Thursday, 31 May 2018

Contamination Control in Pharmaceutical Cleanrooms


Cleanrooms have meticulous levels of contamination (ISO Class) specified by the number of particles per cubic meter at a specified particle size. Air entering a cleanroom from the outside must be filtered to exclude dust. The air inside a cleanroom must be constantly recirculated through high-efficiency particulate air (HEPA) filters to control contaminants that are generated inside the room. Particle levels are tested using a particle counter and microorganisms are detected and counted using environmental monitoring methods.

An overview of good cleanroom design is provided by Juan Miguel Cana Lopez and Kim Zurawski, Grifols, in Controlled Environments magazine.

Here is an extract: “Consideration must also be given to cleanroom features such as nonporous and smooth surfaces, including walls and ceilings that can withstand routine decontamination; proper pressure differentials between rooms, the most positive pressure being in the aseptic processing rooms or areas; use of unidirectional airflow in the immediate vicinity of exposed product or components; sufficient air change frequency; appropriate humidity and temperature environmental controls; and a documented sanitization program.”

To access the article, see: Controlled Environments

Posted by Dr. Tim Sandle

Wednesday, 30 May 2018

Catalase test


The catalase test is used to detect the presence of catalase enzyme by the decomposition of hydrogen peroxide to release oxygen and water as shown by the following reaction:

2 H2O2 → 2H2O + O2

The catalase reaction is evident by the rapid formation of bubbles. Hydrogen peroxide is formed by some bacteria as an oxidative end product of the aerobic breakdown of sugars. If allowed to accumulate, it is highly toxic to bacteria and can result in cell death. Catalase either decomposes hydrogen peroxide or oxidises secondary substrates, but it has no effect on other peroxides.

There are method variations of the catalase test and these include the slide test method, the tube or bottle method and the agar slant method. However, the commonly used methods in microbiology laboratories are the tube or bottle method and the agar slant method because it limits catalase aerosols, which have been shown to carry viable bacterial cells, that if inhaled could cause infections as well as contamination in other laboratory work being set up and work surface areas.

Public Health England has opened a consultation on the standard test method. For details see: UK Government Gateway: https://www.gov.uk/government/consultations/uk-smi-tp-8-catalase-test

Posted by Dr. Tim Sandle

Tuesday, 29 May 2018

Sizing Up the Benefits of Sterile Drug Manufacturing Techniques


A comparison of several sterile processing techniques, including aseptic isolators, restricted access barrier systems and aseptic Split Butterfly Valve technology has been undertaken by Christian Dunne, ChargePoint Technology.

The article appears in Pharmaceutical Manufacturing magazine. Here is an extract:

“The aseptic Spilt Butterfly Valve (SBV) provides a safe method of transferring product from one container, process vessel, Isolator or RABS to another while ensuring the sterility of the transfer is not compromised.

Fundamentally, the SBV consists of two halves, the active (Alpha) unit and the passive (Beta) unit. Each half consists of half of the ‘butterfly’ disc. The active unit is attached to the stationary process equipment such as a mixing vessel, while the passive unit is attached to the mobile container such as an intermediate bulk container (IBC) or flexible bag. When the two parts are brought together, the discs halves join to form a single disc, sealing any surfaces which may have been exposed to a compound during transfer. The two discs then operate as one disc and can be opened to allow transfer of product from one location to another.”

To access the article, see: Pharmaceutical Manufacturing

Posted by Dr. Tim Sandle

Monday, 28 May 2018

Impact of draft Annex 1 on cleanrooms


The December 2017 draft of EU GMP Annex 1 carries implications for cleanrooms. In a new article, Tim Sandle assesses the impact on cleanrooms and controlled environments. The article appears in the journal Clean Air and Containment Review.

The abstract for the article is:

Annex 1 of EU GMP has set the standard for sterile products manufacture for over 25 years. A new draft of the Annex was issued in December 2017 for a targeted stakeholder consultation. Given that the last major update to the guidance was almost ten years ago, considerable changes have taken place in terms of technology (the increased use of RABS and isolators, for example) and contamination control philosophies. This article assesses the primary changes set out in the draft Annex, with a focus on cleanrooms and cleanroom technology.”

