Regeneration of Capto Core 700 resin through high throughput and laboratory scale studies and impact on production of a SARS-CoV-2 vaccine candidate.

During the development of a SARS-CoV-2 vaccine candidate, at the height of the COVID-19 pandemic, raw materials shortages, including chromatography resins, necessitated the determination of a cleaning in place (CIP) strategy for a multimodal core-shell resin both rapidly and efficiently. Here, the deployment of high throughput (HT) techniques to screen CIP conditions for cleaning Capto Core 700 resin exposed to clarified cell culture harvest (CCCH) of a SARS-CoV-2 vaccine candidate produced in Vero adherent cell culture are described. The best performing conditions, comprised of 30% n-propanol and ≥0.75 N NaOH, were deployed in cycling experiments, completed with miniature chromatography columns, to demonstrate their effectiveness. The success of the CIP strategy was ultimately verified at the laboratory scale. Here, its impact was assessed across the entire purification process which also included an ultrafiltration/diafiltration step. It is shown that the implementation of the CIP strategy enabled the re-use of the Capto Core 700 resin for up to 10 cycles without any negative impact on the purified product. Hence, the strategic combination of HT and laboratory-scale experiments can lead rapidly to robust CIP procedures, even for a challenging to clean resin, and thus help to overcome supply shortages.

Defining a sample preparation workflow for advanced virus detection and understanding sensitivity by next-generation sequencing.

The application of next-generation sequencing (also known as deep sequencing or massively parallel sequencing) for adventitious agent detection is an evolving field that is steadily gaining acceptance in the biopharmaceutical industry. In order for this technology to be successfully applied, a robust method that can isolate viral nucleic acids from a variety of biological samples (such as host cell substrates, cell-free culture fluids, viral vaccine harvests, and animal-derived raw materials) must be established by demonstrating recovery of model virus spikes. In this report, we implement the sample preparation workflow developed by Feng et. al. and assess the sensitivity of virus detection in a next-generation sequencing readout using the Illumina MiSeq platform. We describe a theoretical model to estimate the detection of a target virus in a cell lysate or viral vaccine harvest sample. We show that nuclease treatment can be used for samples that contain a high background of non-relevant nucleic acids (e.g., host cell DNA) in order to effectively increase the sensitivity of sequencing target viruses and reduce the complexity of data analysis. Finally, we demonstrate that at defined spike levels, nucleic acids from a panel of model viruses spiked into representative cell lysate and viral vaccine harvest samples can be confidently recovered by next-generation sequencing.

Kinetic magnetic resonance imaging of the cervical spine: a review of the literature.

Study Design Literature review. Objective The purpose of this study is to compile and review the body of literature related to kinetic magnetic resonance imaging (kMRI) of the cervical spine. Methods A review of literature related to kMRI was performed using PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. Results We included 16 prospective and retrospective studies of symptomatic and asymptomatic patients who underwent kMRI of the cervical spine. Conclusions Data suggest that kMRI is able to provide meaningful information regarding changes in the cervical spine in both normal and pathologic segments. A prospective study comparing magnetic resonance imaging and kMRI is needed to confirm clinically utility of this technology.

Prevalence and motion characteristics of degenerative cervical spondylolisthesis in the symptomatic adult.

STUDY DESIGN: Retrospective analysis of kinetic magnetic resonance images. OBJECTIVE: To define the prevalence of degenerative cervical spondylolisthesis in symptomatic patients and to analyze the motion characteristics and influence on the spinal canal at the affected level. SUMMARY OF BACKGROUND DATA: When compared with lumbar spondylolisthesis, there are few studies evaluating cervical spondylolisthesis, and the prevalence and motion characteristics of cervical spondylolisthesis are not well defined. METHODS: Four hundred sixty-eight symptomatic patients underwent upright cervical kinetic magnetic resonance images in neutral, flexion, and extension positions. Segmental displacement and intervertebral angles were measured in 3 positions using computer analysis software. Spondylolisthesis was defined as the vertebral displacement more than 2 mm, and graded based on the magnitude into 2 groups at each level: grade 1 (2-3 mm), grade 2 (>3 mm). Instability was defined as segmental translational motion exceeding 3 mm. RESULTS: Grade 1 and 2 spondylolisthesis at a minimum of 1 level were observed with a prevalence of 16.4% and 3.4% of all patients, respectively. The most affected levels were C4-C5 (6.2%) and C5-C6 (6.0%) followed by C3-C4 (3.6%) and C6-C7 (3.0%). Translational motion was greater in levels with grade 1 as compared with segments without spondylolisthesis, but there was no difference in angular motion between the 3 groups. Translational instability was observed with a prevalence of 16.7% in grade 2, 4.3% in grade 1, and 3.4% in segments without spondylolisthesis. Space available for the cord at the affected level was decreased and spinal cord compression grade was higher in grade 1 and grade 2 as compared with levels without spondylolisthesis. CONCLUSION: Cervical spondylolisthesis of at least 2 mm was observed in 20% of patients and was most common at C4-C5 and C5-C6. The presence of spondylolisthesis was associated with increased translational motion and decreased segmental spinal canal diameter. LEVEL OF EVIDENCE: N/A.

