Optimising cell-based bioassays via integrated design of experiments (ixDoE) - A practical guide.

For process optimisation Design of Experiments (DoE) has long been established as a more powerful strategy than a One Factor at a Time approach. Nevertheless, DoE is not widely used especially in the field of cell-based bioassay development although it is known that complex interactions often exist. We believe that biopharmaceutical manufacturers are reluctant to move beyond standard practices due to the perceived costs, efforts, and complexity.

Vapor-Phase Hydrogen Peroxide Uptake by Silicone Tubing and Primary Packaging Components during Protein Drug Product Aseptic Filling: Impact of Pretreatment and Sterilization Process.

In the vapor-phase hydrogen peroxide (VPHP)-sanitized environment, VPHP uptake by product-contacting components could eventually lead to undesired oxidation of biological drug products. Silicone tubing and primary packaging materials are prominent examples of such product-contacting surfaces that are typically processed/sterilized prior to use. This study investigated the VPHP-uptake tendency of these components and how their respective processing/sterilization methods affect the uptake behaviors.

Stability Evaluation of Hydrogen Peroxide Uptake Samples from Monoclonal Antibody Drug Product Aseptically Filled in Vapor Phase Hydrogen Peroxide-Sanitized Barrier Systems: A Case Study.

During the manufacture of a monoclonal antibody drug product, which was aseptically filled within a vapor phase hydrogen peroxide-sanitized isolator, samples were taken to investigate the hydrogen peroxide uptake behaviors. Surprisingly, the samples had no detectable hydrogen peroxide (most results below the limit of detection). This finding was later attributed to hydrogen peroxide decomposition after the samples were stored frozen at -20°C for two weeks before testing.

Vapor Phase Hydrogen Peroxide Decontamination or Sanitization of an Isolator for Aseptic Filling of Monoclonal Antibody Drug Product-Hydrogen Peroxide Uptake and Impact on Protein Quality.

A monoclonal antibody drug product manufacturing process was transferred to a different production site, where aseptic filling took place within an isolator that was decontaminated (sanitized) using vapor phase hydrogen peroxide (VPHP). A quality-by-design approach was applied for study design to understand the impact of VPHP uptake on drug product quality. Both small-scale and manufacturing-scale studies were performed to evaluate the sensitivity of the monoclonal antibody to hydrogen peroxide (HO) and characterize VPHP uptake mechanisms in the filling process.

Acute anti-allodynic action of gabapentin in dorsal horn and primary somatosensory cortex: Correlation of behavioural and physiological data.

Neuropathic pain is a debilitating consequence of neuronal injury or disease. Although first line treatments include the alpha-2-delta (α2δ)-ligands, pregabalin and gabapentin (GBP), the mechanism of their anti-allodynic action is poorly understood. One specific paradox is that GBP relieves signs of neuropathic pain in animal models within 30min of an intraperitoneal (IP) injection yet its actions in vitro on spinal dorsal horn or primary afferent neurons take hours to develop.

Novel insecticidal peptides from Tegenaria agrestis spider venom may have a direct effect on the insect central nervous system.

Fractionation of venom from an agelenid spider, Tegenaria agrestis, resulted in the isolation of a family of three peptides with potent insecticidal activity. These peptide toxins, TaITX-1, -2, and -3, whose sequences were revealed from cloned cDNAs, each consist of 50 amino acid residues, six of which are cysteines. They appear to be amidated at their C-termini and exhibit greater than 90% sequence identity.

Characterization and cloning of insecticidal peptides from the primitive weaving spider Diguetia canities.

Three potent insecticidal peptide toxins were purified from the venom of the primitive weaving spider, Diguetia canities. The toxins share significant homology (> 40%) in their amino acid sequences and are of related size (masses of 6371-7080 Da). In lepidopteran larvae, the toxins cause a progressive spastic paralysis, with 50% paralytic doses (PD50S) ranging from 0.

Molecular genetic analysis of bacteriophage P22 gene 3 product, a protein involved in the initiation of headful DNA packaging.

Bacteriophage P22 DNA packaging events occur in processive series on concatemeric phage DNA molecules. At the point where such series initiate, the DNA is recognized at a site called pac, and most molecular left ends are generated within six short regions called end sites, which are present in a 120 base-pair region surrounding the pac site. The bacteriophage P22 genes 2 and 3 proteins are required for successful generation of these ends and DNA packaging during progeny virion assembly.

Bacteriophage P22 portal protein is part of the gauge that regulates packing density of intravirion DNA.

The complex double-stranded DNA bacteriophages assemble DNA-free protein shells (procapsids) that subsequently package DNA. In the case of several double-stranded DNA bacteriophages, including P22, packaging is associated with cutting of DNA from the concatemeric molecule that results from replication. The mature intravirion P22 DNA has both non-unique (circularly permuted) ends and a length that is determined by the procapsid.

Nucleotide sequence of the bacteriophage P22 genes required for DNA packaging.

The mechanism of DNA packaging by dsDNA viruses is not well understood in any system. In bacteriophage P22 only five genes are required for successful condensation of DNA within the capsid. The products of three of these genes, the portal, scaffolding, and coat proteins, are structural components of the precursor particle, and two, the products of genes 2 and 3, are not.

Fine structure genetic and physical map of the gene 3 to 10 region of the bacteriophage P22 chromosome.

The mechanism by which dsDNA is packaged by viruses is not yet understood in any system. Bacteriophage P22 has been a productive system in which to study the molecular genetics of virus particle assembly and DNA packaging. Only five phage encoded proteins, the products of genes 3, 2, 1, 8 and 5, are required for packaging the virus chromosome inside the coat protein shell.

Nucleotide sequence of the bacteriophage P22 gene 19 to 3 region: identification of a new gene required for lysis.

The nucleotide sequence of a 2558-bp region of bacteriophage P22 at the right end of the genetic map between genes 19 and 3 was determined. A new gene that is partially required for lytic growth, named gene 15, was noted. P22 mutants were constructed which lack gene 15 function, and the gene 15 product was found to be required for lysis in the presence of some divalent cations.