The RMM Blog - Rapid Microbiology and Rapid Microbiological Methods
Updated routinely by Dr. Michael J. Miller, our RMM blog will keep you informed of new and noteworthy technologies, reviews of recent publications and presentations, upcoming conferences and training events, and what's changing in the RMM world.
Researchers in Carnegie Mellon University’s Department of Chemical Engineering (ChemE) are collaborating with leading biotechnology company Genentech, a member of the Roche Group, and LumaCyte, a biotechnology instrumentation company based in Charlottesville, VA, to develop an advanced biomanufacturing technology for adventitious agent testing, or testing for unexpected viral infections during the production of biopharmaceuticals.
The research recently received funding from the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) to develop and test technologies for improving the safety testing of biologic medicines during production and prior to release. This project, which aims to rapidly and accurately detect viral infectivity in biopharmaceuticals, was one of the first four proposals funded by NIIMBL. The team, which includes Carnegie Mellon, Genentech, and LumaCyte, will receive $1.5 million in funding from NIIMBL over 18 months.
When using biological materials such as mammalian cell lines to produce pharmaceuticals, manufacturers face the risk of viruses infecting the batch. Currently, testing for adventitious agents such as viruses happens late in the manufacturing process—but the research team, which includes ChemE Professor Jim Schneider and Adjunct Professor Todd Przybycien, are developing technologies to test biopharmaceutical batches while they are being produced.
“If you don’t find out about infection until very late in the process, you will have wasted a lot of time and money as more downstream equipment and product becomes infected,” says Schneider. “Current infection detection techniques, such as cell-based assays and polymerase chain reaction, can take days to complete. Our methods can provide readout in less than 15 minutes, which enables a routine, continuous type of testing that could detect infections almost as soon as they take hold.”
Rapid DNA analysis has been in development for a number of years by Schneider and Przybycien, who is also a professor of chemical and biological engineering at Rensselaer Polytechnic Institute. Using a rapid DNA analysis technique developed in Schneider’s lab, the team is detecting viruses and bacteria in process streams used to make biologic pharmaceutical projects. By performing rapid electrophoresis, the researchers can separate tagged and untagged DNA in a sample, indicating the presence of virus or bacteria in biologic process streams.
The researchers aim to combine their method with LumaCyte’s LFC and Radiance technology for a faster, more reliable, and more cost-effective solution. LumaCyte’s Radiance and Carnegie Mellon’s patented rapid DNA analysis platform will combine to rapidly detect the presence of virus and/or bacteria in bio process streams.
“The focus of NIIMBL is to translate existing technologies into biomanufacturing contexts,” said Schneider. “One of the top priorities that the industry has identified is rapid adventitious agent screening. As one of the first four projects funded by NIIMBL, this research with LumaCyte and Genentech shows our commitment to collaboration between academia and the pharmaceutical industry."
NIIMBL is an Innovation Institute designed to revolutionize domestic biopharmaceutical manufacturing. Funded through a $70 million cooperative agreement with the National Institute of Standards and Technology (NIST) in the U.S. Department of Commerce, NIIMBL funds and collaborates on innovative manufacturing technologies that bring life-saving and life-enhancing products to market faster and at reduced cost, while maintaining safety and efficacy.
LumaCyte is an analytical instrument development company headquartered in Charlottesville, VA. LumaCyte produces a label-free cell analysis and sorting instrument called Radiance that does not require the use of antibody or genetic labelling for analysis of cells. Applications of LumaCyte’s label-free platform technology include viral infectivity for vaccine manufacturing, cell and gene therapy, cancer biology, infectious disease, and pre-clinical drug discovery.
A number of companies will be highlighting their new technologies at the 29th Annual European Congress of Clinical Microbiology and Infectious Diseases (ECCMID). ECCMID is being held on April 13 – 16, 2019 in Amsterdam, Netherlands. Below you will find prep releases from some of these companies.
Mesa Biotech to Introduce Expanded Molecular POC Testing Portfolio at the European Congress of Clinical Microbiology and Infectious Diseases
Mesa Biotech Inc., a privately-held, molecular diagnostic company that has developed an affordable and easy to operate PCR (polymerase chain reaction) testing platform designed specifically for point-of-care (POC) infectious disease diagnosis, today announced it will demonstrate its expanded, novel Accula™ Test System at the 29th Annual European Congress of Clinical Microbiology and Infectious Diseases (ECCMID). ECCMID is being held on April 13 – 16, 2019 in Amsterdam, Netherlands and the Accula System will be on exhibit in Booth 1.1C. Mesa Biotech has obtained CE Mark in the European Union (EU), as well as 501(k) clearance and CLIA Waiver from the U.S. Food and Drug Administration (FDA) on both its Accula Flu A/Flu B and RSV tests.
The Accula System, recently named 2019 Frost & Sullivan Price/Performance Global Value Leader, is a palm-sized, reusable dock with disposable test cassettes. The novel molecular test system offers the simplicity, convenience and procedural familiarity of traditional POC rapid immunoassays, while providing the superior sensitivity, specificity and information content of laboratory-based PCR testing. Test results are available in approximately 30 minutes to guide same day treatment decisions. Both the Accula Flu A/Flu B and RSV tests are indicated for use with nasal swab collection that is less invasive than nasopharyngeal swabs and allows for a more comfortable specimen collection experience for the patient.
