Manufacturing processes of peanut (Arachis hypogaea) allergen powder-dnfp.

Background: Important components of drug safety, efficacy, and acceptability involve manufacturing and testing of the drug substance and drug product. Peanut flour sourcing/processing and manufacturing processes may affect final drug product allergen potency and contamination level, possibly impacting drug safety, quality, and efficacy. We describe key steps in the manufacturing processes of peanut (Arachis hypogaea) allergen powder-dnfp (PTAH; Palforzia®), a drug used in oral immunotherapy (OIT) for the treatment of peanut allergy. Methods: Established criteria for source material must be met for manufacturing PTAH drug product. Degree of roasting was determined with a Hunter colorimeter. Protein/allergen content, identity, potency, safety, and quality of each batch of PTAH drug substance were assessed with a combustion analyzer, allergen-specific Western blot (immunoblotting), ELISA, and HPLC. Contaminants (ie, aflatoxin) were measured by UPLC. Results: Roasting degree beyond "light roast" was associated with variable degrees of protein allergen degradation, or potentially aggregation. Relative potency and amounts of protein allergens showed variability due in part to seasonal/manufacturing variability. Proportion of lots not meeting aflatoxin limits has increased in recent years. Up to 60% of peanut flour source material failed to meet screening selection acceptance criteria for proceeding to drug substance testing, mostly because of failure to meet potency acceptance criteria. Other lots were rejected due to safety (ie, aflatoxin) and quality. Influence of potency variation, within specification parameters, on safety/tolerability observed in trials was considered low, in part due to stringent controls placed at each step of manufacturing. Conclusions: Extensive variability in allergen potency is a critical issue during immunotherapy, particularly during OIT initial dose escalation and up-dosing, as it may result in lack of efficacy or avoidable adverse allergic reactions. Based on EU and US regulatory requirements, the production of PTAH includes manufacturing controls to ensure drug product safety, potency, and quality. For example, although PTAH contains all peanut allergens, each lot has met strict criteria ensuring consistent allergenic potency of Ara h 1, Ara h 2, and Ara h 6. The rigor of PTAH's manufacturing process ensures reliable dose consistency and stability throughout its shelf life.

Impact of deglycosylation methods on two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry for proteomic analysis.

Glycosylation is a common post-translational modification that can add complexity to the proteome of many cell types. We used enzymatic and chemical methods of deglycosylation to treat a heavily glycosylated exoproteome sample from the filamentous fungus Trichoderma reesei. Deglycosylated samples were resolved on one-dimensional (1-D) and two-dimensional (2-D) gels in order to determine the effect of deglycosylation on the electrophoresis patterns and on the ability to identify proteins by peptide mass matching using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of in-gel tryptic digests. We found that deglycosylation of the protein sample resulted in different protein patterns on 1-D and 2-D gels, reduced the complexity of gel patterns, and enhanced the protein identification of some proteins via MALDI-TOF-MS. Deglycosylation with trifluoromethanesulfonic acid (TFMS) was found to be more effective than enzymatic treatments. These deglycosylation techniques may be employed in whole proteome analysis to locate glycosylated proteins and assist in their identification by MS.

Role of a ubiquitin-like modification in polarized morphogenesis.

Type I ubiquitin-like proteins constitute a family of protein modifiers. Here we report the identification of a posttranslational protein modifier from Saccharomyces cerevisiae, Hub1. Overexpression of Hub1 resulted in enhanced conjugate formation when its carboxyl-terminal residue was deleted, suggesting that mature Hub1 may be produced by proteolytic processing. In vivo targets of Hub1 conjugation included cell polarity factors Sph1 and Hbt1. In the hub1Delta mutant, the subcellular localization of both Hbt1 and Sph1 was disrupted, and cell polarization during the formation of mating projections was defective. Consistent with these polarization defects, the hub1Delta mutant was deficient in mating.