Sunday, 28 May 2017

Biosimilars (EDQM News)

EDQM Biosimilars: Ph. Eur. monographs are flexible and evolving standards During a seminar co-organised with the European Medicines Agency (EMA), the EDQM clarified further the role that Ph. Eur. monographs play in the assessment of biosimilars. As public standards for the quality of medicines in Europe, monographs ensure the quality of biosimilar and other biotherapeutic products, but compliance with them is not sufficient for demonstrating biosimilarity.

However, while Ph. Eur. monographs provide specifications in the form of tests and acceptance criteria for all medicines, they are dynamic documents that can be adapted to scientific progress.

Dr Peter Richardson, Head of Quality at the EMA, provided information on EU legislation in the field of biosimilars, and Dr Niklas Ekman, Senior Researcher at the Finnish Medicines Agency (FIMEA) shared his experience as an assessor.

See: biosimilars seminar from which a video recording of the EDQM-EMA session on biosimilars can be viewed

Posted by Dr. Tim Sandle

Saturday, 27 May 2017

Synthetic biologists engineer inflammation-sensing gut bacteria

Synthetic biologists at Rice University have engineered gut bacteria capable of sensing colitis, an inflammation of the colon, in mice. The research points the way to new experiments for studying how gut bacteria and human hosts interact at a molecular level and could eventually lead to orally ingestible bacteria for monitoring gut health and disease.

The research, published in a new study in Molecular Systems Biology, involved a series of breakthroughs in the lab of Jeffrey Tabor, assistant professor of bioengineering and of biosciences at Rice, and key contributions from collaborators Robert Britton and Noah Shroyer at Baylor College of Medicine. Tabor's team, including lead co-author and postdoctoral researcher Kristina Daeffler, identified the first genetically encoded sensor of a novel biomarker linked to inflammation, inserted the genes for the sensor into a well-studied gut bacterium and collaborated with Shroyer and Britton to use the engineered bacteria to detect colon inflammation in mice.

"The gut harbors trillions of microorganisms that play key roles in health and disease," Tabor said. "However, it is a dark and relatively inaccessible place, and few technologies have been developed to study these processes in detail. On the other hand, bacteria have evolved tens of thousands of genetically encoded sensors, many of which sense gut-linked molecules. Thus, genetically engineered sensor bacteria have tremendous potential for studying gut pathways and diagnosing gut diseases."

Synthetic biologists like Tabor specialize in programming single-celled organisms like bacteria in much the same way an engineer might program a robot. In particular, Tabor's team is working to develop bacterial sensors that can detect disease signals in the gut. Like electrical engineers who build circuits from wires and electronic components, Tabor's team uses genetic circuits to program single-celled creatures to carry out complex information processing.

Previous work has suggested that alterations to the gut microbiota, genetic predisposition and other environmental factors may play key roles in inflammatory bowel disease, a condition that includes Crohn's disease and ulcerative colitis and which affects as many as 1.6 million Americans.

"Based on a number of previous studies, we hypothesized that the molecule thiosulfate may be elevated during colitis," Daeffler said. "It has been difficult for scientists to study this link because there aren't tools for reliably measuring thiosulfate in living animals. Our first goal in this project was to engineer such a tool."

From the outset of the project in 2015, Daeffler said, the idea was to use sensor bacteria, in this case an engineered form of Escherichia coli, to sense thiosulfate and related sulfur-containing compounds that may also be biomarkers of colitis. There were well-understood methods for programming E. coli to produce a fluorescent green protein in response to specific stimuli, but there were no known genes -- in any organism -- that were used to sense thiosulfate, and few for the other compounds.

"There's a link between gut sulfur metabolism and inflammation, and we knew that we needed to be able to measure sulfur metabolites accurately to diagnose colon inflammation," she said.

Tabor said study co-author Ravi Sheth, an undergraduate researcher in the group in 2015, used a computer program to identify potential sensors of thiosulfate and other sulfur compounds in the genome of Shewanella, a type of bacteria that live in marine sediment. Tabor's group believes that Shewanella likely breathe these molecules and use the sensors to turn on the proper enzymes in their presence.

Daeffler spent one year engineering E. coli to express the sensor genes, validate their function and optimize them to respond to the potential biomarkers by producing a green fluorescent protein signal. It took another year to prove that the system worked and detected colon inflammation in mice.