"We are excited to introduce our expanded PCR test platform at ECCMID," said Hong Cai, Co-founder and Chief Executive Officer, Mesa Biotech, Inc. "As our product offerings continue to increase, we are carefully selecting additional strategic distributors to add to our growing international network."
About Mesa Biotech Inc.
Mesa Biotech designs, develops, manufactures and commercializes next generation molecular diagnostic tests, bringing the superior diagnostic performance of nucleic acid PCR amplification to the point-of-care (POC). Mesa Biotech's Accula™ System consists of a portable, palm-sized dock and disposable, assay-specific test cassettes. This patented system enables healthcare professionals to access actionable, laboratory-quality results at the POC with greater sensitivity and specificity than current infectious disease rapid immunoassay tests. The Accula Flu A/Flu B and the Accula RSV tests have obtained CE Mark in the EU and 510(k) clearance and Clinical Laboratory Improvements Amendments (CLIA) waiver from the U.S. Food and Drug Administration (FDA). Both products are distributed in the US by Sekisui Diagnostics under the Silaris™ brand. Mesa Biotech has also secured a number of strategic agreements for distribution in Europe and Asia.
T2 Biosystems and Clinicians to Share Clinical Data in Integrated Symposium and Poster Presentations at the European Congress of Clinical Microbiology & Infectious Diseases (ECCMID)
T2 Biosystems, Inc., an emerging leader in the development of innovative medical diagnostic products for critical unmet needs in healthcare, announced today that the Company will host an integrated symposium highlighting key clinical data about the T2Bacteria® and T2Candida® panels at the European Congress of Clinical Microbiology & Infectious Diseases (ECCMID) in Amsterdam on Monday, April 15, at 16:00-18:00 Central European Time (CET). ECCMID will take place April 13-16 at the Amsterdam RAI Exhibition and Convention Center.
Additionally, seven leading clinicians and users of T2Direct Diagnostics™ will offer scientific presentations during ECCMID that will highlight the most recent scientific data on the Company’s FDA-cleared T2Bacteria and T2Candida Panels. The Company will also present the first patient case studies with the T2Resistance™ Panel, which recently received FDA Breakthrough designation and is pending CE mark for commercial availability in Europe. All presentations will demonstrate the potential for these panels to significantly improve infectious disease management for patients in real clinical settings.
“I have seen firsthand how the rapid detection of bacterial and fungal pathogens with T2Direct Diagnostics can improve patient outcomes, better manage broad-spectrum antimicrobial usage and combat antibiotic resistance,” said Neil Clancy, MD, University of Pittsburgh Medical Center, who is one of the integrated symposium speakers.
The T2Bacteria and T2Candida Panels are able to identify sepsis-causing pathogens within 3 to 5 hours directly from whole blood, instead of days required with blood culture based diagnostics. This gives clinicians actionable information much earlier than was previously possible, allowing them to make more informed treatment plans for escalation or de-escalation of antimicrobial therapy.
Dr. Clancy continued, “When diagnosing and treating infectious diseases, time is of the essence. I am proud to be one of the clinicians here at ECCMID using T2Direct Diagnostics and believe that we must continue to spread awareness about this rapid diagnostic technology to improve patient care.”
T2 Biosystems will showcase its latest innovations at Booth #1.22. The Company will also host an educational event, “Rapid Diagnostics Direct from Whole Blood: A Solution for Fast and Appropriate Antimicrobial therapy,” which will be co-chaired by Prof. Karsten Becker, MD, University Hospital Münster and Prof. Emmanuel Roilides, MD Aristotle University of Thessaloniki; and it will include the following presentations:
-- “Rapid Diagnostics Direct from Whole Blood: A Solution for Fast and Appropriate Antimicrobial Therapy,” on Monday, April 15 from 16:00-18:00 CET in Hall D; presenters include: • Prof. Michael Bauer, MD, Jena University (Jena, Germany) • Dr. Cornelius (Neil) Clancy, University of Pittsburgh Medical Center (Pittsburgh, PA) • Dr. Giulia De Angelis, Institute of Microbiology, UniversitàCattolica del Sacro Cuore, Fondazione Policlinico Universitario Agostino Gemelli (Rome, Italy) • Dr. Thomas Walsh, New York Presbyterian Hospital (New York, NY)
-- Development of a highly sensitive assay for the detection of carbapenem-resistance genes from whole blood by T2 magnetic resonance, on Sunday, April 14, from 13:30-14:30 CET (Tom Lowery) -- Real-life diagnostic performance of T2Candida among ICU patients with risk factors for invasive candidiasis, on Tuesday, April 16 from 12:30-13:30 CET (Maiken Arendrup) -- The T2Bacteria assay is a sensitive and rapid detector of bacteraemia that can be initiated in the emergency department and has potential to favourably influence subsequent therapy, on Tuesday, April 16 from 12:30-13:30 CET (Christopher Voigt) -- The T2Bacteria Panel is a rapid detector of bacteraemia and has potential to guide therapy in patients with haematological malignancies and haematopoietic stem cell transplantation: a pilot study of non-culture molecular diagnosis, on Tuesday, April 16 from 12:30-13:30 CET (Thomas J. Walsh) -- Evaluation of a molecular technology magnetic resonance for the direct identification of pathogens from blood samples in paediatric patients with suspected sepsis, on Tuesday, April 16 from 12:30-13:30 CET (Paola Bernaschi)
About T2 Biosystems
T2 Biosystems, a leader in the development and commercialization of innovative medical diagnostic products for critical unmet needs in healthcare, is dedicated to improving patient care and reducing the cost of care by helping clinicians effectively treat patients faster than ever before. T2 Biosystems’ products include the T2Dx® Instrument, T2Candida® Panel, and T2Bacteria® Panel and are powered by the proprietary T2 Magnetic Resonance (T2MR®) technology. T2 Biosystems has an active pipeline of future products, including products for the detection of additional species and antibiotic resistance markers of sepsis pathogens, and tests for Lyme disease.
