Ensuring Sterility in Pharmaceutical Milling Processes

 

Particle size is critical to a pharmaceutical product’s effectiveness. Consequently, milling is a crucial process for drug manufacturers, but any amount of physical contact introduces cross-contamination concerns in this industry. As drugmakers consider how these workflows meet particle size and efficiency needs, they must also ensure sterility.

 

By Ellie Gabel  

Historically, meeting high standards of cleanliness and sanitation has led to complications and high costs in pharmaceutical manufacturing. Today, new technologies let pharma companies ensure sterility while minimizing production time and expenses. These innovations can apply to milling before, during and after the actual grinding phase.

Preventing Cross-Contamination Before Milling

Pharmaceutical sterility begins with preventing contaminants from entering the workspace in the first place. Contact between equipment and materials cannot produce cross-contamination if no microbes, dirt or other pollutants ever reach the machinery.

Creating a reliable clean room environment is crucial. Airflow is one of the most critical considerations here. Laminar flow will keep contaminants out by pushing inside air in one direction to move it away from sensitive equipment and toward filters. All clean room HVAC systems should include HEPA filters, which can remove 99.97% of airborne pollutants of 0.3 microns or larger.

Internet of Things (IoT) sensors can help by monitoring pollutants in real time. As a result, it becomes easier to ensure protective measures are working correctly or enforce sanitary workflow policies. IoT solutions can also track equipment maintenance to help HEPA filters and other sanitation machinery remain in acceptable condition for longer.

Using Contamination-Resistant Milling Methods

The pharmaceutical milling process itself can adapt to become more contamination-resistant, too. While manufacturers often choose methods based on particle size and efficiency needs, some systems provide sterility benefits over their alternatives.

Conical grinding may be the most common option, but micronization minimizes contamination risks by producing no heat and can achieve smaller particle sizes. In instances where drug classes are incompatible with this method, manufacturers could use cryogenic grinding, which has similar temperature-related benefits. These more advanced solutions may incur higher upfront costs, but they often produce better results and higher efficiency, which can make up for the investment over time.

The optimal method depends on the drug and manufacturing process in question. Generally speaking, though, wet techniques are preferable to dry alternatives, as they require a contained system. This containment, in turn, minimizes cross-contamination and material loss.

Post-Milling Sterilization

Even with thorough preparatory steps, contamination is still possible. Consequently, pharma manufacturers must also apply post-milling sterilization measures to counteract any sanitation issues that may have arisen in the previous steps.

Physical filtration is a reliable way to remove contaminants from drug solutions after milling. Studies show that 200-nanometer particles easily pass through most readily available sterile filtration solutions, while most bacteria are larger than that. As a result, manufacturers can mill their pharmaceutical ingredients as small as possible so they can pass through such pores while the filters catch pollutants.

Chemical and thermal sterilization may provide additional benefits, but manufacturers should approach them carefully. Many pharmaceuticals are sensitive to heat, and chemicals introduce the risk of unwanted reactions. The optimal solution will depend on the kinds of materials a process is dealing with, and organizations will likely need to use various methods to serve different products’ needs.

Pharma Manufacturers Must Address Sterility at Every Point

Contaminants can enter the pharmaceutical production process at many points. Consequently, manufacturers in this industry must ensure high standards of sterility at each step throughout the workflow. Failure to account for any area could result in far-reaching consequences if the pollution is significant enough.

Technologies like the IoT, novel milling equipment and advanced filtration make sterility easier to achieve than ever before. Businesses must recognize the potential such solutions hold and take advantage of them to stay compliant with rising demands and regulations. Getting ahead of tech trends will also keep organizations competitive in a fast-changing environment.

 

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Tattoo inks and microbial contamination

This week's article looks at microbial contamination risks associated with tattoo inks and the tattoo process and assesses recently published US FDA guidance: 

https://www.linkedin.com/pulse/tattoos-microbial-infection-risks-tim-sandle-ph-d-cbiol-fisct-eqiue/

Save 50.0% on select products from Colourbing with promo code 50LQ68OG, through 1/5 while supplies last. 

Many tattoo inks are contaminated, although different rounds of laboratory analysis show a degree of variation according to locale and ink source. However, there has been a commonality over the past decade for ~50% of inks found to contain microorganisms (10–80% of unopened commercial tattoo inks are contaminated with microorganisms according to my review of the literature. The populations recovered are up to 3·6 × 10^8 CFU per gram. It should be noted that studies tend to pinpoint higher rates of contamination in North America compared with Europe.