The researchers administered orally two drops containing about a billion sensor bacteria to both healthy mice and to mice with colitis. They measured the activity of the sensor bacteria in each group six hours later. The tell-tale green fluorescent protein showed up in the feces of the mice. Though it was not visible to the unaided eye, it could easily be measured with a standard laboratory instrument called a flow cytometer.

The team found that the thiosulfate sensor was activated in the mice with inflammation, and was not activated in the healthy mice. Furthermore, the researchers found that the more inflammation the mouse had, the more the sensor was activated.

Tabor said the study shows that gut bacteria can be outfitted with engineered sensors and used to noninvasively measure specific metabolites and that this result could open the door to many new studies that could help elucidate a wide range of gut processes.

Though it would likely take several additional years of development, and it remains unknown if thiosulfate is a biomarker of human colitis, the sensor bacteria might eventually be re-engineered to function as a diagnostic of human colitis, Tabor said. In particular, the green fluorescent protein could be replaced with an enzyme that makes a colored pigment.

"We'd like to develop a home inflammation test where a person prone to colitis flare-ups would eat yogurt that contained the engineered bacteria and see blue pigment in the toilet if they were sick," he said.

Tabor said such a test could reduce unneeded and costly trips to the doctor and unneeded colonoscopy procedures, which are both expensive and invasive. He said his team has begun collaborations with gastroenterologists at Baylor to achieve this goal.


Kristina NM Daeffler, Jeffrey D Galley, Ravi U Sheth, Laura C OrtizVelez, Christopher O Bibb, Noah F Shroyer, Robert A Britton, Jeffrey J Tabor. Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation. Molecular Systems Biology, 2017; 13 (4): 923 DOI: 10.15252/msb.20167416

Posted by Dr. Tim Sandle

Friday, 26 May 2017

EMA: risk based prevention of cross contamination in production

The European Medicines Agency has issued a new document, with the lengthy title of “Questions and answers on implementation of risk based prevention of cross contamination in production and ‘Guideline on setting health based exposure limits for use in risk identification in the manufacture of different medicinal products in shared facilities’ (EMA/CHMP/CVMP/SWP/169430/2012).”

The document deals with highly hazardous products are those that can cause serious adverse effects at low doses and that therefore would benefit from a full toxicological assessment in order to derive a safe Health Based Exposure Limits. A health based exposure limit is a limit (permitted daily exposure (PDE) or equivalent) at which a product is regarded to be safe in humans. It can be based on either clinical dose or non-clinical safety data, depending on which would give the lower exposure limit.

Such products are:

  1. Genotoxic (specifically mutagenic) compounds that are known to be, or highly likely to be, carcinogenic to humans. Compounds of this group are easily identifiable, since genotoxicity would be related to the pharmacology, e.g. as DNA alkylating cytostatics, and their use is usually restricted to oncology indications with respective warning statements in the Summary of Product Characteristics.
  2. Compounds that can produce reproductive and/or developmental effects at low dosages, for example where evidence exists of such effects being caused by a clinical dose of <10 mg/day (veterinary dose equivalent 0.2 mg/kg/day) or dosages in animal studies of ≤1 mg/kg/day.
  3. Compounds that can produce serious target organ toxicity or other significant adverse effects at low doses, for example where evidence exists of such effects being caused by a clinical dose of <10 mg/day (veterinary dose equivalent 0.2 mg/kg/day) or dosages in animal studies of ≤1 mg/kg/day.
  4. Compounds with a high pharmacological potency i.e. recommended daily dose of <1 mg (veterinary dose equivalent 0.02 mg/kg)

Compounds with a high sensitising potential.

The document can be accessed here: EMA

Posted by Dr. Tim Sandle

Thursday, 25 May 2017

New assay for bioterrorism threat

Researchers in Spain are using RPA as the foundation for a highly sensitive and specific solid-phase optical assay that can detect the potential biowarfare agent, Y. pestis, in less than an hour. The enzyme-linked oligonucleotide assay (ELONA) approach developed by Ioanis Katakis and Ciara K. O’Sullivan, at the Universitat Rovira i Virgili’s Interfibio Research Group, and the ICREA (Catalan Institution for Research and Advanced Studies), uses conventional PCR primers to amplify both single- and double-stranded Y. pestis DNA. Their work provides proof of concept for applying RPA in a heterogeneous format, with one primer immobilized onto a solid surface. The researchers aim to further develop the technology into an integrated, portable lateral flow-type test device for the rapid amplification and detection of Y. pestis in resource limited and field settings.

