PDE concept for controlling cleaning agent residues in pharmaceuticals- A critical analysis.

Cleaning agents (CAs) are used in multipurpose facilities to control carryover contamination of active pharmaceutical ingredients (APIs) to scientifically justified limits. While this is often done with the PDE methodology used for API impurities, it is unclear if it is justifiable and necessary for cleaning agents, which generally represent a comparatively lower health risk. Comparing calculated oral PDE values for CA ingredients (CAIs) from four companies with PDEs of a selected number of small-molecule APIs showed that the toxicity of CAIs is several orders of magnitude lower. Furthermore, a critical review of the toxicity and everyday exposure to the general population of the main CAIs functional groups showed that the expected health risks are generally negligible. This is particularly true if the associated mode of actions cause local toxicity that is usually irrelevant at the concentration of potential residue carryover. This work points towards alternative approaches to the PDE concept to control CAIs' contamination and provides some guidance on grouping and identifying compounds with lower health risks based on exposure and mode of action reasoning. In addition, this work supports the concept that limit values should only be set for CAIs of toxicological concern.

Isolation and characterization of type IIS restriction endonuclease from Neisseria cuniculi ATCC 14688.

Neisseria cuniculi produces the restriction enzyme NcuI which is an isoschizomer of MboII. We have demonstrated that NcuI recognizes a pentanucleotide sequence (5'-GAAGA-3'/3'-CTTCT-5'), and cleaves the DNA 8 and 7 nucleotides downstream from the recognition site leaving a single 3'-protruding nucleotide. We have purified this enzyme to electrophoretic homogeneity using a four-step chromatographic procedure. NcuI endonuclease is a monomeric protein with a M(r)=48,000+/-1000 under denaturing conditions. The properties of NcuI are consistent with those for MboII, the position of the cleavage site being identical and the pH profile and divalent cation requirements being similar. Moreover, NcuI cross-reacts strongly with anti-MboII serum suggesting the presence of similar antigenic determinants. We have determined the sequence of 20 N-terminal amino acids for NcuI and concluded that this sequence is identical to the N-terminal portion of the MboII enzyme.