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

The Role of Robotics Programming In Science

The world is changing faster than ever, and robots are leading the charge! They're building our cars, performing surgeries, and even exploring the depths of space. But how do these incredible machines know what to do? The answer lies in the fascinating world of robotics programming, which is the secret code that brings robots to life."

What is Robotics Programming?

Picture that you have a robot friend, but it does not know how to walk or talk. You need to teach it! Robotics programming is like giving your robot friend instructions. These instructions are written in special languages that robots can understand.

To get started with robotics programming, you'll need to learn a programming language. C/C++ are powerful languages that give programmers a lot of control over the robot's hardware. They're often used for tasks that need speed and efficiency, such as controlling robot movements. Python is great for tasks that involve artificial intelligence (AI), like helping a robot recognize objects or make decisions, and Java is good at handling lots of data and complex tasks. It's often used for programming robots that interact with the real world. Some examples include self-driving cars or robots that work in warehouses. Even cooler, new 'no-code' platforms are popping up, making it easier than ever for anyone to try their hand at robotics programming, even without knowing complex languages! To illustrate, Pepper is a humanoid robot often used in customer service roles. PandaSuite allows users to create interactive behaviors for Pepper, such as greeting customers, answering questions, and providing directions, all without coding.

The Role of Robotics Programming in Science

Robotics programming is super important in science because it helps us do incredible things. Scientists use robots to explore dangerous places, such as the deep sea or outer space, where humans cannot easily go. These automatons can also help us with difficult tasks, such as manufacturing products or assisting in surgeries. In car factories, robotic arms weld parts together with pinpoint accuracy to make sure every car is built tough, and when it comes to your phone or computer, tiny robots place the super-small parts onto circuit boards way faster and better than any human could. Even in giant warehouses, robots zoom around, lifting and stacking those huge boxes of stuff like it's nothing. Robots are also being used in STEM education.

What You Should Know About Programming in Robotics

If you think robotics programming sounds exciting, here are some things to keep in mind. First, it involves problem-solving. You need to figure out the best way to tell a robot to complete a task. This can be tricky, but it is also extremely rewarding when you find a solution. Additionally, robotics programming requires creativity. You can program these machines to do all sorts of things, from playing music to drawing pictures. The possibilities are endless! Finally, do not be afraid to make mistakes. Everyone makes mistakes when they are learning something new.

Robotics Programming is Fun!

Robotics programming may sound complicated, but it can be a lot of fun. There are many resources available to help you learn, such as books, websites, and online courses. On top of that, you can even join a robotics club or team to learn from others and work on projects together.

So, there you have it! Robotics programming is a fascinating field that combines technology, science, and creativity. If you are curious and love to solve problems, robotics programming might be the perfect thing for you.

Written by Taylor McKnight, Author for Automation Integration Solutions, LLC
 

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Diet matters less than evolutionary relationships in shaping gut microbiome

 

Gut microbes provide many services for their hosts, including digesting their food. Researchers have long known that mammals with specialized diets, such as carnivores and anteaters, have special types of gut microbes that allow them to eat that diet.

Is the same true in primates? In the largest published comparative dataset of non-human primate gut microbiomes to date, a new Northwestern University study set out to find whether leaf-eating primates have similar gut microbes that help them break down their leafy diet, which is full of fiber and toxins.

A common theme in the microbiome field is that host diet has large effects on the gut microbiome — both across lifespans (week-to-week changes in host diet change the gut microbiome) and across evolution (mammals with similar diets have similar gut microbes regardless of their evolutionary histories), said Katherine Amato, lead author of the study and assistant professor of anthropology in the Weinberg College of Arts and Sciences at Northwestern.

Therefore, they expected to see many similarities between leaf-eating primates, regardless of how closely related they were to each other. Rather, the researchers discovered that diet mattered much less than host evolutionary relationships in shaping the gut microbiome.

“Our data suggest that, across evolution, the effect of primate diet on the primate gut microbiome is not large,” Amato said. “Evolutionary relationships between primates are much more important for predicting microbiome composition and function.” 

The study is the first cross-species comparison of the gut microbiota that exclusively uses samples from wild animals.

“We conclude that although gut microbes play a critical role in supporting host dietary specializations, their impact is regulated through host physiology,” Amato said.

