Efficient extraction of adventitious virus nucleic acid using commercially available methods.

An essential step in pharmaceutical product development is screening for contamination with adventitious agents, and there is desire to develop highly sensitive assays to detect adventitious viral nucleic acid. This study sought to examine the nucleic acid extraction efficiency of three viral candidates in relevant background matrices using four different extraction methods. Three model adventitious viruses, Minute virus of Mice, Porcine Circovirus, and Feline Leukemia Virus, were diluted within a variety of background matrices relevant to pharmaceutical production methods. Upon extraction, the nucleic acid was quantified using droplet digital PCR methods. Four nucleic acid extraction methods were assessed, including commercially available kits and manual extraction methods. Each method recovered nucleic acid post-extraction for each of the model viruses within the tested background matrices. The silica-column based method recovered a greater amount of viral nucleic acid, compared to the other methods tested. Similar trends were observed when model virus was diluted in bioreactor supernatant, which replicates industry testing conditions and provides details on which extraction methods might be used in Next Generation Sequencing and PCR methods for detecting contamination within pharmaceutical products.

Purification of a recombinant oncolytic virus from clarified cell culture media by anion exchange monolith chromatography.

The use of viral vectors for vaccine, gene therapy, and oncolytic virotherapy applications has received increased attention in recent years. Large-scale purification of viral vector-based biotherapeutics still presents a significant technical challenge. Chromatography is the primary tool for the purification of biomolecules in the biotechnology industry; however, the majority of chromatography resins currently available have been designed for the purification of proteins. In contrast, convective interaction media monoliths are chromatographic supports that have been designed and successfully utilized for the purification of large biomolecules, including viruses, viruslike particles, and plasmids. We present a case study on the development of a purification method for recombinant Newcastle disease virus directly from clarified cell culture media using strong anion exchange monolith technology (CIMmultus QA, BIA Separations). Resin screening studies showed at least 10 times higher dynamic binding capacity of CIMmultus QA compared to traditional anion exchange chromatography resins. Design of experiments was used to demonstrate a robust operating window for the purification of recombinant virus directly from clarified cell culture without any further pH or conductivity adjustment of the load material. The capture step was successfully scaled up from 1 mL CIMmultus QA columns to the 8 L column scale and achieved a greater than 30-fold reduction in process volume. Compared to the load material, total host cell proteins were reduced by more than 76%, and residual host cell DNA by more than 57% in the elution pool, respectively. Direct loading of clarified cell culture onto a high-capacity monolith stationary phase makes convective flow chromatography an attractive alternative to centrifugation or TFF-based virus purification procedures.

Using a noninfectious MVM surrogate for assessing viral clearance during downstream process development.

Viral contamination is an inherent risk during the manufacture of biopharmaceuticals. As such, biopharmaceutical companies must demonstrate the viral clearance efficacy of their downstream process steps prior to clinical trials and commercial approval. This is accomplished through expensive and logistically challenging spiking studies, which utilize live mammalian viruses. These hurdles deter companies from analyzing viral clearance during process development and characterization. We utilized a noninfectious minute virus of mice-mock virus particle (MVM-MVP) as a surrogate spiking agent during small scale viral filtration (VF) and anion exchange chromatography (AEX) studies. For VF experiments, in-process mAb material was spiked and processed through Asahi Kasei P15, P20, P35, and BioEX nanofilters. Across each filter type, flux decay profiles and log reduction values (LRVs) were nearly identical for either particle. For AEX experiments, loads were conditioned with various amounts of sodium chloride (9, 20, 23, and 41 mS/cm), spiked with either particle and processed through a Q-SFF packed column. LRV results met our expectations of predicting MVM removal.

Effects of pH, conductivity, host cell protein, and DNA size distribution on DNA clearance in anion exchange chromatography media.