The reference is:

Sandle, T. (2018) Draft revision of EU GMP Annex 1 Manufacturing of Sterile Medicinal Products for targeted consultation: implications for cleanrooms, Clean Air and Containment Review, Issue 33, pp16-18

Posted by Dr. Tim Sandle

Sunday, 27 May 2018

Deoxyribonuclease test


The test is used primarily as a supplementary presumptive test to distinguish pathogenic staphylococci which produce large quantities of extracellular DNase. The DNase reacts with media containing DNA with the resulting hydrolysis of the DNA. The oligonucleotides liberated by the hydrolysis are soluble in acid and in a positive reaction, the addition of hydrochloric acid results in a clear zone around the inoculum. Due to the precipitation of DNA by hydrochloric acid, in a negative reaction, the solution becomes cloudy. In contrast to hydrochloric acid, toluidine blue produces much more clearly delineated zones of DNase activity.

Most strains of Staphylococcus aureus hydrolyse DNA and give positive reactions in this test, but some MRSA strains do not and some strains of the coagulase negative staphylococci may give weak reactions such as Staphylococcus capitis. Some strains of Staphylococcus intermedius are DNase positive. Subspecies of Staphylococcus schleiferi are DNase positive and produce heat stable nucleases.

This test also aids in the differentiation of closely related genera within the Klebsiella/Enterobacter-Serratia division of Enterobacteriaceae and several other pathogens, including Pseudomonas aeruginosa. Serratia and Moraxella species also produce deoxyribonuclease.

Public Health England has opened a consultation on the standard test method. For details see: UK Government Gateway: https://www.gov.uk/government/consultations/uk-smi-tp-12-deoxyribonuclease-test


Posted by Dr. Tim Sandle

Saturday, 26 May 2018

Coagulase test


Coagulase is a protein enzyme produced by several microorganisms that enables the conversion of fibrinogen to fibrin. Coagulase binds plasma fibrinogen, causing the organisms to agglutinate or plasma to clot. Coagulase exists in two forms: “bound coagulase” (or clumping factor) which is bound to the cell wall and “free coagulase” which is liberated by the cell wall. Bound coagulase is detected by the slide coagulase test, whereas free coagulase is detected by the tube coagulase test.

Bound coagulase adsorbs fibrinogen from the plasma and alters it so it precipitates on the staphylococci, causing them to clump resulting in cell agglutination. The tube coagulase test detects both bound and free coagulase. Free coagulase reacts with a substance in plasma to form a fibrin clot.

Public Health England has opened a consultation on the standard test method. For details see: UK Government Gateway: https://www.gov.uk/government/consultations/uk-smi-tp-10-coagulase-test

Posted by Dr. Tim Sandle

Friday, 25 May 2018

New version of ISO 14024 on ecolabeling


Consumers have high concerns about what they buy and environmental labels and declarations can help them identify those products or services proven “environmentally preferable”.

News from ISO:

“Ecolabelling found its origins in the growing global concern for environmental protection by government, business and the general public. As companies have come to recognize that environmental concerns may be translated into a market advantage for certain products and services, various environmental declarations, claims and labels have emerged, such as natural, recyclable, eco-friendly, low-energy, recycled content, and so forth.

These have exerted a powerful attraction on consumers looking for ways to reduce environmental impacts through their purchasing choices, but they have also led to some confusion and scepticism. Hence why a new version of ISO 14024, Environmental labels and declarations – Type I environmental labelling – Principles and procedures, was needed to help make sense of it all.”

For details, see ISO

Posted by Dr. Tim Sandle

Thursday, 24 May 2018

Pyrogen Detection: Want to stop using animal based methods and get higher sensitivity?

Pyrogen Detection: Want to stop using animal based methods and get higher sensitivity? 