“Leaf-eating primates shared very few gut microbial characteristics. Instead, New World monkeys shared the most gut microbial characteristics with each other, regardless of diet. The same was true for Old World monkeys, lemurs and apes. These patterns appear to be the result of host physiological traits such as how the gastrointestinal tract is built.”

Cross-mammal examinations of the gut microbiome have been performed, Amato said, but they all had weaknesses in that species with similar diets also had similar evolutionary histories or physiology. Many studies also mixed captive and wild animals, and captivity is known to change the gut microbiome.

“This study was able to eliminate these issues due to the fact that leaf-eating evolved independently multiple times in the order Primates and is associated with a different physiology in each part of the primate tree,” Amato said. “We also only used wild primates, which compared to captive primates, are more likely to have gut microbiomes like those they evolved with.”

Further research could involve looking at more primate species with more varied diets and understanding how the human gut microbiome fits into this bigger evolutionary picture and what it can tell us about our physiology and health, Amato said.

“Evolutionary trends in host physiology outweigh dietary niche in structuring primate gut microbiomes” published online earlier this month in the ISME Journal: Multidisciplinary Journal of Microbial Ecology.

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Unmasked: The Science Powering Human, Humanized, Chimeric, and Single-Domain Antibodies

 Antibodies, the immune system’s frontline fighters, are nature’s way of keeping pathogens in check. Thanks to biotech innovation, these proteins have been reimagined to tackle everything from cancer to autoimmune diseases. Let’s unpack the fascinating world of engineered antibodies—human, humanized, chimeric, and single-domain—and see how they’re changing the medical game.  

By Bella Smith, Creative Biolabs

Human Antibodies: The Real Deal  

Human antibodies are as close as it gets to the natural defenders produced by your immune system. Fully derived from human genes, they boast unmatched compatibility, significantly reducing the risk of adverse immune responses. Advances like transgenic mice and phage display tech have made producing these antibodies a breeze, making them MVPs in treating chronic conditions and infections.  

Humanized Antibodies: The Perfect Blend  

When it comes to balancing efficacy and safety, humanized antibodies steal the show. Initially generated in animals, these antibodies are modified to replace non-human components with human ones, keeping only the antigen-binding regions intact. This reduces the risk of rejection while maintaining functionality.  

They’ve become a go-to for autoimmune and cancer therapies, ensuring cutting-edge solutions that won’t make your immune system go haywire.  

Chimeric Antibodies: A Best-of-Both-Worlds Hybrid  

Chimeric antibodies are the ultimate mash-up, blending human and non-human regions to maximize both safety and efficacy. By combining a human constant region with a variable region from another species (often mouse-derived), researchers create highly effective antibodies for tough targets.  

This hybrid approach has seen massive success, with drugs like rituximab paving the way for cancer immunotherapies. Chimeric antibody products illustrate the versatility of this design in the clinic.  

Single-Domain Antibodies: Tiny Titans  

Single-domain antibodies, are a stripped-down, no-frills version of the traditional antibody. Sourced from camelids like llamas and alpacas, these mini marvels are small but incredibly mighty. Their compact size lets them access hard-to-reach spots and bind with precision.  

Applications in imaging, diagnostics, and targeted therapies are expanding fast, with products like single-domain antibodies pushing the boundaries of what these tiny proteins can do.  

Antibody Engineering: What’s Next?  

From lab bench to bedside, engineered antibodies are shaking up the medical world. These biotechnological breakthroughs are unlocking new possibilities for personalized medicine, improving outcomes, and delivering hope to patients battling complex diseases.  

With experts like Creative Biolabs offering solutions that span the antibody spectrum, researchers have the tools to push innovation further than ever before. Whether it’s human, humanized, chimeric, or single-domain antibodies, these scientific marvels are rewriting the rules of modern medicine.  
 

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Innovations in rapid microbiological methods

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Introduction to sterility assurance (video)

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Spirulina: Quest for new medicines

 Image: Spirulina powder, from the genus Arthrospira 

( By John Alan Elson - http://www.3dham.com/protist/spirulina.htm, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=47098390)

A biotechnology company has sought to re-invent how biologic drugs are invented by using patented technology to adapt the food algae spirulina to deliver therapeutic proteins1. Spirulina (Arthrospira platensis (Nordstedt) Gomont, formerly Spirulina platensis and Spirulina maxima) is a species belonging to the Cyanobacteria class that lives in freshwater lakes with alkaline and warm waters (an oxygenic photosynthetic bacterium). 

