneous nucleation are determined. The number of nucleation centers for both mechanisms are estimated and from the density of nucleation centers we obtain for the activation barrier of adsorption ∼3.8 kJ mol−1. No step-step interaction is observed for interstep distances >70 nm. Theoretical fits of step velocity data suggest surface diffusion makes a non-negligible contribution to surface kinetics. From the temperature dependence of the step kinetic coefficient the activation barrier for crystallization was determined to be <22.4 kJ mol−1.

The biopharmaceutical industry is evolving toward process intensification that can offer increased productivity and improved economics without sacrificing process robustness. A semi‐continuous downstream process linking purification/polishing unit operations in series can reduce or eliminate intermediate holding tanks and reduce overall processing time. Accordingly, we have developed a therapeutic monoclonal antibody polishing template comprised of a connected flow‐through polishing technologies that include activated carbon, cation exchange, and anion‐exchange chromatography. In this report, we evaluated fully‐connected pool‐less polishing with three flow‐through technologies, operating as a single skid to streamline and improve an mAb purification platform. Laboratory‐scale pool‐less processing was achieved without utilizing in‐line pH adjustment and conductivity dilution based on the previously optimized single process parameter. Two connected flow‐through configurations of polishing steps were evaluated: a two‐step process using anion exchange and cation exchange and a three step process using activated carbon, anion exchange and cation exchange chromatography. Laboratory‐scale proof of concept studies showed comparable performance between the batch purification process and the pool‐less process configuration. Three step polishing highly intensified the processes and provided higher process loading and achieved bulk drug specification with higher impurity clearance (>95%) and high overall mAb yield (>95%).

Pan-microbial inactivation technologies that do not require high temperatures, reactive chemical compounds, or UV radiation could address gaps in current infection control strategies and provide efficient sterilization of biologics in the biotechnological industry. Here, we demonstrate that femtosecond (fs) laser irradiation of resonant gold nanoparticles (NPs) under conditions that allow for E-field mediated cavitation and shockwave generation achieve an efficient plasmon-enhanced photonic microbial pathogen inactivation. We demonstrate that this NP-enhanced, physical inactivation approach is effective against a diverse group of pathogens, including both enveloped and nonenveloped viruses, and a variety of bacteria and mycoplasma. Photonic inactivation is wavelengthdependent and in the absence of plasmonic enhancement from NPs, negligible levels of microbial inactivation are observed in the near-infrared (NIR) at 800 nm. This changes upon addition of resonant plasmonic NPs, which provide a strong enhancement of inactivation of viral and bacterial contaminants. Importantly, the plasmon-enhanced 800 nm femtosecond (fs)pulse induced inactivation was selective to pathogens and was obtained without specific targeting of the NPs to the pathogens. No measurable damage was observed for antibodies included as representative biologics under identical conditions.

Confocal Raman microscopy is a nondestructive analytical technique that combines the chemical information from vibrational spectroscopy with the spatial resolution of confocal microscopy. It was applied, for the first time, to measure conformation and distribution of protein adsorbed in wetted chromatographic particles. Monoclonal antibody was loaded into the Fractogel EMD SO(3) (M) cation exchanger at 2 mS/cm or 10 mS/cm. Amide I and III frequencies in the Raman spectrum of the adsorbed protein suggest that there are no detectable changes of the original β-sheet conformation in the chromatographic particles. Protein depth profile measurements indicate that, when the conductivity is increased from 2 mS/cm to 10 mS/cm, there is a change in mass transport mechanism for protein adsorption, from the shrinking-core model to the homogeneous-diffusion model. In this study, the use of confocal Raman microscopy to measure protein distribution in chromatographic particles fundamentally agrees with previous confocal laser scanning microscopic investigations, but confocal Raman spectroscopy enjoys additional advantages: use of unlabeled protein to eliminate fluorescent labeling, ability for characterization of protein secondary structure, and ability for spectral normalization to provide a nondestructive experimental approach to correct light attenuation effects caused by refractive index (RI) mismatching in semiopaque chromatographic particles.

An ambitious 10‐year collaborative program is described to invent, design, demonstrate, and support commercialization of integrated biopharmaceutical manufacturing technology intended to transform the industry. Our goal is to enable improved control, robustness, and security of supply, dramatically reduced capital and operating cost, flexibility to supply an extremely diverse and changing portfolio of products in the face of uncertainty and changing demand, and faster product development and supply chain velocity, with sustainable raw materials, components, and energy use. The program is organized into workstreams focused on end‐to‐end control strategy, equipment flexibility, next generation technology, sustainability, and a physical test bed to evaluate and demonstrate the technologies that are developed. The elements of the program are synergistic. For example, process intensification results in cost reduction as well as increased sustainability. Improved robustness leads to less inventory, which improves costs and supply chain velocity. Flexibility allows more products to be consolidated into fewer factories, reduces the need for new facilities, simplifies the acquisition of additional capacity if needed, and reduces changeover time, which improves cost and velocity. The program incorporates both drug substance and drug product manufacturing, but this paper will focus on the drug substance elements of the program.