Ready-to-use PyroMAT™ in vitro system 

We are glad to inform you that our new in vitro solution for pyrogen detection is now available: our PyroMAT™ system is the only cell-line based Monocyte Activation Test (MAT) provided as a ready-to-use kit on the market.

The PyroMAT™ system is based on the use of the Mono-Mac-6 cell line: it is designed to provide a standardized reactivity. Once these human monocytes come into contact with pyrogens from a contaminated sample, they produce cytokines such as interleukin-6 (IL-6), which can be detected with enzyme-linked immunosorbent assay (ELISA).

With the PyroMAT™ System, we offer a new robust and sensitive solution for pyrogen detection supported by international regulations and guidelines to reduce animal consumption for pyrogen tests. Make the move to in vitro methods, and benefit from our expertise:

The advantages of monocyte activation test… 

  • Detection of the full range of pyrogens to ensure patient safety. Like the rabbit pyrogen test, MAT is effective in the detection of both endotoxins and Non-Endotoxin Pyrogens (NEP). 
  • Extended range of testable products: the most frequently applied methods are limited in the product types they are able to test. The MAT offers more flexibility regarding its applications. 
  • In vitro assay that mimics the human immune reaction: for a robust predictive model that reduces animal consumption. • Compliance with international regulations and guidelines in line with ethical trends of industry and regulatory authorities to decrease the use of animal based testing. ... Combined with the benefits of using a cell line • Standardized reactivity and high sensitivity (LOD 0.05 EU/mL) 
  • Convenience of a ready-to-use cell line to avoid laborious lab work to maintain the cell line and avoids the need of a culture cell laboratory. • Qualified cells: in addition to being cited in the international validation of MAT, MonoMac-6 cells are qualified for the detection of all types of pyrogens via testing of the expression of all surface Toll-Like Receptors (TLRs). 

Click here to learn more

Mechanism of action of new class of antibiotics in molecular Cell


Nosopharm, a company dedicated to the research and development of new anti-infective drugs, and the University of Illinois at Chicago (UIC) today announce the publication of a study in Molecular Cell on the mechanism of action of odilorhabdins, a new class of antibiotics to combat antibiotic resistance.

The antibiotic, first identified by Nosopharm, is unique and promising on two fronts: its unconventional source and its distinct way of killing bacteria, both of which suggest the compound may be effective at treating drug-resistant or hard to treat bacterial infections.

Called odilorhabdins, or ODLs, the antibiotics are produced by symbiotic bacteria found in soil-dwelling nematode worms that colonize insects for food. The bacteria help to kill the insect and, importantly, secrete the antibiotic to keep competing bacteria away. Until now, these nematode-associated bacteria and the antibiotics they make have been largely understudied.

The odilorhabdin program was launched by Nosopharm in 2011. To identify the antibiotic, Nosopharm’s researchers team screened eighty cultured strains of the bacteria for antimicrobial activity. They then isolated the active compounds, studied their chemical structures and their structure-activity relationships, investigated their pharmacology and engineered more potent derivatives.

The study, published in Molecular Cell, describes the new antibiotic and, for the first time, how it works. Nosopharm’s Maxime Gualtieri, UIC's Alexander Mankin and Yury Polikanov are corresponding authors on the study and led the research on the antibiotic's mechanism of action. They found that ODLs act on the ribosome – the molecular machine of individual cells that makes the proteins it needs to function – of bacterial cells.

"Novel classes of antibiotics like the odilorhabdins are very difficult to discover, but then very interesting to investigate", said Maxime Gualtieri, co-founder and chief scientific officer of Nosopharm. "Our research at Nosopharm is focused on the understanding of the pharmacology of this new class, and the elucidation of their mode of action is a part of this work. We accumulated several early evidences that the bacterial translation was the target of the ODLs. At this point, we needed complementary scientific expertise to investigate much more in detail the mechanism of action of the molecules. This was the purpose of our collaboration with the UIC."

"Like many clinically-useful antibiotics, ODLs work by targeting the ribosome," said Yury Polikanov, assistant professor of biological sciences in the UIC College of Liberal Arts and Sciences, "But ODLs are unique because they bind to a place on the ribosome that has never been used by other known antibiotics."