 

Spirulina is most commonly utilised as a food supplement in special algal farms in outdoor tanks and bioreactors. Tim Sandle spoke with Lumen Bioscience CoFounder and CEO, Brian Finrow. 

Sandle, T. (2024) Biologic Drugs Are Reinvented To Treat Infectious Disease, Pharmig News #97, pp11-13

 

Read the article here.

 

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Bacillus spizizenii

 

The American Type Culture Collection (ATCC) #6633 bacterium Bacillus subtilis subsp. spizizenii (commonly Bacillus subtilis) has been reclassified as Bacillus spizizenii.

The change was the result of whole genome sequencing and the paper triggering the change was issued in 2020. However, several culture collections (and providers of cultures) were slow to adopt the taxonomic change.

Bacillus subtilis encompassed four subspecies: Bacillus subtilis subsp. subtilis, Bacillus subtilis subsp. inaquosorum, Bacillus subtilis subsp. spizizenii and Bacillus subtilis subsp. stercoris.

As a result of the research, each has become a separate species. Bacillus spizizenii is retained as the strain commonly used by the world’s culture collections for activities including growth promotion testing.

Reference:

Christopher A. Dunlap . Michael J. Bowman . Daniel R. Zeigler. Promotion of Bacillus subtilis subsp. inaquosorum, Bacillus subtilis subsp. spizizenii and Bacillus subtilis subsp. stercoris to species status. Antonie van Leeuwenhoek (2020) 113:1–12
 

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

John Neiger

John Neiger, who was one of the leading technical experts in the field of cleanrooms, clean air devices, and contamination control has sadly passed away.

John was a founder of the company Envair, which was one of the early pioneering firms specialising in clean air devices (including some of the first isolators).

In 1979, John joined the British Standards committee for microbiological safety cabinets and remained an active participant in national and international cleanroom standards development for around 40 years.

As a technical writer, John co-wrote the book ‘Pharmaceutical Isolators' which was published in 2004 (and subsequently updated). John was also the editor of the essential industry journal Clean Air and Containment Review  - CACR (published by Euromed). CACR recorded over 50 issues.

John wrote two chapters for books that I have edited and in turn I was a regular contributor to CACR. He was a wise, incredibly detailed, and knowledgeable professional. He will be sorely missed.
 

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Surface Particulate Monitor and Analyser for Clean Environments

Surface Particulate Monitor and Analyser for Clean Environments by Tim Sandle on Scribd

 

This real-time, in-situ, automated surface particulate monitoring system tracks
the accumulation of particulates over time, alerts the operators and people responsible for quality control to contamination incidents early on and also allows classification of contaminants according to physical characteristics. In this paper, we describe the PFO1000 unit, its operating principles and measurements, and provide the first results of field trials of the unit in a real-world situation.

PFO 1000 MONITOR: A REAL-TIME, IN-SITU, AUTOMATED INSTRUMENT FOR MONITORING PARTICULATE FALL-OUT IN CLEAN... by Tim Sandle on Scribd

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Microbiology and Infectious Disease

Reviewed title: 'Dimer-monomer transition defines a novel hyper-thermostable peptidoglycan hydrolase mined from bacterial proteome'

DOI link: https://doi.org/10.7554/eLife.98266.1

Summary: This study details a method to identify new antimicrobial drugs with therapeutic promise from bacterial datasets, providing clues for discovering alternatives to traditional antibiotics. eLife's editors describe it as a valuable new strategy for identifying novel lysins (a type of enzyme) with antimicrobial activity, and say that it provides solid evidence for the therapeutic potential of two such lysins discovered during the work.

Full eLife press release for further details: 'Harnessing big data helps scientists hone in on new antimicrobials' – https://elifesciences.org/for-the-press/a444a8f0/harnessing-big-data-helps-scientists-hone-in-on-new-antimicrobials

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

The Future of Clean Room Design: Integrating Tech and Innovation for Optimal Performance

                                                                                                    Cleanroom - designed by Tim Sandle

People specializing in clean room design for pharmaceutical plants have eagerly explored possibilities that include tech and innovation, believing that those attributes will prepare the facilities ready for current and future needs. However, creating a well-designed and purposeful space is about more than just ensuring it has a cutting-edge look and shows the company as a well-equipped entity. Which trends have supported these goals? 