Sherlock Biosciences, an Engineering Biology company dedicated to making diagnostic testing better, faster and more affordable, announced its launch and initial financing of $35 million. The financing includes a $17.5 million non-dilutive grant and an investment from the Open Philanthropy Project with support from additional undisclosed investors.
Sherlock is using Engineering Biology tools, including CRISPR and Synthetic Biology, to create a new generation of molecular diagnostics that can rapidly deliver accurate and inexpensive results for a vast range of needs in virtually any setting.
“Our founders have created some of the most important breakthroughs in modern science through advances in the field of Engineering Biology, the practice of designing and building biological systems into tools that can enhance human health,” said Rahul K. Dhanda, Sherlock’s co-founder, president and CEO. “We are building Sherlock to transform these breakthroughs into a new and powerful generation of molecular diagnostics that will enable users to make more effective decisions in both clinical and non-clinical settings worldwide – including hospitals, industrial settings, low-resource settings and at home.”
The company takes its name from one of its foundational platform technologies, SHERLOCK™ (Specific High-sensitivity Enzymatic Reporter unLOCKing), which is licensed from the Broad Institute of MIT and Harvard. SHERLOCK was developed by a team led by co-founder and chair of Sherlock’s scientific advisory board Feng Zhang, Ph.D., and collaborators, as a method for identifying specific genetic targets using CRISPR. SHERLOCK can detect genetic fingerprints across multiple organisms or sample types and has been described in four papers published in the journal Science.
The company is also developing INSPECTR™ (INternal Splint-Pairing Expression Cassette Translation Reaction), a Synthetic Biology-based molecular diagnostics platform developed by a team led by co-founder James J. Collins, Ph.D., at the Wyss Institute for Biologically Inspired Engineering at Harvard University. The technology, licensed from Harvard’s Office of Technology Development, can be programmed to distinguish targets based on a single nucleotide without an instrument, at room temperature.
Used as stand-alone tools or in combination, these platforms allow for the detection and quantification of targets without complex instruments and in a variety of potential settings. The flexibility and modularity of these platform technologies open a wide range of potential applications and actionable insights in areas including precision oncology, infection identification, food safety, at-home testing, and disease detection in the field.
The company will employ a strategy of selective partnering and direct product development to apply these technologies into a wide range of settings and applications. The financing will be used to advance development programs and design new assays.
“Engineering Biology-based tools have broad potential to transform not just the treatment of disease but also how diseases are diagnosed,” said co-founder James Collins, Ph.D. “Sherlock Biosciences will make a significant difference in the world by bringing the power of Synthetic Biology and CRISPR to diagnostic development.”
“We believe Sherlock Biosciences offers an enormous opportunity to improve human health worldwide by delivering fast, accurate and simple diagnostic testing. It is especially encouraging that the broad potential of its technologies is matched by co-founders and a team who are deeply experienced scientists, entrepreneurs and clinicians,” said Heather Youngs, program officer for scientific research at the Open Philanthropy Project. “Development of this technology could both reduce viral pandemic threats and benefit healthcare more broadly. We are excited to support Sherlock’s efforts to realize the potential of diagnostics and propel this technology into the mainstream.”
“Our team has the expertise and technology to transform diagnostics with a powerful set of Engineering Biology tools to enable rapid test design and deployment, an essential component of addressing many healthcare needs, including the growing problem of resistant bacteria,” said co-founder Deborah Hung, M.D., Ph.D. “After early experiments, our tools were quickly used in a wide range of geographies with real patient samples, confirming that we can swiftly respond to urgent healthcare needs.”
“We founded Sherlock Biosciences to improve health worldwide through the development of disruptive molecular diagnostics. We are delighted to have the support of the Open Philanthropy Project and our investors as we develop Sherlock’s platforms to achieve that goal,” said co-founder David Walt, Ph.D. “Existing molecular diagnostic tools are often limited in their effectiveness because they are costly, labor-intensive, and are not mobile. We believe that Sherlock is poised to overcome those challenges by creating tests that are faster, less expensive and easier to use than currently available molecular diagnostics.”