For further details see:

Researchers uncover how bacteria burst our cells

Scientists based in Vienna unveil the complex molecular structure that causes lethal infections by Mycobacterium tuberculosis (Mtb). Their findings might have implications for potential therapies against antibiotic-resistant tuberculosis. The researchers have described the overall architecture of an assembly of proteins known as Type VII (T7SS) secretion systems found in a group of bacteria which cause diseases such as tuberculosis.

T7SS-systems play a key role in tuberculosis infections and might present important targets for much needed new drugs: blocking these systems could prevent the bacteria from bursting the host cells and could thus alleviate the infection.

In addition to the core body of T7SS, some of the proteins extend down into the bacterial cell. The team collected Small Angle X-ray Scattering (SAXS) data at the EMBL SAXS beamline on the DESY campus in Hamburg to help understand what they look like and how these parts of the secretion system might move. "We believe these arm-like proteins help to move the molecules of different shapes and sizes from the inside of the bacterial cell towards the pore of the secretion system for them to be transported out of the cell," says first author Kate Beckham from EMBL Hamburg.

Now further biochemical and genetic experiments will be carried out to support the structural data and to provide in vivo insights into the components required for assembly of the T7 secretion system.


Katherine S. H. Beckham, Luciano Ciccarelli, Catalin M. Bunduc, Haydyn D. T. Mertens, Roy Ummels, Wolfgang Lugmayr, Julia Mayr, Mandy Rettel, Mikhail M. Savitski, Dmitri I. Svergun, Wilbert Bitter, Matthias Wilmanns, Thomas C. Marlovits, Annabel H. A. Parret, Edith N. G. Houben. Structure of the mycobacterial ESX-5 type VII secretion system membrane complex by single-particle analysis. Nature Microbiology, 2017; 2: 17047 DOI: 10.1038/nmicrobiol.2017.47

Posted by Dr. Tim Sandle

Wednesday, 24 May 2017

Too much pressure: a behavioral approach to Data Integrity

The MHRA has published an interesting article on the behavioral factors that shape data integrity.

Data integrity refers to maintaining and assuring the accuracy and consistency of data over its entire life-cycle, and is a critical aspect to the design, implementation and usage of any system which stores, processes, or retrieves data.

Here is an extract:

“Implementing a quality culture and ensuring job satisfaction is easier said than done, but relatively simple actions taken by management can make big differences in everyday operations.  Informal senior management visits to the shop floor enable an understanding of operational issues which are invisible from the boardroom. The ability of an individual to justify their decision to manipulate data can be reduced by making sure that all employees have ‘visibility to the patient’ and understand the impact of their actions. Senior management has the power to fix problematic test methods from the beginning, upgrade outdated equipment and software, encourage open reporting of deviations, and reward good behavior rather than speed.”

The full article can be accessed here.

Tim Sandle’s article on data integrity for the microbiology laboratory can be read here.

Posted by Dr. Tim Sandle

Tuesday, 23 May 2017

How a beneficial gut microbe adapted to breast milk

Breast milk provides vital nutrients not only to infants, but also to beneficial microbes that inhabit the gastrointestinal tract. A new study shows that a bacterial species called Bifidobacterium longum has successfully adapted to the unique niche of the infant gut by producing an enzyme called LnbX, which enables this microbe to grow on a sugar that is abundant only in human milk.

Gut microbes in early life are thought to have long-lasting effects on human health, and studies have shown that diet strongly influences the composition of this population. For example, human milk sugars are known to selectively promote the growth of beneficial gut microbes such as Bifidobacteria, which prevent diarrhea and pathogenic infection in infants. One major component of human milk is a sugar called lacto-N-tetraose, which is virtually absent in the milk of other mammals. Bifidobacteria produce enzymes that break down this sugar, strongly suggesting that a symbiotic relationship recently evolved between these microorganisms and humans.

While investigating how this symbiotic relationship evolved, Katayama and co-senior study author Shinya Fushinobu of the University of Tokyo previously characterized LnbB and isolated LnbX -- enzymes that degrade lacto-N-tetraose in Bifidobacterium bifidum and Bifidobacterium longum, respectively. In the new study, the researchers set out to build on these findings by determining the X-ray crystal structure of the catalytic domain of LnbX. The crystal structure, in combination with mutation and pharmacological experiments, revealed that LnbX has a distinct structure and catalytic mechanism from LnbB and therefore belongs to a novel family of glycoside hydrolase enzymes called GH136.