 

By Emily Newton

Using AI-Enabled Digital Twins in Clean Room Design

One emerging trend involves executives relying on digital twins to evaluate differences between potential design choices. Changing the location of an entrance or running through various airflow simulations can help people make informed decisions rather than realizing too late that specific ideas they had were more effective in their minds than reality.

Digital twins are highly realistic representations of actual assets or physical spaces, so people can quickly alter these virtual copies to familiarize themselves with the likely effects. Versions with artificial intelligence features have become more recently available. The most advanced versions can provide prescriptive analyses that recommend which actions people should take. Such data can steer them in the right direction when differentiating between various choices, including many that seem extremely similar.

Additionally, AI-powered digital twins can help people determine if now is the right time to invest in certain innovations they believe will positively impact their adaptability and profits. Since a digital twin provides a consistent environment for running simulations, enables users to experiment and see the likely outcomes before finalizing decisions.

For example, a pharmaceutical clean room classification is a numerical designation based on the size and amount of particulate matter. However, the classification system varies on factors such as the associated regulatory body and the country where the company operates. On one commonly used scale, the lower the number, the stricter the associated contamination control. However, other methods concern a letter grade on a four-point scale. Suppose leaders wanted to investigate the technologies required for a Class 3 clean room versus one classified as a Class 4 facility.

Digital twins can clarify what is needed to make a clean room achieve a particular class ranking. It is then easier for executives to plan and justify their investments. 

Centering Clean Room Design on Quality Control

As a facility’s design teams ponder specifics such as pharmaceutical clean room classification and airflow models, they must also examine the bigger picture. How can they make strategic choices that will increase the company’s likelihood of maintaining high quality control?

One possibility is to implement room features to reduce electrostatic discharge. It causes the destruction or degradation of sensitive components. It is particularly problematic for pharmaceutical companies operating both medication and device divisions. If a device such as an insulin pump performs unexpectedly due to an electrostatic discharge issue that occurred in the factory, the associated brand could experience severe reputation-based repercussions.

Some control programs include measures to safeguard equipment or components at risk of damage from electricity that is at least to 100 volts on the human body model, which measures electrostatic discharge from people. That is the most common source, making it necessary for designers to find practical solutions for curbing it. Possibilities such as flooring materials that dissipate or neutralize electrostatic discharge are excellent foundational options in clean room design.

Another innovative quality control-related measure is to install various connected sensors that give managers and other designated personnel real-time statistics about particle counts, occupancy levels and more. Then, they can immediately see if specific conditions could interfere with quality control and respond accordingly before costly outcomes occur. 

 Cleanroom workers - designed by Tim Sandle

Prioritizing Sustainability and Waste Reduction

Many pharmaceutical executives want to capitalize on the many ways to operate more sustainably. That might mean installing solar panels on some of their buildings. Since statistics show the prices for such systems have dropped by 88% in 11 years, that opportunity is an increasingly affordable one. However, resource conservation can occur inside clean rooms, too.

A commonly utilized option is to make the facilities well-insulated, and more energy-efficient as a result. Additionally, decision-makers can select products made from recycled materials and buy used equipment rather than new items when applicable. All those seemingly small measures add up to create meaningful sustainability gains that can inspire peers and position pharmaceutical brands as eco-friendly pioneers to watch.

Alternatively, leaders may determine that automation investments are among the best ways to operate sustainably by reducing waste and remaining more mindful of resource usage. In such cases, people can achieve impactful results without making all-encompassing changes. For example, one high-tech clean room in a pharmaceutical factory has an automated filling station. It palletizes, labels and straps products without human intervention, improving workflows and freeing people up for other tasks.

Although many individuals think of waste as a physical thing to minimize, it also manifests as delays or underutilized skills. When people choose automation to support their sustainability aims in pharmaceutical facilities, they typically find the advantages span further than initially envisioned. 

A Bright Future for Clean Room Design

Although these are some of the most widely applied trends by clean room designers, people can expect to see more of them for the foreseeable future. The performance-centric efforts explored here connect to executives’ desire to remain competitive and meet high expectations in a demanding industry.
 

Pharmaceutical Microbiology Resources (https://www.pharmamicroresources.com/)

Understanding steam sterilisation failures

Dr. Tim Sandle discusses some of the common issues encountered in the sterilisation of surgical instruments, using autoclave technologies. He argues that to make a thorough evaluation of sterilisation, reliance cannot be restricted to chemical or biological indicators, and a complete understanding of hazards and physical operating parameters is required.