The U.S. Food and Drug Administration, Centers for Disease Control and Prevention (CDC) and the Centers for Medicare and Medicaid Services (CMS) announced the launch of the Tri-Agency Task Force for Emergency Diagnostics. This task force has been created to help leverage the expertise of each agency to advance rapid development and deployment of diagnostic tests in clinical and public health laboratories during public health emergencies.
“Public health emergencies, like Ebola outbreaks, remind us that we’re a global community when it comes to public health protection. Bacteria and viruses don’t respect territorial boundaries. It takes a sustained, robust and globally coordinated effort to protect our nation and the global community from various infectious disease threats. We’re all in this together. To that end, the FDA knows that collaborating with our federal partners to employ our collective expertise, experiences from previous incidents, and resources will better assist in a global response. We also believe that this task force could lead to more innovation for diagnostic tests as developers will see a more predictable federal regulatory response through the agencies’ coordination,” said Jeffrey Shuren, M.D., J.D., director of the FDA’s Center for Devices and Radiological Health. “This task force will help our agencies better collaborate to prepare for, and respond to, public health threats, including identifying threats and ensuring the appropriate diagnostics are in place to support efforts in the field.”
The FDA, CDC and CMS each play a critical role in responding to public health emergencies, including identifying threats, regulating medical products, and providing oversight for laboratories. The agencies have robust teams of scientists, researchers and policy experts that are dedicated to preparing the U.S. for rapid disaster response.
Diagnostic tests—such as those that can detect pathogens like the Ebola and Zika viruses—can be quickly made available to meet response needs during a crisis through the Emergency Use Authorization (EUA) process. The FDA has authority to issue an EUA for the use of diagnostic tests during public health emergencies, provided criteria are met. The CDC is responsible for providing agent-specific subject matter expertise in epidemiology, laboratory expertise and guidance to clinicians and laboratories responding to the emergency. The CDC and other federal laboratories are often the ones developing new tests to respond to emergency needs. CMS has authority to ensure quality testing at laboratories through the Clinical Laboratory Improvement Amendments (CLIA). CMS provides guidance, even during public health emergencies, to laboratories on meeting CLIA requirements to ensure laboratories produce accurate, reliable and timely results.
Prior to this partnership, feedback from the clinical laboratory community indicated that there was uncertainty about how to implement the diagnostic tests once they received an EUA; particularly, the community was uncertain about meeting CLIA regulations under an EUA to allow labs to start testing specimens.
“During public health emergencies, ensuring the health and safety of patients through quality laboratory testing will remain the focus of CMS,” said Kate Goodrich, director of the Center for Clinical Standards and Quality and CMS Chief Medical Officer. “Timely implementation of EUA diagnostic assays in the US healthcare system is dependent upon laboratories understanding the instructions for use and applying them to the patient samples received for testing. As part of this taskforce, it is our goal to provide clear and consistent guidance to laboratories on the application of CLIA requirements for these emergency assays.”
By standardizing collaboration efforts, the federal partners hope to address issues related to implementation of diagnostic tests authorized for emergency use under an EUA, as well as other unmet needs and gaps in preparing and responding to global health threats. The task force will provide a forum for each agency to coordinate, provide consultation, and improve the availability of diagnostic tests during public health emergencies. In addition, to assist in public health preparedness, the task force will work to define, refine and streamline interagency approaches for the implementation of EUA diagnostic tests. The hope is that the task force will enable an even more efficient federal government response for making diagnostic tests available in the event of a public health emergency.
“Time and time again, we’re reminded that disease knows no borders. While our globalized world and modern transportation help promote economic prosperity, these features also facilitate the spread of emerging infectious diseases,” said Chesley Richards, CDC’s Deputy Director for Public Health Science and Surveillance. “In the past 15 years alone, we’ve faced serious global outbreaks of deadly pathogens. During public health emergencies, it is critical for diagnostic tests to be made available and adopted quickly into clinical and public health laboratories for rapid patient care.”
The FDA, an agency within the U.S. Department of Health and Human Services, protects the public health by assuring the safety, effectiveness, and security of human and veterinary drugs, vaccines and other biological products for human use, and medical devices. The agency also is responsible for the safety and security of our nation’s food supply, cosmetics, dietary supplements, products that give off electronic radiation, and for regulating tobacco products.
Bacteria have been around for over a billion years and have developed remarkable capabilities to not only cause deadly infections but also to survive adverse environmental conditions (1, 2). This has led to a rich cellular diaspora with diverse geno- and phenotypic variability that is important to characterize owing to its dire implications on mankind.
Researchers are constantly evolving strategies that allow them to study complex cellular processes like cell-cell interactions, cellular metabolic activity, gene function, response to external stimuli (3-7), host-pathogen interactions, and surface-associated redox activities (8) at a single cell level. The foremost requirements of these single cell test platforms are to distinguish, isolate, manipulate, immobilize, and characterize cells in an easy and reproducible manner.