"When ODLs are introduced to the bacterial cells, they impact the reading ability of the ribosome and cause the ribosome to make mistakes when it creates new proteins," said Alexander Mankin, director of the Center for Biomolecular Sciences in the UIC College of Pharmacy. "This miscoding corrupts the cell with flawed proteins and causes the bacterial cell to die."

While many antibiotics can slow bacterial growth, Mankin says antibiotics that actually kill bacteria, called bactericidal antibiotics, are rare.

"The bactericidal mechanism of ODLs and the fact that they bind to a site on the ribosome not exploited by any known antibiotic are very strong indicators that ODLs have the potential to treat infections that are unresponsive to other antibiotics," said Mankin, who is also professor of medicinal chemistry and pharmacognosy in the UIC College of Pharmacy.

According to the World Health Organization, antimicrobial resistance is one of the biggest threats to global health today and a significant contributor to longer hospital stays, higher medical costs and increased mortality.

In France, the Nosopharm researchers tested the ODL compounds against bacterial pathogens, including many known to develop resistance.

"We found that the ODL compounds cured mice infected with several pathogenic bacteria and demonstrated activity against both Gram-negative and Gram-positive pathogens, notably including carbapenem-resistant Enterobacteriacae," said co-corresponding author Maxime Gualtieri, co-founder and chief scientific officer of Nosopharm.

Carbapenem-resistant Enterobacteriacae, or CRE, are a family of germs that have high levels of resistance to antibiotics. One study suggests that CRE, which are the common culprits in bloodstream and surgical site infections, contribute to death in up to 50 percent of patients who become infected.

The researchers say this study is a testament to the growing trend of international and cross-disciplinary collaboration, which is needed to combat the growing and global threat of antibiotic resistance.

"As a biotech company, Nosopharm has to focus on the pharmaceutical development of the ODLs,” said Philippe Villain-Guillot, co-founder and chief executive officer of Nosopharm. “Collaborations with academia with renown expertise in antibiotics like the UIC team help us for this preclinical development and add credibility to the research".

NOSO-502, the first clinical candidate of the odilhorhabdins, is the most advanced molecule in Nosopharm’s portfolio. The company plans to begin the first clinical trials in humans in 2020.

Wednesday, 23 May 2018

Sugar-Coated Nanosheets Hit Specific Pathogen Targets


Researchers have developed a process for creating ultrathin, self-assembling sheets of synthetic materials that can function like designer flypaper in selectively binding with viruses, bacteria, and other pathogens.

The sheets were designed to present simple sugars in a patterned way along their surfaces, and these sugars, in turn, were demonstrated to selectively bind with several proteins, including one associated with the Shiga toxin, which causes dysentery. Because the outside of our cells are flat and covered with sugars, these 2-D nanosheets can effectively mimic cell surfaces.

The nanosheets could also potentially be used in environmental cleanups to neutralize specific toxins and pathogens, and the sheets could potentially be scaled to target viruses like Ebola and bacteria like E. coli, and other pathogens.

In the latest study, the researchers confirmed that the bindings with the targeted proteins were successful by embedding a fluorescent dye in the sheets and attaching another fluorescent dye on the target proteins. A color change indicated that a protein was bound to the nanosheet.

The intensity of this color change can also guide researchers to improve them, and to discover new nanosheets that could target specific pathogens.

See: ACS Nano “Glycosylated Peptoid Nanosheets as a Multivalent Scaffold for Protein Recognition.”

Posted by Dr. Tim Sandle

Tuesday, 22 May 2018

Cleanroom Cleaning and Disinfection: Eight Steps for Success

The CDC Handbook: A Guide to Cleaning and Disinfecting Cleanrooms


Cleanrooms in healthcare and pharmaceutical facilities must be kept in a state of microbiological control. This article outlines eight key steps for keeping a cleanroom clean.