Staff working in decontamination services will ensure that reusable medical devices, such as endoscopes and other surgical instruments and equipment are cleaned, sterilised, and repackaged to high standards. 

This article can be read at the Clinical Services Journal webpage (registration is required).

The reference is: 

Sandle, T. (2024) Understanding steam sterilisation failures, Clinical Services Journal, 23 (10): 33-37

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Endospores and mechanisms of resistance

 Image created by Tim Sandle

Endospores present a concern in controlled environments due to their resistance and indefinite survivability. The production of a spore is part of a sophisticated stress response. Here, the bacterial genome is copied and transferred into the safety of a spore (sporulation).

The spore remains dormant until environmental conditions improve. When conditions are favorable, the spore will germinate (generally rapidly) and become a functioning, vegetative cell.

This week’s article looks at what endospores are, how they are formed, and their relative resistance as part of improving our understanding of contamination control.

See:  https://www.linkedin.com/pulse/resistance-so-futile-endospores-mechanisms-tim-sfaxe/  

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Evolution of Pseudomonas aeruginosa

 

Two hundred years ago, give or take the odd decade, Pseudomonas aeruginosa was an environmental bacterium (1), apparently not one, as far as medical records in the pre-microbiology days can be discerned, associated as a human pathogen (2).

Today, P. aeruginosa is associated with a high number of multidrug-resistant infections (3), many of which are nosocomial. Those especially vulnerable to the bacterium are people with underlying lung conditions.

It is estimated that P. aeruginosa is responsible for communicable diseases leading to over 500,000 deaths per year around the world, of which over 300,000 are associated with antimicrobial resistance (AMR). People who are immunocompromised as a result of conditions such as COPD (smoking-related lung damage), cystic fibrosis (CF), and non-CF bronchiectasis, are particularly susceptible.

This week’s article looks at the bacterium and also highlights some new research that charts how the organism evolved rapidly and then proceeded to spread globally over the last 200 years. At the heart of this are changes in human behavior. 

See: https://www.linkedin.com/pulse/200-year-old-problem-evolution-multi-drug-resistant-tim-lqn4e/  

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Oligonucleotide Analysis

The 15-crown-5 crown ether, a cyclic oligomer, and its monomer, ethylene oxide (By Jorge Stolfi CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=92112505)

What are oligonucleotides?

Oligonucleotides, which cover both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are increasingly demonstrating their central value in clinical practice. A notable example is the introduction of customized DNA sequences into immune cells that are genetically engineered to drive these cells to express chimeric antigen receptors (CARs), triggering a new era of cell-based mediated immunotherapy. At the same time, a variety of RNA molecules, such as messenger RNA (mRNA) and small interfering RNA (siRNA), are also cleverly used to achieve instant protein synthesis and specific protein expression inhibition, respectively, opening up new ways of therapeutic intervention. In view of the increasing role of oligonucleotides in medical intervention, it is becoming increasingly important to adopt effective methods for purification, analysis and characterization of oligonucleotides.

Oligonucleotide purification method

Polyacrylamide gel electrophoresis (PAGE)

Polyacrylamide gel electrophoresis is a standard purity analysis method, which is particularly suitable for the separation of oligonucleotides based on fragment length. The process begins with the polymerization of polyacrylamide with the aid of a crosslinker to produce a three-dimensional gel matrix. Subsequently, under the action of an electric field, the oligonucleotide fragment carried by the negative charge migrates towards the anode. Due to spatial structural limitations, small molecules exhibit faster penetration rates due to lower resistance compared to larger molecules. Over time, oligonucleotides of various sizes exhibit differentiated migration distances in the gel matrix, which makes it possible to classify and purify them by length.

Ion pair reversed phase high performance chromatography (IP-RP-HPLC)

IP-RP-HPLC is the most popular oligonucleotide purification technique, in which low concentrations of long-chain alkylamines are added to bind negatively charged oligonucleotides in the LC mobile phase. The retention and elution of oligonucleotides in LC columns are influenced by the charge and ion of the oligonucleotides on the alkyl chain length in reagents such as triethylammonium acetate. For example, the retention time usually increases in proportion to the charge of the oligonucleotide and the hydrophobicity of the long alkyl chain in the ion-pairing reagent. A key advantage of IP-RP-HPLC is that it can also be coupled directly to a mass spectrometer for detailed mass characterization of oligonucleotides.