The conventional methods widely used for single cell isolation include fluorescence- or magnetic-activated cell sorting (FACS/MACS), micromanipulation, microdissection, and manual cell picking. While FACS/MACS need high cellular loads, large sample volumes and specific treatment of cells prior to analysis, the remaining techniques have quite a low throughput and require highly-skilled labor for carrying out the experiments. Other methods for cell capture include ink-jet printing on molecularly-patterned templates (9) or dip-pen nanolithography (10), but they again, suffer from poor reproducibility, high cost, and our inability to reuse the substrate.
With recent advances in the field of microfluidics, manipulation and separation of cells on the basis of their dielectric properties, using external electric fields has garnered significant attention. This approach offers numerous advantages such as a high extent of miniaturization, low sample volume, controlled fluid dynamics, ease of sample handling, high throughput, great precision, automation, and label-free operation.
Motivated by these factors, we exploited an electrokinetic phenomenon called alternating current (AC) dielectrophoresis to collect live bacterial cells into grid-like patterns, with each node containing a single cell (see image below). This electric field-assisted assembly not only provided high spatio-temporal control over several thousands of cells simultaneously, but the dynamically-formed cellular arrays were also fully reversible meaning that both the cells and the chips could be retrieved at the end of each capture cycle. This study was recently published in Biosensors and Bioelectronics (111, 2018, 159–165) and showed that model Gram-negative bacteria S.typhi and E.coli can be trapped inside micropatterned conductive circular well electrodes by applying AC electric fields (5 MHz, 5-20 Vpp) across a few microliters of cell suspension.
Dynamic electropatterning of live bacteria into single microwell arrays using AC electrokinetics. The bacteria were fluorescently tagged to allow visualization. Image courtesy Meenal Goel & Shalini Gupta
Shrinking the size of the individual electrodes down to 5 μm allowed single cells to be captured per well with 90% efficiency. This trapping of cells took place due to positive dielectrophoresis that attracted them toward regions of high electric field intensity (microwells in our case). The cells remained alive during the one-hour-long operation, and the overall collection efficiency (from bulk to the surface) was also found to be around 90%.
The potential biotechnological application of our chip was demonstrated in two ways. First, the two cell types were mixed in suspension and collected together using the chip. Counting their relative concentration in the 2D matrix using their phenotypic traits allowed us to directly estimate cellular bulk concentrations in the mixture without generating any calibration curves. Both the total time taken and the volume of the sample used was a fraction of the conventional methods. In the second case, the chip was integrated with an impedance spectroscope to carry out rapid cell viability testing. The response of dielectrophoretically-trapped bacteria against a known antimicrobial peptide was recorded at different drug concentrations and found to be ultrasensitive, reducing the overall time for drug susceptibility testing to just under an hour. This is a significant reduction over standard clinical methods that take several hours to produce outcomes.
In the future, our technology could be used for a wide range of applications including multiplexed sensing, rapid single bacterial profiling in heterogeneous populations, and ultrafast drug susceptibility testing to reduce the use of broad-spectrum antibiotics and, hence, the emergence of new antibiotic-resistant strains.
Researchers from Eindhoven University of Technology (The Netherlands) and Keio University (Japan) present a practicable and reliable way to test for infectious diseases: All you need are a special glowing paper strip, a drop of blood and a digital camera, as they write in the scientific journal Angewandte Chemie. Not only does this make the technology very cheap and fast -- after twenty minutes it is clear whether there is an infection -- it also makes expensive and time-consuming laboratory measurements in the hospital unnecessary. In addition, the test has a lot of potential in developing countries for the easy testing of tropical diseases.
The image above shows research leader Maarten Merkx with one copy of the 'glow-in-the-dark' test. Photo: Bart van Overbeeke.
The test shows the presence of infectious diseases by searching for certain antibodies in the blood that your body makes in response to, for example, viruses and bacteria. The development of handy tests for the detection of antibodies is in the spotlight as a practicable and quick alternative to expensive, time-consuming laboratory measurements in hospitals. Doctors are also increasingly using antibodies as medicines, for example in the case of cancer or rheumatism. So this simple test is also suitable for regularly monitoring the dose of such medicines to be able to take corrective measures in good time.
Paper gives light
The use of the paper strip developed by the Dutch and Japanese researchers is a piece of cake. Apply a drop of blood to the appropriate place on the paper, wait twenty minutes and turn it over. "A biochemical reaction causes the underside of paper to emit blue-green light," says Eindhoven University of Technology professor and research leader Maarten Merkx. "The bluer the color, the higher the concentration of antibodies." A digital camera, for example from a mobile phone, is sufficient to determine the exact color and thus the result.
This paper strip (extremely zoomed in) contains two copies of the test. The three glowing dots per test indicate that you can check on three different antibodies within one test. Credit: Bart van Overbeeke.
The color is created thanks to the secret ingredient of the paper strip: a so-called luminous sensor protein developed at TU/e. After a droplet of blood comes onto the paper, this protein triggers a reaction in which blue light is produced (known as bioluminescence). An enzyme that also illuminates fireflies and certain fish, for example, plays a role in this. In a second step, the blue light is converted into green light. But here comes the clue: if an antibody binds to the sensor protein, it blocks the second step. A lot of green means few antibodies and, vice versa, less green means more antibodies.