Cleanrooms in healthcare and pharmaceutical facilities must be kept in a state of microbiological control. This is achieved in a number of ways, including the physical operation of Heating, Ventilation, and Air Conditioning (HVAC) systems, control of materials, properly gowned and trained personnel, and through the use of defined cleaning techniques, together with the application of detergents and disinfectants.

The object of cleaning and disinfection is to achieve appropriate microbiological cleanliness levels for the class of cleanroom for an appropriate period of time. Thus the cleaning and disinfection of cleanrooms is an important part of contamination control.

This article examines the eight key steps to be followed, in relation to cleaning and disinfection, in helping to keep cleanrooms “clean.”

See Tim Sandle’s article in Controlled Environments magazine here.



Posted by Dr. Tim Sandle

Monday, 21 May 2018

Risk considerations for the installation of a new pharmaceutical facility autoclave


Tim Sandle has written an article for the website sterilize.it, the article covers:

This article considers some of the risk considerations that need to be accounted for when replacing a steam sterilization autoclave within a pharmaceutical processing facility. In outlining the key risk considerations, the article adopts a risk assessment approach – Failure Modes and Effects Analysis (FMEA).

Risk management and risk assessment principles should be applied as early as possible during the design and construction of steam sterilization devices. With steam sterilization devices the most critical functions are, arguably, the steam sterilization of direct and indirect the product contact parts. A second important aspect relates to air removal. All of the trapped air must be removed from the autoclave before activation. This is because trapped air is a very poor medium for achieving sterility.

The reference is:

Sandle, T. (2018) Risk considerations for the installation of a new pharmaceutical facility autoclave, Sterilize.IT at: https://www.sterilize.it/process/risk-considerations-for-the-installation-of-a-new-pharmaceutical-facility-autoclave/

Posted by Dr. Tim Sandle

Sunday, 20 May 2018

Artificial Enzymes Use Light to Kill Bacteria


Researchers at RMIT University have developed a new artificial enzyme that uses light to kill bacteria. The artificial enzymes could one day be used in the fight against infections, and to keep high-risk public spaces like hospitals free of bacteria like E coli and Golden Staph.

Made from tiny nanorods — 1,000 times smaller than the thickness of the human hair — the “NanoZymes” use visible light to create highly reactive oxygen species that rapidly break down and kill bacteria.

Lead researcher Professor Vipul Bansal said: “For a number of years we have been attempting to develop artificial enzymes that can fight bacteria, while also offering opportunities to control bacterial infections using external 'triggers' and 'stimuli'. Now we have finally cracked it.”


He adds: “Our NanoZymes are artificial enzymes that combine light with moisture to cause a biochemical reaction that produces OH radicals and breaks down bacteria. Nature’s antibacterial activity does not respond to external triggers such as light. We have shown that when shined upon with a flash of white light, the activity of our NanoZymes increases by over 20 times, forming holes in bacterial cells and killing them efficiently. This next generation of nanomaterials are likely to offer new opportunities in bacteria free surfaces and controlling spread of infections in public hospitals.”

The NanoZymes work in a solution that mimics the fluid in a wound. This solution could be sprayed onto surfaces. The NanoZymes are also produced as powders to mix with paints, ceramics, and other consumer products. This could mean bacteria-free walls and surfaces in hospitals.

While the NanoZymes currently use visible light from torches or similar light sources, in the future they could be activated by sunlight. The researchers have shown that the NanoZymes work in a lab environment. The team is now evaluating the long-term performance of the NanoZymes in consumer products.

See: the journal ACS Applied Nano Materials “Visible-Light-Triggered Reactive-Oxygen-Species-Mediated Antibacterial Activity of Peroxidase-Mimic CuO Nanorods.”

Posted by Dr. Tim Sandle

Saturday, 19 May 2018

The CDC's Plan to Combat 'Nightmare' Bacteria


One of the biggest threats comes from carbapenemases, molecules that can make microbes impervious to carbapenems, which are some of the most potent antibiotics. The Enterobacteriaceae family of bacteria is the one that causes the most infections in hospitals, and that family began to pick up resistance to carbapenems. In 1988 these microbes, called CRE for carbapenem-resistant Enterobacteriaceae, were first identified. Plasmids spread carbapenem resistance, and efficiently enough that by 2001, the carbapenem-resistant bacteria were becoming impervious to other antibiotics as well. It became evident that more aggressive strategies were needed.