Separation of a 10-30 mer heterooligonucleotide ladder using three different ion-pairing buffers. (Gilar, M., 2002)

Qualitative characterization of oligonucleotides

One way to analyze the purity of oligonucleotides is to analyze their quality using mass spectrometry (MS). One common method is matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) MS. The technique uses a laser with a chemical matrix to ionize a sample of oligonucleotides and then accelerates the ions through a flight tube to a detector, which measures the particle count as a function of time. TOF is proportional to the mass of the molecule. MALDI TOF MS has a high throughput and is well suited for analyzing oligonucleotides below 50 bases, as ionization efficiency and separation resolution are reduced. There is also a risk that photosensitive modified oligonucleotides may be damaged by strong laser sources.

Structural analysis of oligonucleotides

X-ray crystallography

X-ray crystallography is undoubtedly the most authoritative and detailed means to reveal the structure of oligonucleotides, and its brilliant history is inseparable from the birth of several Nobel prizes. Given that the wavelength of X-rays is similar to the length of the internal bonds of molecules – about 1.5 angstroms – this technique provides unmatched structural detail with high precision. Through the precise interpretation of X-ray diffraction patterns, scientists were able to reconstruct the three-dimensional layout of oligonucleotide molecules and gain insight into their electron density distribution.

Nuclear magnetic resonance spectroscopy (NMR)

Another popular approach to structural analysis is nuclear magnetic resonance (NMR) spectroscopy, which is unique in that it can be analyzed without the need for the sample to form crystals. In a stable strong magnetic field, the nucleus is excited by a weak fluctuating magnetic field and releases electromagnetic waves reflecting the characteristic frequency of its magnetic field. When the oscillation frequency of the external magnetic field coincides with the natural frequency of the nucleus, that is, the resonance phenomenon is produced. The NMR spectra are rich in specific resonance information from the different nuclei in the sample, and the NMR Settings can be flexibly adjusted according to the resolution required for the experiment.

Circular dichroism (CD) spectrum

In the exploration of secondary structure of oligonucleotides, circular dichroism (CD) spectrum plays a key role. The technique is particularly suitable for identifying structures such as double helices, hairpins, and G-quadruplets, each of which presents a unique peak shape and signal on the CD spectrum. CD spectroscopy is not only good at identifying different secondary structures, but also tracking the conformational evolution of oligonucleotides in response to environmental changes (such as temperature, pH, salt concentration). Especially under temperature-controlled conditions, the melting behavior of oligonucleotides can be accurately described and their thermodynamic stability can be quantified. These insights are invaluable in elucidating the biological activity mechanism of oligonucleotides.

About the Author:

Carrie Taylor
R & D Director of BOCSCI

2014 - Present, working in BOCSCI
2012-2014 Study in Rice University, MBA
2004-2008 Study in Rice University,Pharmacy
Linkedin profile: https://www.linkedin.com/in/carrier-taylor/ 

Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Chemical control: Chlorine and the disinfection of water

 

Heat or different biocidal products can be used to reduce microbial contamination in water. This includes strong oxidizing agents like chlorine dioxide and ozone. Historically, the introduction of chlorination to municipal water supplies led to a reduction in cholera and Salmonella (a process regularly in place, for the first time, in England from 1905) (1).

Chlorination can be applied to mains water and initial stages of water generation, for pharmaceuticals; however, it is generally not used thereafter with the preferred methods being ozone or heat. Chlorination is an important step for ensuring public drinking water and shared services, like swimming pools, remain suitable for human use.

This week’s article looks specifically at chlorine and its use as an antimicrobial agent in a water system. 

See:  https://www.linkedin.com/pulse/chemical-control-chlorine-disinfection-water-tim-cibhe/?trackingId=63gnZL34S0GL3HpIRZ2EeQ%3D%3D 

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

How Calibration and Regular Maintenance Ensure Accurate Pharmaceutical Testing

 Cleanroom calibration (designed by Tim Sandle)

Pharmaceutical testing is a critical quality control step that ensures the produced products can withstand specific activities and will perform as patients expect. Friability testing examines tablets’ strength, verifying that they will not break or crumble during transport or handling. Drug assays reveal whether the strength of active ingredients in a tested sample matches its label. Performing these tests and others on the required schedules allows pharmaceutical manufacturers to operate within regulatory requirements.