Market launched within a few years
The ratio of blue and green light can be used to derive the concentration of antibodies. "So not only do you know whether the antibody is in the blood, but also how much," says Merkx. By measuring the ratio precisely, they suffer less from problems that other biosensors often have, such as the signal becoming weaker over time. In their prototype, they successfully tested three antibodies simultaneously, for HIV, flu and dengue fever. Merkx expects the test to be commercially available within a few years.
Now the production of the paper strips is still handicraft. Photo: Bart van Overbeeke.
This work reports on fully integrated “sample‐in‐signal‐out” microfluidic paper‐based analytical devices (μPADs) relying on bioluminescence resonance energy transfer (BRET) switches for analyte recognition and colorimetric signal generation. The devices use BRET‐based antibody sensing proteins integrated into vertically assembled layers of functionalized paper, and their design enables sample volume‐independent and fully reagent‐free operation, including on‐device blood plasma separation. User operation is limited to the application of a single drop (20–30 μL) of sample (serum, whole blood) and the acquisition of a photograph 20 min after sample introduction, with no requirement for precise pipetting, liquid handling, or analytical equipment except for a camera. Simultaneous detection of three different antibodies (anti‐HIV1, anti‐HA, and anti‐DEN1) in whole blood was achieved. Given its simplicity, this type of device is ideally suited for user‐friendly point‐of‐care testing in low‐resource environments.
This week, FDA’s Center for Biologics Evaluation and Research (CBER) published six draft guidances relating to gene therapy, three of which cover products for specific disease categories (hemophilia, rare diseases, and retinal disorders), and three of which address manufacturing and clinical study design issues related to gene therapy: chemistry, manufacturing and control (CMC) information in INDs, long term follow-up study design, and testing of retroviral vector-based products.
In a press release, FDA Commissioner Scott Gottlieb, M.D., highlighted “rapid advancements” and “great promise” in the gene therapy space, saying the guidances “are aimed at fostering developments in this innovative field.” Dr. Gottlieb acknowledged that for some gene therapies, FDA “may need to accept some level of uncertainty” at the time of approval regarding questions related to durability of response, as well as product manufacturing and quality. He acknowledged the need, however, to assure patient safety and to assure that potential risks are adequately characterized and benefits are adequately demonstrated.
The purpose of the draft guidance is to inform sponsors how to provide sufficient CMC information required to assure product safety, identity, quality, purity, and strength (including potency) of the human gene therapy investigational product.
When finalized, the draft guidance will supersede the document entitled “Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs),” dated April 2008.
Because the draft guidance recommends the use of rapid microbiological methods (RMM) for in-process and finished product sterility testing, it is important to understand FDA's position in this regard. As such, I will highlight specific sections in the draft guidance and where appropriate, provide additional interpretation and comment.
Human gene therapy products are defined as all products that mediate their effects by transcription or translation of transferred genetic material or by specifically altering host (human)genetic sequences. Some examples of gene therapy products include nucleic acids, genetically modified microorganisms (e.g., viruses, bacteria, fungi), engineered site-specific nucleases used for human genome editing, and ex vivo genetically modified human cells. Gene therapy products meet the definition of “biological product” in section 351(i) of the Public Health Service (PHS) Act (42 U.S.C. 262(i)) when such products are applicable to the prevention, treatment, or cure of a disease or condition of human beings.
The FDA requires all sponsors of investigational new drug products (DPs), including investigational gene therapy products, to describe the CMC information for the drug substance (DS). FDA may place the IND on clinical hold if the IND does not contain sufficient CMC information to assess the risks to subjects in the proposed studies. The CMC information submitted in an IND is a commitment to perform manufacturing and testing of the investigational product, as stated. However, FDA acknowledges that manufacturing changes may be necessary as product development proceeds, and sponsors should submit information amendments to supplement the initial information submitted for the CMC processes. The CMC information submitted in the original IND for a Phase 1 study may be limited, and therefore, the effect of manufacturing changes, even minor changes, on product safety and quality may not be known. Thus, if a manufacturing change could affect product safety, identity, quality, purity, potency, or stability, sponsors should submit the manufacturing change prior to implementation.
What is Required in the IND Application
The IND should include specifications with established acceptance criteria for safety testing at Phase 1. Safety testing includes tests to ensure freedom from extraneous material, adventitious agents, microbial contamination, and replication competent virus. Information on some common safety test methods is provided in more detail in section 3.2.S.4.2, Analytical Procedures.
To maximize the sensitivity of safety testing, it is important that sponsors perform each test at the stage of production at which contamination is most likely to be detected. For example, tests for mycoplasma or adventitious viruses (in vivo or in vitro) should be performed on cell culture harvest material (cells and supernatant) prior to further processing, e.g., prior to clarification, filtration, purification, and inactivation.
Additional testing will depend on the type of gene therapy product and the phase of clinical development. These tests may include assays to assess product characteristics, such as identity, purity (including endotoxin and contaminants, such as residual host cell DNA, bovine serum albumin (BSA), DNase), and potency/strength.