The Centers for Disease Control and Prevention (CDC) issued directives in 2009 to confront the problem, and there were updates in 2013 and 2015. Now the CDC wanted to survey the state of antibiotic resistance in the United States. Data from 2006 to 2015 from the National Healthcare Safety Network showed that there was a decline in the number of CRE infections in hospitals.

The report concludes: “The proportion of Enterobacteriaceae infections that were CRE remained lower and decreased more over time than the proportion that were ESBL phenotype. This difference might be explained by the more directed control efforts implemented to slow transmission of CRE than those applied for ESBL-producing strains. Increased detection and aggressive early response to emerging antibiotic resistance threats have the potential to slow further spread.”

See: Morbidity and Mortality Weekly Report (MMWR) “Vital Signs: Containment of Novel Multidrug-Resistant Organisms and Resistance Mechanisms — United States, 2006–2017.”


Posted by Dr. Tim Sandle

Friday, 18 May 2018

Determining ages of different microbial groups


To learn about the past, paleontologists turn to the fossil record. The occurrence, abundance and diversity of fossils provides a window into the evolutionary history of animal and plant groups, anchoring them in absolute geological time.

But the fossil record is almost no good at all for microbial, single-celled life. Microbes rarely fossilise and, with a few notable exceptions, the available fossils are too indistinct to reveal which groups were already in existence at a particular time.

This is a major problem for students of evolutionary history, because almost all of life's genetic, biochemical and metabolic diversity is microbial -- both today and in the distant past.

While most microbes are invisible to the naked eye, their collective action in recycling nutrients, producing the oxygen we need to breathe, and maintaining the stability of global ecosystems is impossible to ignore.

Microbial dominance was, if anything, even higher in the past. The most familiar groups of large, multicellular life forms that exist today, animals, plants and fungi, are relative newcomers in evolutionary terms, evolving within the last half-billion years or so.

The researchers have developed a new method for working out the relative ages of microbial groups -- which lineages evolved first, and which came later?

Instead of using fossil dates, the method works by looking at events of horizontal gene transfer among ancient microbes, which can be detected by studying the genomes of their modern descendants.

Horizontal gene transfer is a process that many microbes use to obtain new genes from other cells living in the same habitat and it underlies the rapid spread of antibiotic resistance, but is also a more general way in which microbes can adapt to new ecological niches.

See:

Adrián A. Davín, Eric Tannier, Tom A. Williams, Bastien Boussau, Vincent Daubin, Gergely J. Szöllősi. Gene transfers can date the tree of life. Nature Ecology & Evolution, 2018; DOI: 10.1038/s41559-018-0525-3

Posted by Dr. Tim Sandle

Thursday, 17 May 2018

Yeast engineered to manufacture complex medicine


Bioengineers have figured out a way to make noscapine, a non-narcotic cough suppressant that occurs naturally in opium poppies, in brewer's yeast.

The researchers inserted 25 foreign genes into the one-celled fungus to turn it into an efficient factory for producing the drug. Many of the inserted genes came from the poppy, but several came from other plants and even from rats. All those genes were recipes for enzymes: protein machines that, working together, can build complex substances from simple starting materials.

The researchers also modified some of the plant, rat and yeast genes, as well as the medium in which the yeast proliferates, to help everything work better together. The result was an 18,000-fold improvement in noscapine output, compared with what could be obtained by just inserting the plant and rat genes into yeast.

See:

Yanran Li, Sijin Li, Kate Thodey, Isis Trenchard, Aaron Cravens, Christina D. Smolke. Complete biosynthesis of noscapine and halogenated alkaloids in yeast. Proceedings of the National Academy of Sciences, 2018; 201721469 DOI: 10.1073/pnas.1721469115

Posted by Dr. Tim Sandle

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