For further details see:

Chihaya Yamada, Aina Gotoh, Mikiyasu Sakanaka, Mitchell Hattie, Keith A. Stubbs, Ayako Katayama-Ikegami, Junko Hirose, Shin Kurihara, Takatoshi Arakawa, Motomitsu Kitaoka, Shujiro Okuda, Takane Katayama, Shinya Fushinobu. Molecular Insight into Evolution of Symbiosis between Breast-Fed Infants and a Member of the Human Gut Microbiome Bifidobacterium longum. Cell Chemical Biology, 2017; DOI: 10.1016/j.chembiol.2017.03.012

Posted by Dr. Tim Sandle

Monday, 22 May 2017

Pharmig Guide to Cleanrooms

Pharmig has issued a new publication relating to cleanrooms. The guide covers good cleanroom design (together with suitable operational parameters), cleanroom specifications, microbial control, and classification according to the international cleanroom standard ISO 14644 together with EU GMP. The guide includes practical information, including reference to risk assessment.


Sandle, T. (2017) Pharmig Guide to Cleanroom Operation and Contamination Control, Pharmig, Stanstead Abbotts, UK

For further information, see: Pharmig

Also purchase:

Posted by Dr. Tim Sandle

Sunday, 21 May 2017

Giant viruses found in Austrian sewage

Giant viruses are characterized by disproportionately large genomes and virions that house the viruses' genetic material. They can encode several genes potentially involved in protein biosynthesis, a unique feature which has led to diverging hypotheses about the origins of these viruses. But after discovering a novel group of giant viruses with a more complete set of translation machinery genes than any other virus known to date, scientists at the U.S. Department of Energy Joint Genome Institute (DOE JGI), a DOE Office of Science User Facility, believe that this group (dubbed "Klosneuviruses") significantly increases our understanding of viral evolution. Thus the Klosneuviruses contradict the theory that viruses make up a distinct domain of life.
The predicted hosts for the Klosneuviruses are protists (single-celled eukaryotic (nucleus-containing) microorganisms) and while their direct impacts on protists are not yet worked out, these giant viruses are thought to have a large impact on these protists that help regulate the planet's biogeochemical cycles.

Scientists have been fascinated by giant viruses since 2003, when a group of French biologists led by Didier Raoult discovered the Mimiviruses. Since then, a handful of other giant virus groups have been found. The unique ability among them to encode proteins involved in translation (typically DNA to RNA to protein) piqued researchers' interests as to the origin of giant viruses. Since then, two evolutionary hypotheses have emerged. One posits that giant viruses evolved from an ancient cell, perhaps one from an extinct fourth domain of cellular life. Another -- a scenario championed by Koonin -- presents the idea that giant viruses arose from smaller viruses.

The discovery of Klosneuvirus supports the latter idea, according to Tanja Woyke, DOE JGI Microbial Genomics Program lead and senior author of the paper. "In this scenario, a smaller virus infected different eukaryote hosts and picked up genes encoding translational machinery components from independent sources over long periods of time through piecemeal acquisition," she said.

For further details see:

Frederik Schulz, Natalya Yutin, Natalia N. Ivanova, Davi R. Ortega, Tae Kwon Lee, Julia Vierheilig, Holger Daims, Matthias Horn, Michael Wagner, Grant J. Jensen, Nikos C. Kyrpides, Eugene V. Koonin, Tanja Woyke. Giant viruses with an expanded complement of translation system components. Science, 2017; 356 (6333): 82 DOI: 10.1126/science.aal4657

Posted by Dr. Tim Sandle

Pharmig News

The latest Pharmig News letter is now available.

Members will have received a copy, non-members can purchase a copy via the Pharmig website.

No. 67 – May 2017

In this issue:
  • Chairman’s Newsletter
  • Book Reviews
  • The Future Possibilities of Graphene for Microbiology
  • Microbiological Cleaning Validation (Disinfectant Validation)
  • Pharmig News Corner #35

Posted by Dr. Tim Sandle

Saturday, 20 May 2017

Maple syrup extract enhances antibiotic action

Antibiotics save lives every day, but there is a downside to their ubiquity. High doses can kill healthy cells along with infection-causing bacteria, while also spurring the creation of "superbugs" that no longer respond to known antibiotics. Now, researchers may have found a natural way to cut down on antibiotic use without sacrificing health: a maple syrup extract that dramatically increases the potency of these medicines.