Sponsors should provide a description of all the analytical procedures used during manufacturing to assess the manufacturing process and product quality. In the original IND submission, descriptions should have sufficient detail so that FDA can understand and evaluate the adequacy of the procedures. FDA recommends that sponsors develop detailed SOPs for how analytical procedures are conducted at early stages of product development as a part of the sponsor's quality system. FDA acknowledges that, during product development, analytical methods may be modified to improve control and suitability. However, assay control is necessary during all phases of clinical development to ensure product quality and safety and to allow for comparability studies, following manufacturing changes.
Safety testing on the DS should include microbiological testing, such as bioburden (or sterility, as appropriate), mycoplasma, and adventitious viral agent testing, to ensure product quality. Guidelines and/or procedures for many safety tests have been described in detail, elsewhere (e.g., bioburden  sterility  mycoplasma , adventitious agent testing, and tests for specific pathogens ).
Sponsors should list DP specifications in the original IND submission. The testing plan should be adequate to describe the physical, chemical, or biological characteristics of the DP necessary to ensure that the DP meets acceptable limits for identity, strength (potency), quality, and purity. Product lots that fail to meet specifications should not be used in clinical investigation without FDA approval. For early phase clinical studies, FDA recommends that assays be in place to assess safety (which includes tests to ensure freedom from extraneous material, adventitious agents, and microbial contamination) and dose (e.g., vector genomes, vector particles, or genetically modified cells) of the product.
Sponsors should describe the analytical procedures used for testing the DP. If the analytical procedures are the same as those for the DS, sponsors do not need to repeat this information unless there is a matrix effect from the DP on assay performance.
Product Release Testing
FDA recommends that product release assays be performed at the manufacturing step at which they are necessary and appropriate. For example, mycoplasma and adventitious agents release testing is recommended on cell culture harvest material. In addition, sterility, endotoxin, and identity testing are recommended on the final container product to ensure absence of microbial contamination or to detect product mix-ups that might have occurred during the final DP manufacturing steps (e.g., buffer exchange, dilution, or finish and fill steps).
If a DP is frozen before use, FDA recommends that sponsors perform sterility testing on the product prior to cryopreservation so that results will be available before the product is administered to a patient. However, if the product undergoes manipulation after thawing (e.g., washing, culturing), particularly if procedures are performed in an open system, sterility testing may need to be repeated.
FDA also recommends that the results of in-process sterility testing be incorporated into the acceptance criteria for final product specifications.
Alternative Microbiological Methods and Rapid Sterility Tests
Analytical procedures different than those outlined in the United States Pharmacopeia (USP), FDA guidance, or Code of Federal Regulations (CFR) may be acceptable under IND if sponsors provide adequate information on test specificity, sensitivity, and robustness. Examples of alternative methods, which may be needed for live cells, include rapid sterility tests, rapid mycoplasma tests (including PCR-based tests), and rapid endotoxin tests. FDA recommends that sponsors plan to demonstrate equal or greater assurance of the test methodology, compared to a compendial method, prior to licensure, as required under 21 CFR 610.9.
For reference, Sec. 610.9, Equivalent methods and processes, states the following:
Modification of any particular test method or manufacturing process or the conditions under which it is conducted as required in this part or in the additional standards for specific biological products in parts 620 through 680 of this chapter shall be permitted only under the following conditions:
(a) The applicant presents evidence, in the form of a license application, or a supplement to the application submitted in accordance with 601.12(b) or (c), demonstrating that the modification will provide assurances of the safety, purity, potency, and effectiveness of the biological product equal to or greater than the assurances provided by the method or process specified in the general standards or additional standards for the biological product; and
(b) Approval of the modification is received in writing from the Director, Center for Biologics Evaluation and Research or the Director, Center for Drug Evaluation and Research.
FDA recognizes that the compendial sterility test may not be suitable for all products. For example, rapid sterility tests may be needed for ex vivo genetically modified cells administered fresh or with limited hold time between final formulation and patient administration.
For ex vivo genetically modified cells that are administered immediately after manufacturing, in-process sterility testing on sample taken 48 to 72 hours prior to final harvest is recommended for product release. For such products, aside from an in-process sterility test, we also recommend that sponsors perform a rapid microbial detection test, such as a Gram stain, on the final formulated product and a sterility test, compliant with 21 CFR 610.12, on the final formulated product.
Under this approach, the release criteria for sterility would be based on a negative result of the Gram stain and a no-growth result from the 48 to 72 hour in-process sterility test. Although the results of the sterility culture performed on the final product will not be available for product release, this testing will provide useful data. A negative result will provide assurance that an aseptic technique was maintained. A positive result will provide information for the medical management of the subject and trigger an investigation of the cause of the sterility failure. The sterility culture on the final formulated product should be continued for the full duration (usually 14 days) to obtain the final sterility test result, even after the product has been administered to the patient.
In all cases where product release is prior to obtaining results from a full 14-day sterility test, the investigational plan should address the actions to be taken in the event that the 14-day sterility test is determined to be positive after the product is administered to a subject. Sponsors should report the sterility failure to both the clinical investigator and FDA. FDA recommends that sponsors include results of the investigation of cause and any corrective actions in an information amendment submitted to the IND within 30 calendar days after initial receipt of the positive culture test result.
