A QRM Discussion of Microbial Contamination of Non-sterile Drug Products, Using FDA and EMA Warning Letters Recorded between 2008 and 2016.

Microbial quality control of non-sterile drug products has been a concern to regulatory agencies and the pharmaceutical industry since the 1960s. Despite being an old challenge to companies, microbial contamination still affects a high number of manufacturers of non-sterile products. Consequences go well beyond the obvious direct costs related to batch rejections or product recalls, as human lives and a company's reputation are significantly impacted if such events occur. To better manage risk and establish effective mitigation strategies, it is necessary to understand the microbial hazards involved in non-sterile drug products manufacturing, be able to evaluate their potential impact on final product quality, and apply mitigation actions. Herein we discuss the most likely root causes involved in microbial contaminations referenced in warning letters issued by US health authorities and non-compliance reports issued by European health authorities over a period of several years. The quality risk management tools proposed were applied to the data gathered from those databases, and a generic risk ranking was provided based on a panel of non-sterile drug product manufacturers that was assembled and given the opportunity to perform the risk assessments. That panel identified gaps and defined potential mitigation actions, based on their own experience of potential risks expected for their processes. Major findings clearly indicate that the manufacturers affected by the warning letters should focus their attention on process improvements and microbial control strategies, especially those related to microbial analysis and raw material quality control. Additionally, the WLs considered frequently referred to failures in quality-related issues, which indicates that the quality commitment should be reinforced at most companies to avoid microbiological contaminations.LAY ABSTRACT: Microbial contamination of drug products affects the quality of non-sterile drug products produced by numerous manufacturers, representing a major risk to patients. It is necessary to understand the microbial hazards involved in the manufacturing process and evaluate their impact on final product quality so that effective prevention strategies can be implemented. A risk-based classification of most likely root causes for microbial contamination found in the warning letters issued by the US Food and Drug Administration and the European Medicines Agency is proposed. To validate the likely root causes extracted from the warning letters, a subject matter expert panel made of several manufacturers was formed and consulted. A quality risk management approach to assess microbiological contamination of non-sterile drug products is proposed for the identification of microbial hazards involved in the manufacturing process. To enable ranking of microbial contamination risks, quality risk management metrics related to criticality and overall risk were applied. The results showed that manufacturers of non-sterile drug products should improve their microbial control strategy, with special attention to quality controls of raw materials, primary containers, and closures. Besides that, they should invest in a more robust quality system and culture. As a start, manufacturers may consider investigating their specific microbiological risks, adressing their sites' own microbial ecology, type of manufacturing processes, and dosage form characteristics, as these may lead to increased contamination risks. Authorities should allow and enforce innovative, more comprehensive, and more effective approaches to in-process contamination monitoring and controls.

Phylogenetic and clonality analysis of Bacillus pumilus isolates uncovered a highly heterogeneous population of different closely related species and clones.

Bacillus pumilus is a Gram-positive bacterium with a wide range of attributed applications, namely as a plant growth promoting rhizobacteria (PGPR), animal, and human probiotic. However, a rare putative role in human diseases has been reported, namely in food poisoning or as anthrax-like cutaneous infectious agent. This species is difficult to distinguish from its closely related species on the basis of phenotypic or biochemical characteristics and 16S rRNA gene sequences. In this study, the phylogenetic analysis of gyrB and rpoB gene sequences of a collection of isolates previously identified as B. pumilus, assigned most of them (93%, 38 of 41 isolates) to B. safensis or to the new recently described B. invictae. Moreover, we extended the previously reported recognized habitats of these species and unveiled a human health or biotechnological relevance (e.g. as implicated in food poisoning or PGPR) for them. Additionally, we demonstrated that both B. safensis and B. invictae species encompass a clonally diverse population, which can justify their great adaptation ability to different niches, with evidence of clonal-host specificity.

Bacillus invictae sp. nov., isolated from a health product.