To figure out how the extract makes antibiotics work better, the researchers investigated whether the extract changed the permeability of bacterial cells. The extract increased the permeability of the bacteria, suggesting that it helps antibiotics gain access to the interior of bacterial cells. Another experiment suggested that the extract may work by a second mechanism as well, disabling the bacterial pump that normally removes antibiotics from these cells.


American Chemical Society. "No more 'superbugs'? Maple syrup extract enhances antibiotic action.”

Posted by Dr. Tim Sandle

Friday, 19 May 2017

The most beautiful bacteria you’ll ever see

Synthetic biologist Tal Danino manipulates microorganisms in his lab to create eye-catching, colorful patterns. Here’s a look at the process he uses to turn “Oh, yuck” into “Oh, wow.”

TED are hosting a range of beautiful microbial images. These are from Synthetic biologist Tal Danino, who has a new project called Microuniverse. Here he produced a series of dazzling, abstract images created by different species of bacteria, each grown under different conditions for varying lengths of time.

TED (Technology, Entertainment, Design) is a media organization which posts talks online for free distribution, under the slogan "ideas worth spreading". Tal Danino is a synthetic biologistartist and assistant professor of biomedical engineering at Columbia University.

“The project is about getting to see this unseen universe that’s really small and all around us, every day.” 

The images can be accessed here: TED

Posted by Dr. Tim Sandle

European Pharmacopoeia 9th Edition (Supplement 9.1)

The 1st supplement to the 9th edition of the European Pharmacopeia became effective on 1st April 2017.

Of interest to readers:

Water for injections (0169)

Production: revision to include purification processes equivalent to distillation (such as reverse osmosis coupled with appropriate techniques) for producing water for injections (WFI), in addition to distillation; use of non-distillation technologies for the production of WFI requires that notice is given to the supervisory authority of the manufacturer before implementation.

A requirement for regular monitoring of total organic carbon has been added to further emphasize the specific test controls required in the Production section.

Posted by Dr. Tim Sandle

Thursday, 18 May 2017

Bacterial Identification Database Updated with Plant Pathogens

Biolog, Inc. have announced details of its latest GEN III identification database version 2.8, containing 1568 taxa, including 258 plant pathogens. This database update was completed by scientists at Biolog's headquarters in Hayward, CA in collaboration with Dr. Bevan Weir and his colleagues at Landcare Research in Auckland, New Zealand.

The database update reflects Biolog's commitment to serving the needs of the agricultural market by providing state-of-the-art tools for bacteria, yeast and filamentous fungi identification and characterization. Using Biolog's OmniLog® and MicroStation™ Systems, many bacterial plant pathogens can now be identified to the pathovar level. This level of resolution of pathovars, especially within the Pseudomonas and Xanthomonas genera, cannot be easily achieved by current molecular testing technologies
Biolog’s CEO Dr. Barry Bochner stated, “We are pleased to release this updated database and believe that this greatly expanded capability will provide an important benefit to our agricultural customers needing to rapidly and efficiently identify pathogens isolated from plants.”  Dr. Bevan Weir, Landcare Research Scientist and ICMP Culture Collection Curator noted, “Landcare Research is a world leader in plant pathogen identification and taxonomy. Our extensive ICMP culture collection was used to ensure that the data in the GEN III database is scientifically robust and useful for the agriculture sector.”  The GEN III version 2.8 database is compatible with previous versions and platforms configured to run Biolog’s GEN III MicroPlates™.

Wednesday, 17 May 2017

Giant virus viewing required retrofitted microscope

In order to map one of the world’s largest viruses, scientists took a DIY approach to build a retrofitted cryo-electron microscope.

“If the common cold virus is scaled to the size of a ladder, then the giant Samba virus is bigger than the Washington Monument,” says Kristin Parent, assistant professor of biochemistry and molecular biology at Michigan State University and coauthor of the paper in the journal Viruses. “Cryo-EM allowed us to map this virus’ structure and observe the proteins it uses to enter, or attack, cells.”

It seems counterintuitive that bigger organisms are harder to see, but they are when using cryo-electron microscopy. That’s because scientists usually use these microscopes to look at thin specimens. The microscopes can’t decipher larger organisms to reveal their biological mechanisms. For thick samples, scientists see only dark gray or black blobs instead of seeing the molecular framework.