In addition, be aware that a product may sometimes interfere with the results of sterility testing. For example, a product component or manufacturing impurities (e.g., antibiotics) may have mycotoxic or anti-bacterial properties. Therefore, FDA recommends that sponsors assess the validity of the sterility assay using the bacteriostasis and fungistasis testing, as described in USP , Sterility Tests.
The draft guidance is a welcome indication that the FDA accepts and encourages the use of alternative and rapid microbiological methods, specifically for sterility testing of short-lived products, such as advanced therapy medicinal products (ATMP; gene and cell therapy). It is appropriate that the guidance document aligns fairly well with 21 CFR 610.12; however, there are some areas that require further clarification.
For example, the use of the 19th Century Gram Stain should be viewed only as a measure of gross contamination because low levels of microbial contaminants would never be observed from a loopful of finished product.
Next, a finished product sterility test that is compliant with 21 CFR 610.12 does not require a 14 day incubation via the compendial USP Sterility Test.
In fact, 21 CFR 610.12 states the following:
Advances in technology in recent years have allowed the development of new sterility test methods that yield accurate and reliable test results in less time and with less operator intervention than the currently prescribed methods. Some examples of novel methods include the Adenosine Triphosphate (ATP) bioluminescence, chemiluminescence, and carbon dioxide head space measurement. Manufacturers may benefit from using such sterility test methods with rapid and advanced detection capabilities. Accordingly, we have amended § 610.12 to promote improvement and innovation in the development of sterility test methods, to address the challenges of novel products that may be introduced to the market in the future, and to potentially enhance sterility testing of currently approved products. This final rule provides manufacturers the flexibility to take advantage of methods as they become available, provided that these methods meet certain criteria.
Because some of these new technologies do not reply on microbial growth, or more raid growth-based methods may be validated to provide an equivalent sterility test result in less than 14 days, the full USP incubation period may not be necessary.
The teachings in 21 CFR 610.12 do allow for the sterility testing of material other than the finished drug product in its final container. The May 3, 2012 Federal Register Vol. 77, No. 86, Page 26162 – 26175 provides additional guidance on what alternative material may be comprised of, e.g., bulk material or active pharmaceutical ingredient (API), in- process material, stock concentrate material), as appropriate, and as approved in the biologics license application (BLA) or BLA supplement.
Additional guidance was provided in the same CFR reference (see page 26165 in the CFR):
As discussed in the preamble to the proposed rule (76 FR 36019 at 36021), certain allergenic and cell and gene therapy products may need to be tested for sterility at an in- process stage or some other stage of the manufacturing process (e.g., intermediate, API, bulk drug substance) instead of the final container material because the final container material may interfere with the sterility test. Likewise, as discussed in the preamble to the proposed rule, some cell therapy products and cell-based gene therapy products may need to be tested for sterility at an in-process stage or some other stage of manufacturing process because low production volumes may result in an insufficient final container material sample for sterility testing or a short product shelf-life may necessitate administration of the final product to a patient before sterility test results on the final container material are available.
Therefore, based on 21 CFR 610.12, it may be appropriate to test an alternative material and not necessarily the finished product, as long as this strategy is justified and acceptable to FDA. For these reasons, the draft guidance should be clearer on whether a sterility test on the final formulated product is a recommendation or an expectation.
Furthermore, the statement regarding demonstrating the validity of the sterility assay using the bacteriostasis and fungistasis testing, as described in USP , should be extended to alternative or rapid sterility tests. These studies are, in fact, an expected part of the qualification of the alternative method and is usually conducted during the Method Suitability phase of the validation process.
Overall, the draft guidance is further assurance from the FDA that rapid and alternative methods are encouraged for use, especially on product that is short-lived or needs to be administered prior to obtaining results from the compendial sterility test. Although the document is specifically focused on gene therapy products in support of IND applications, the recommendations can be used for the routine release of the same or similar ATMPs.
Training on Rapid Sterility Testing of Gene and Cell Therapy Products
I will be speaking on this same topic during the PDA Europe Conference on Pharmaceutical Microbiology, which will be held in Berlin, Germany on October 15-16. More importantly, I will provide a comprehensive overview of rapid methods, including validation strategies, technology reviews, regulatory acceptance and applications during a two-day training course immediately following the conference on October 17-18. For more information, please visit https://www.pda.org/global-event-calendar/event-detail/rapid-microbiology-methods.
USP describes membrane filtration, plate count, and most probable number methods that can be done to quantitatively determine the bioburden of non-sterile DPs. Although 21 CFR 211.110(a)(6) does not specify a test method, it requires that bioburden in-process testing be conducted pursuant to written procedures during the manufacturing process of DPs.
Sterility testing may be performed on the DS when it cannot be performed on the DP, as outlined in the final rule: Amendments to Sterility Test Requirements for Biological Products (May 3, 2012; 77 FR 26162 at 26165). Sterility tests are described in 21 CFR 610.12 and USP Sterility Tests.