Flowthrough anion exchange chromatography is commonly used as a polishing step in downstream processing of monoclonal antibodies and other therapeutic proteins to remove process-related impurities and contaminants such as host cell DNA, host cell proteins, endotoxin, and viruses. DNA with a wide range of molecular weight distributions derived from Chinese Hamster Ovary cells was used to advance the understanding of DNA binding behavior in selected anion exchange media using the resin (Toyopearl SuperQ-650M) and membranes (Mustang® Q and Sartobind® Q) through DNA spiking studies. The impacts of the process parameters pH (6-8), conductivity (2-15 mS/cm), and the potential binding competition between host cell proteins and host cell DNA were studied. Studies were conducted at the least and most favorable experimental conditions for DNA binding based on the anticipated electrostatic interactions between the host cell DNA and the resin ligand. The resin showed 50% higher DNA binding capacity compared to the membrane media. Spiking host cell proteins in the load material showed no impact on the DNA clearance capability of the anion exchange media. DNA size distributions were characterized based on a "size exclusion qPCR assay." Results showed preferential binding of larger DNA fragments (>409 base pairs). © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:141-149, 2018.

Major families of multiresistant plasmids from geographically and epidemiologically diverse staphylococci.

Staphylococci are increasingly aggressive human pathogens suggesting that active evolution is spreading novel virulence and resistance phenotypes. Large staphylococcal plasmids commonly carry antibiotic resistances and virulence loci, but relatively few have been completely sequenced. We determined the plasmid content of 280 staphylococci isolated in diverse geographical regions from the 1940s to the 2000s and found that 79% of strains carried at least one large plasmid >20 kb and that 75% of these large plasmids were 20-30 kb. Using restriction fragment length polymorphism (RFLP) analysis, we grouped 43% of all large plasmids into three major families, showing remarkably conserved intercontinental spread of multiresistant staphylococcal plasmids over seven decades. In total, we sequenced 93 complete and 57 partial staphylococcal plasmids ranging in size from 1.3 kb to 64.9 kb, tripling the number of complete sequences for staphylococcal plasmids >20 kb in the NCBI RefSeq database. These plasmids typically carried multiple antimicrobial and metal resistances and virulence genes, transposases and recombinases. Remarkably, plasmids within each of the three main families were >98% identical, apart from insertions and deletions, despite being isolated from strains decades apart and on different continents. This suggests enormous selective pressure has optimized the content of certain plasmids despite their large size and complex organization.

The dynamic genome of Hydra.

The freshwater cnidarian Hydra was first described in 1702 and has been the object of study for 300 years. Experimental studies of Hydra between 1736 and 1744 culminated in the discovery of asexual reproduction of an animal by budding, the first description of regeneration in an animal, and successful transplantation of tissue between animals. Today, Hydra is an important model for studies of axial patterning, stem cell biology and regeneration. Here we report the genome of Hydra magnipapillata and compare it to the genomes of the anthozoan Nematostella vectensis and other animals. The Hydra genome has been shaped by bursts of transposable element expansion, horizontal gene transfer, trans-splicing, and simplification of gene structure and gene content that parallel simplification of the Hydra life cycle. We also report the sequence of the genome of a novel bacterium stably associated with H. magnipapillata. Comparisons of the Hydra genome to the genomes of other animals shed light on the evolution of epithelia, contractile tissues, developmentally regulated transcription factors, the Spemann-Mangold organizer, pluripotency genes and the neuromuscular junction.

The diploid genome sequence of an individual human.

Presented here is a genome sequence of an individual human. It was produced from approximately 32 million random DNA fragments, sequenced by Sanger dideoxy technology and assembled into 4,528 scaffolds, comprising 2,810 million bases (Mb) of contiguous sequence with approximately 7.5-fold coverage for any given region. We developed a modified version of the Celera assembler to facilitate the identification and comparison of alternate alleles within this individual diploid genome. Comparison of this genome and the National Center for Biotechnology Information human reference assembly revealed more than 4.1 million DNA variants, encompassing 12.3 Mb. These variants (of which 1,288,319 were novel) included 3,213,401 single nucleotide polymorphisms (SNPs), 53,823 block substitutions (2-206 bp), 292,102 heterozygous insertion/deletion events (indels)(1-571 bp), 559,473 homozygous indels (1-82,711 bp), 90 inversions, as well as numerous segmental duplications and copy number variation regions. Non-SNP DNA variation accounts for 22% of all events identified in the donor, however they involve 74% of all variant bases. This suggests an important role for non-SNP genetic alterations in defining the diploid genome structure. Moreover, 44% of genes were heterozygous for one or more variants. Using a novel haplotype assembly strategy, we were able to span 1.5 Gb of genome sequence in segments >200 kb, providing further precision to the diploid nature of the genome. These data depict a definitive molecular portrait of a diploid human genome that provides a starting point for future genome comparisons and enables an era of individualized genomic information.