Development of a high-throughput rubella virus infectivity assay based on viral activation of caspases.

Rubella virus (RV, German measles) is a teratogenic agent that can lead to serious congenital defects after maternal infection during early pregnancy. Currently, the disease can be prevented effectively by available live attenuated vaccines. An important requisite for the manufacture and release of a safe and potent live virus vaccine is the measurement of the vaccine titer (potency), to ensure the correct dose and efficacy of the vaccine. One historical method for measuring potency is the endpoint dilution TCID(50) assay. Traditionally, RV TCID(50) titers are calculated after visual inspection of cells for presence of cytopathic effect (CPE). Such visual scoring is tedious and labor intensive. The development of a new TCID(50) readout method, based on a fluorescent molecular marker of RV-induced apoptosis, is described in this report. Further, in order to calculate TCID(50) potency a novel mathematical model was established to convert the numerical fluorescence measurements into categorical data. Finally, the assay parameters such as signal-to-noise ratio, robustness, variability and bias were optimized. This new readout method demonstrated strong concordance with the standard manual scoring of CPE, and therefore can provide a practical, objective and higher-throughput alternative to the traditional TCID(50) readout used for calculating titers of rubella virus.

Rapid automation of a cell-based assay using a modular approach: case study of a flow-based Varicella Zoster Virus infectivity assay.

Vaccine manufacturing requires constant analytical monitoring to ensure reliable quality and a consistent safety profile of the final product. Concentration and bioactivity of active components of the vaccine are key attributes routinely evaluated throughout the manufacturing cycle and for product release and dosage. In the case of live attenuated virus vaccines, bioactivity is traditionally measured in vitro by infection of susceptible cells with the vaccine followed by quantification of virus replication, cytopathology or expression of viral markers. These assays are typically multi-day procedures that require trained technicians and constant attention. Considering the need for high volumes of testing, automation and streamlining of these assays is highly desirable. In this study, the automation and streamlining of a complex infectivity assay for Varicella Zoster Virus (VZV) containing test articles is presented. The automation procedure was completed using existing liquid handling infrastructure in a modular fashion, limiting custom-designed elements to a minimum to facilitate transposition. In addition, cellular senescence data provided an optimal population doubling range for long term, reliable assay operation at high throughput. The results presented in this study demonstrate a successful automation paradigm resulting in an eightfold increase in throughput while maintaining assay performance characteristics comparable to the original assay.

A robust approach for the quantitation of viral concentration in an adenoviral vector-based human immunodeficiency virus vaccine by real-time quantitative polymerase chain reaction.

A real-time quantitative polymerase chain reaction (PCR)-based method was developed to measure the concentration of recombinant adenoviral vector genomes in purified virus bulks and final container samples of monovalent and multivalent human immunodeficiency virus (HIV) adenoviral vector vaccine candidates. This method, referred to as the genome quantitation assay (GQA), was optimized through a rigorous approach for evaluating PCR detection chemistries, designing a robust assay format, and establishing a properly calibrated reference standard. In addition, the use of a simplified lysis procedure, automated liquid transfer system, and parallel-line data analysis contribute to an accurate, precise, reliable, and high-throughput assay procedure that can be used for process monitoring, final formulation, and release of vaccine products. A variance component analysis study indicated that the GQA typically produces results with an interassay precision of less than 10% relative standard deviation (RSD), allowing generation of final results (average of three runs) with associated interassay precision of 6% RSD or less. The precision, accuracy, specificity, and robustness of the GQA demonstrate its utility for analytical characterization of a wide variety of viral vector- and DNA plasmid- based vaccines or gene therapy products. In addition, we also evaluated the Adenovirus Reference Standard generated by the Adenovirus Reference Material Working Group in the GQA to provide a common point-of-reference for our analytical method.