A Gram-positive, rod-shaped, endospore-forming Bacillus isolate, Bi.(FFUP1) (T), recovered in Portugal from a health product was subjected to a polyphasic study and compared with the type strains of Bacillus pumilus, Bacillus safensis, Bacillus altitudinis and Bacillus xiamenensis, the phenotypically and genotypically most closely related species. Acid production from cellobiose, D-glucose and D-mannose and absence of acid production from D-arabinose, erythritol, inositol, maltose, mannitol, raffinose, rhamnose, sorbitol, starch and L-tryptophan discriminated this new isolate from the type strains of the most closely related species. Additionally, a significant different protein and carbohydrate signature was evidenced by spectroscopic techniques, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and Fourier transform IR spectroscopy with attenuated total reflectance. Using a chemometric approach, the score plot generated by principal component analysis clearly delineated the isolate as a separate cluster. The quinone system for strain Bi.(FFUP1) (T) comprised predominantly menaquinone MK-7 and major polar lipids were diphosphatidylglycerol, an unidentified phospholipid and an unidentified glycolipid. Strain Bi.(FFUP1) (T) showed ≥ 99% 16S rRNA gene sequence similarity to B. safensis FO-036b(T), B. pumilus (7061(T) and SAFR-032), B. altitudinis 41KF2b(T) and B. xiamenensis HYC-10(T). Differences in strain Bi.FFUP1 (T) gyrB and rpoB sequences in comparison with the most closely related species and DNA-DNA hybridization experiments with Bi.FFUP1 (T) and B. pumilus ATCC 7061(T), B. safensis FO-036b(T), B. altitudinis 41KF2b(T) and B. xiamenensis HYC-10(T) gave relatedness values of 39.6% (reciprocal 38.0%), 49.9% (reciprocal 42.9%), 61.9% (reciprocal 52.2%) and 61.7% (reciprocal 49.2%), respectively, supported the delineation of strain Bi.(FFUP1) (T) as a representative of a novel species of the genus Bacillus, for which the name Bacillus invictae sp. nov. is proposed, with strain Bi.(FFUP1) (T) ( =DSM 26896(T) =CCUG 64113(T)) as the type strain.

Identification of carbapenem-resistant Acinetobacter baumannii clones using infrared spectroscopy.

In this work we assessed the discriminatory ability of Fourier-transform Infrared Spectroscopy (FTIR) in 22 representative isolates from a collection of 318 carbapenem-hydrolyzing class D ß -lactamases (CHDL)-producing Acinetobacter spp. (5 hospitals; 2001-2008) previously characterized by DNA-based typing methods. FTIR spectra were acquired with a Bruker spectrometer and analyzed with support of several chemometric tools. The results showed that FTIR spectroscopy was able to distinguish the main CHDL-producing Acinetobacter baumannii lineages causing infection in Portugal, the ST103 carrying blaOXA-58 , ST98 carrying blaOXA-24/40 and ST92 carrying blaOXA-23 . Moreover, this study revealed distinctive phenotypic features of A. baumannii lineages causing infections that might justify different epidemic potential. Spectroscopy may arise as a low cost and easily to perform alternative for typing A. baumannii isolates.

Burkholderia cenocepacia J2315 acyl carrier protein: a potential target for antimicrobials' development?

This work describes the isolation and characterization of an acyl carrier protein (ACP) mutant from Burkholderia cenocepacia J2315, a strain of the Burkholderia cepacia complex (Bcc). Bcc comprises at least 9 species that emerged as opportunistic pathogens able to cause life-threatening infections, particularly severe among cystic fibrosis patients. Bacterial ACPs are the donors of the acyl moiety involved in the biosynthesis of fatty acids, which play a central role in metabolism. The mutant was found to exhibit an increased ability to form biofilms in vitro, a more hydrophobic cell surface and reduced ability to colonize and kill the nematode Caenorhabditis elegans, used as a model of infection. The B. cenocepacia J2315 ACP protein is composed of 79 amino acid residues, with a predicted molecular mass and pI of 8.71kDa and 4.08, respectively. The ACP amino acid sequence was found to be 100% conserved within the genomes of the 52 Burkholderia strains sequenced so far. These data, together with results showing that the predicted structure of B. cenocepacia J2315 ACP is remarkably similar to the Escherichia coli AcpP, highlight its potential as a target to develop antibacterial agents to combat infections caused not only by Bcc species, but also by other Burkholderia species, especially B. pseudomallei and B. mallei.

Exceptionally high representation of Burkholderia cepacia among B. cepacia complex isolates recovered from the major Portuguese cystic fibrosis center.

Burkholderia cepacia, a species found infrequently in cystic fibrosis (CF), was isolated from 85% of patients infected with bacteria of the B. cepacia complex that visited the major Portuguese CF center, in Lisbon, during 2003 to 2005. A detailed molecular analysis revealed that this was mainly due to two B. cepacia clones. These clones were indistinguishable from two strains isolated from intrinsically contaminated nonsterile saline solutions for nasal application, detected during routine market surveillance by the Portuguese Medicines and Health Products Authority.