Cryo-EM allowed Parent’s team to image the giant Samba virus and understand the structures that allow it to enter an amoeba. Once inside, Samba opens one of its capsid layers and releases its nucleocapsid—which carries the genetic cargo that sparks an infection. While Samba isn’t known to cause any diseases in humans, its cousin, the mimivirus, may be a culprit for causing some respiratory ailments in humans.

For more on this news item see: Laboratory Roots

Posted by Dr. Tim Sandle

Tuesday, 16 May 2017

European Pharmacopoeia 9th Edition: Review of Supplement 9.2

Below is a list of monographs and general chapters that are new, or that have been revised, corrected or deleted for the 9th Edition (supplement 9.2). The implementation date is 1st July 2017.

Revised texts
2.2.1    Clarity and degree of opalescence of liquids              

General revision to restructure the text and eliminate unnecessary repetition. The requirements for accuracy and repeatability of the instrument have been changed.

2.6.30  Monocyte-activation test                                                         
As a result of a survey distributed by the EDQM in 2013 to users of the Ph.Eur., the following improvements have been made:

Introduction: the wording ‘very steep dose-response curves’ has been changed to ‘very steep or non-linear dose-response curves’ as the latter better characterises the responses when non-endotoxin contaminants are present.

Definitions: clarification added that calculation of the maximum valid dilution (MVD) is based on the endotoxin reference standard; the possibility of using an estimated limit of detection (LOD) based on historical data when calculating MVD has been introduced.

Cell sources and qualification - Additional cross-references to sections relating to the qualification of cells according to their origin, preparation and/or intended use (i.e. for the detection of endotoxin and/or nonendotoxin contaminants) have been included in sections 5-1, 5-2, 5-4, 5-5 and 5-6.

Section 5-4. Qualification of cells pooled from a number of donors: a caution statement has been added regarding the need to consider the averaging effect when cells are pooled.

Section 5-5. Qualification of cryo-preserved cells: the repetitive description regarding the preparation of cell pools has been deleted.

Section 5-6. Monocytic continuous cell lines: a statement regarding the limited use of monocytic cell lines for the detection of non-endotoxin pyrogens has been introduced. Preparatory testing

Section 6-1. Assurance criteria for the endotoxin standard curve: numerical example provided to illustrate the term ‘as low as possible’, which defines the blank.

Section 6-2. Test for interfering factors: text revised so that the concentration of added endotoxin in the preparation or the diluent is to be justified and can be estimated before starting the test. In Method C, it is stated that the type of analysis used to compare the test and reference lots must be justified and validated for each preparation, that assay validity criteria are to be included and that the dilutions tested depend on the type of analysis used. In addition, more information is given on how to test preparations with an inherently high pyrogen content.

Section 6-3. Method validation for non-endotoxin monocyte-activating contaminants: text revised to note that during preparatory testing, at least 2 non-endotoxin ligands for toll-like receptors must be used to validate the test system, 1 of which is also used to spike the test preparation, and that the choice of non-endotoxin pyrogens used should reflect the most likely contaminant(s) of the test preparation. In addition, more information is given regarding the available ligands that can be used.

Methods: Section 7-1-1. Method A, Test procedure: regarding the qualification procedure applied to monocytes of different origin, the term ‘qualified cells’ has now been introduced throughout the text. Changes to Table 2.6.30.-1 have been made so that all 3 test solutions (A, B and C) are to be spiked and not just the highest concentration. Solution D has therefore been deleted and replaced by solutions AS, BS and CS (i.e. spiked solutions A, B and C).

Section 7-1-2. Calculation and interpretation: text reworded to reflect the changes in Table 2.6.30.-1. In addition, the text now states that dilutions with an invalid spike recovery are deleted from further analyses and that at least 1 valid dilution is required for a valid test.

Section 7-1-3. Pass/fail criteria of the preparation: conditions for the use of monocytic cell lines have been deleted.

Section 7-2-1/2. Method B, Table 2.6.30.-2: text updated accordingly, as above for Method A.

Section 7-3. Method C. Reference lot comparison test: although there is flexibility on the type of analysis used, the analysis must be justified and validated for each product and is to include assay validity criteria; the text has been changed to reflect this. A statement has also been included to emphasise that the description of the test method includes just an example of a type of analysis which could be used.

Section 7-3-2. Calculation and interpretation: numerical example provided to show a possible acceptance value.