 

By Emily Newton

 

However, the testing equipment must be properly calibrated and maintained for reliable results. Maintenance and calibration-related shortcomings can have far-reaching effects that disrupt operations and cause the affected companies to receive unwanted scrutiny.

Calibration and Maintenance Flagged by Inspectors

Pharmaceutical companies are in a tightly regulated industry, which means periodic checks occur to confirm that the entities follow all required procedures. When inspectors visited the New Jersey site of an American multinational pharmaceutical company, they found eight problems, including some related to the calibration and maintenance of equipment.

 

More specifically, the inspection report showed some of the plant staff who tested its products did not have adequate training. Since knowing how to perform a test means understanding when and how to calibrate the associated equipment, the lack of role-related education could have serious consequences. Additionally, inspectors discovered stability samples were missing for some drugs, suggesting workers did not perform them on the appropriate schedule.

 

Another testing-related issue was that problems associated with the company’s electronic data-tracking system left test information open to tampering. In any case, when maintenance and calibration do not happen as required, people cannot trust that the results are accurate.

 

Inspectors want to see that the parties handling the calibration, testing and recording of the outcomes have the proper training and that all steps have occurred consistently and at the right times. Otherwise, all the results could become untrustworthy, making it impossible to ensure the safety of those taking the affected drugs.

Sensors Installed to Maintain Consistency

Pharmaceutical companies often rely on sensors to maintain consistent conditions in demanding environments. The sensor data can also alert workers to an abnormality that could render test results inaccurate. For example, nitrogen is a carrier gas used with some chromatography testing equipment. The pharmaceutical industry typically uses a type with a 99.99% gas concentration, marketed as ultra-high-purity nitrogen. If the gas is less pure for any reason, an alert is necessary.

 

Pharmaceutical tests need specific environments and correctly calibrated, well-maintained equipment to achieve accurate results. Fortunately, connected sensors can generate notifications when they encounter unusual conditions or reminders for maintenance. Some sensor equipment also connects to recordkeeping platforms, registering when individuals perform particular steps.

 

Even highly observant pharmaceutical factory workers cannot manually check all environmental parameters. However, sensors can work in the background and alert staff when readings fall outside of preset parameters.

 

Similarly, sensors can verify when machines are correctly calibrated, eliminating or reducing many of the manual checks. Even the most observant workers sometimes get tired or become distracted. Strategically deployed sensors increase awareness, so poor calibration or inadequate maintenance cannot disrupt the production environment. Some pharmaceutical executives already use sensors for supply chain monitoring. This decision can halve associated costs, making it worth pursuing.

Automation Used to Reduce Manual Work

Many pharmaceutical decision-makers have realized automated technologies are essential for helping calibration and testing go more smoothly and efficiently. One reason automation is so beneficial is because it boosts productivity by handling many of the steps workers formerly did manually. Additionally, automated sensors with predictive algorithms measure machine operations in real time, detecting abnormal vibrations, temperatures out of an acceptable range or other unusual details.

 

Some businesses also use robots to test injectable drugs, such as verifying the volume of the delivered medicine or the force a patient must apply to remove the injector’s safety cap. Since robots excel at repetitive tasks, they are ideal for these quality control verifications. Also, people who formerly oversaw manual tests can reskill to set up and supervise the automated testing equipment. Then, there is a reduced likelihood of testing mistakes that degrade accuracy.

 

Moreover, sensors that automate calibration give people more confidence in their results. One example measures pH and automatically performs a standard calibration before transferring all the associated data to the process control system. That approach creates a reliable audit trail and shows that the pharmaceutical company has prioritized recordkeeping. Additionally, when calibration requires adjusting the machine, the necessary steps always occur in the same sequence.

 

Leaders in other industries — such as oil and gas — have used robots to check equipment and take readings for validation. Applying that option to pharmaceutical facilities could save time while ensuring accuracy because there is a lower chance of humans accidentally contaminating the environment when they take measurements by hand.

Accuracy Solidifies Trust

The pharmaceutical industry is highly competitive and tightly regulated. Patients, those who prescribe medications and other stakeholders must trust that the products made within this sector are safe and effective. Well-maintained, carefully calibrated testing equipment fits leaders’ quality control goals while bolstering brand reputation and loyalty.

 

Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)