Guidance notes

Section 2-1. Information regarding the choice of methods: further clarification is given on the inappropriateness of Method A if the dose-response curve for the preparation to be examined is not parallel to that of the standard endotoxin curve. In addition, a notice has been added regarding the product specific validation and capacity of the chosen method to identify
non-responders along with low and high responders to a particular product/contaminant(s) combination(s).

Section 2-5. Cross-validation has been added. Regarding the presence of non-endotoxin pyrogens in the product, a recommendation to perform cross-validation of the monocyteactivation test together with the bacterial endotoxins test has been introduced. In the context of the 3Rs, the rabbit pyrogen test can be performed for cross-validation purposes where the monocyte-activation test cannot be validated.

A new entry has been included in Table 2.6.30.-4 for ‘Parenteral formulations administered per square metre of body surface’, in accordance with the recently revised general chapter 5.1.10. Guidelines for using the test for bacterial endotoxins.

It is now specified that MAT is considered as a replacement for the rabbit pyrogen test

5.1.1. Methods of preparation of sterile products                   

This text has undergone a general revision and has been completely rewritten. The sections on the different sterilisation processes, where appropriate, now have the same format: principle, equipment, sterilisation cycle, cycle effectiveness and routine control; where required, specific information has been added.

Sterility assurance level: the reference to exponential inactivation has been removed as membrane filtration is not a first-order process.

Steam sterilisation: modern concepts for validation have been added.

Dry heat sterilisation: a wider description of the suitable equipment has been provided.

Ionising radiation sterilisation: the reference to European Notes for Guidance has been removed.

Gas sterilisation: 2 types of agents are defined: alkylating agents and oxidising agents; the establishment of the cycle effectiveness has been described in more detail.

Membrane filtration: the description of the microbial challenge test has been moved to general chapter.

5.1.2. Biological indicators and related microbial preparations used in the manufacture of sterile products (previously Biological indicators of sterilization)

Aseptic assembly: freeze-drying under aseptic conditions is added.

The general chapter has undergone significant revision as listed below.

Title: it has been adapted to take into account microbial preparations used for sterilization grade filtration.

Introduction: describes when biological indicators (BIs) are intended to be used and what is outside the scope of the general chapter, including that BIs are in most cases only to be used for development of the sterilisation process and are not to be employed for routine monitoring unless otherwise stated in this general chapter. A definition of BIs is given and the processes in which they can be used are described. Importantly, the Introduction section introduces the concept of the use of reduced sterilisation process conditions in order to ensure the validity of the sterilisation process. It is also made clear that there should be no surviving microorganisms when the biological indicator is subject to a full sterilisation process.

BIs for sterilisation processes: this section gives guidance on how BIs are selected and how they are used to characterise sterilisation processes.

A description is provided of 4 types of BIs for sterilisation processes: inoculated carriers, self-contained BIs, characterised spore suspensions and custom-made BIs.

Information regarding the quality requirements for BIs and user requirement specifications have been introduced.

BIs for heat sterilisation: the parameters of BIs for heat sterilisation are described and how a validation cycle is established. Further information on biological validation with reduced sterilisation cycles has been included.

BIs for moist heat sterilisation: it is recognised that Geobacillus stearothermophilus may not be suitable for sterilisation processes delivering an F0 between 8 and 15, therefore a different test micro-organism may be used.

BIs for dry heat sterilisation: description of the reference conditions and an example of how survivor rates of typical BIs are affected by temperature variations are given.

BIs for gas sterilisation: this section sets out that the use of gas sterilisation for disinfection is outside the scope of the general chapter. There are a number of different types of gas sterilisation processes and no reference cycles, therefore no criteria to which the BIs shall comply have been defined. Suitable micro-organisms for ethylene oxide sterilisation are given. It is, however, the responsibility of the user to define the cycle and the suitability of any BI used.

BIs for ionising radiation sterilisation: it is recognised that BIs are not considered to be necessary for defining the suitability of the radiation sterilising dose, but their use may be required for the development and validation of ionising radiation sterilisation in specific cases. Information on test micro-organisms is given.

Microbial preparations for sterilisation grade filtration: information on test microorganisms is now given for the validation of retention of micro-organisms using a membrane.

Indicators for depyrogenation processes: this section has been removed from this general chapter and will be published elsewhere in the Ph. Eur.

Posted by Dr. Tim Sandle