Rapid Detection of Bacteria and Viruses in Bioprocess Samples: Justification, Regulation, Requirements and Technologies — How Can Industry Achieve Broad Adoption?

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RAPID DETECTION OF BACTERIA AND VIRUSES IN BIOPROCESS SAMPLES: JUSTIFICATION, REGULATION, REQUIREMENTS AND TECHNOLOGIES — HOW CAN INDUSTRY ACHIEVE BROAD ADOPTION?

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1 Contents

1.0 Executive summary...... 6

2.0 Introduction ...... 8

3.0 Current practices ...... 10

3.1 Sterility testing...... 10

3.2 Mycoplasma testing...... 11

3.3 Virus testing...... 13

4.0 Drivers for change...... 15

5.0 Overcoming barriers to change ...... 17

5.1 Technical/operational attributes and impediments...... 20

5.2 Cost–benefit ratio...... 21

5.3 Regulatory acceptance...... 21

5.4 Behavioral...... 22

5.5 Summary ...... 23

6.0 Advocacy for improvement and change...... 24

7.0 Conclusions and path forwards...... 25

References...... 27

Definitions/glossary...... 30

Acronyms/abbreviations...... 35

Appendix: Different types of PCR...... 36

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 2 List of tables

Table 1: Use and limitation of adventitious virus testing (adapted from ICH Q5A)...... 14

Table2: Analysis of potential behavioral barriers to the adoption of new technologies...... 23

List of figures

Figure 1: Areas of dissatisfaction with current GMP lot release tests...... 17

Figure 2: Impediments to implementing alternative rapid microbiology methods for GMP release testing...... 18

Figure 3: Activities to drive regulatory acceptance of alternative rapid microbiology methods...... 19

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 3 Authors

The following member company participants are acknowledged for their efforts and contributions in the production of this document:

AstraZeneca Merck & Co., Inc., Kenilworth, NJ, USA Jennifer Anderson Scott Hooper Rick Lu Novo Nordisk Biogen Corinna Cavan Pedersen Cassandra Braxton Maria Rathmann Sørensen Chugai Pharma Manufacturing Co., Ltd Pall Biotech Kunihiro Kamata Morven McAlister Fujifilm Diosynth Biotechnologies Roche Williams Nkrumah Sven Deutschmann Kevin McCarthy Lonza Eva Tur Garcia Thermo Fisher Scientific Rita Tommasi Michael Brewer Merck BioPhorum Cristina Barbirato Bela Green Audrey Chang

Contributor

The team would like to thank the following non-member contributor:

Bioprocessing Technology Institute, Agency for Science, Technology and Research Andre Choo

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 4 About BioPhorum The BioPhorum Operations Group’s (BioPhorum’s) mission is to create environments where the global biopharmaceutical industry can collaborate and accelerate its rate of progress, for the benefit of all. Since its inception in 2004, BioPhorum has become the open and trusted environment where senior leaders of the biopharmaceutical industry come together to openly share and discuss the emerging trends and challenges facing their industry. Growing from an end-user group in 2008, BioPhorum now comprises 53 manufacturers and suppliers deploying their top 2,800 leaders and subject matter experts to work in seven focused Phorums, articulating the industry’s technology roadmap, defining the supply partner practices of the future, and developing and adopting best practices in drug substance, fill finish, process development and manufacturing IT. In each of these Phorums, BioPhorum facilitators bring leaders together to create future visions, mobilize teams of experts on the opportunities, create partnerships that enable change and provide the quickest route to implementation, so that the industry shares, learns and builds the best solutions together.

BioPhorum Technology Roadmapping BioPhorum Technology Roadmapping establishes a dynamic and evolving collaborative technology management process to accelerate innovation by engaging and aligning industry stakeholders to define future needs, difficult challenges and potential solutions. The Phorum involves biomanufacturers, supply partners, academia, regional innovation hubs and agencies, serving to communicate the roadmap broadly while monitoring industry progress. The project through which this paper has been developed is part of a broad portfolio of collaborative technology implementation projects, mobilized to impact the most critical challenges identified by the first edition of BioPhorum’s BioManufacturing Technology Roadmap https://www.biophorum.com/ phorum/technology-roadmapping/trm-projects-2. This paper is an example of how the Phorum is continuing to deliver tangible results on its mission to accelerate industry innovation. For more information on the Technology Roadmapping mission and membership, please see https://www.biophorum.com/phorum/technology-roadmapping/overview/

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 5 1.0

Executive summary The need for absence of adventitious agents (fungi, viruses, bacteria, including mycoplasma) is inherent to every pharmaceutical manufacturing process, but is of critical concern to the biopharmaceutical industry, where a contamination event can arise from a contaminated cell line, raw materials, cell substrates, equipment, facilities or operators. Biological therapeutics must be demonstrated to be devoid of adventitious agents. This is enforced through extensive selection and testing of cell lines and raw materials, incorporation of dedicated manufacturing steps to remove or inactivate any adventitious agents, and in-process and final product testing.

The expectations for assurance of microbial safety are clearly stated by global or regional regulatory guidelines and the United States, European and Japanese Pharmacopeias. However, many of these mandatory methods have remained unchanged for decades and most, if not all, are performed off-line. In the first edition of BioPhorum’s Biomanufacturing Technology Roadmap it was recognized that in/on/at-line monitoring of biopharmaceutical processes, along with real-time release of the product, are critical to the success of future bioprocessing applications. Therefore, this white paper has been written to address the key factors preventing implementation of alternative rapid microbiology methods (RMM) to assure sterility and absence of adventitious agents, and move the industry from a product release time of weeks towards real-time release. A focused survey was completed by representative biopharmaceutical end-users, covering a range of job functions. This revealed that the biggest hurdles to adoption of new rapid methods were regulatory acceptance of these alternative methods, filing a regulatory change control and the time taken to validate a non-compendial method. Further, there was a consensus that provision of a guidance document that enabled sharing of best validation practices within the industry and included regulatory involvement, would have the greatest impact on widespread industry adoption of new methods.

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 6 Adoption of new technologies to detect adventitious therapy medicinal products (ATMPs) or continuous agents for already approved biopharmaceuticals bioprocessing applications), a standardized means may be viewed as a barrier to change due to the of adopting alternative RMM and subsequently complexity of post-approval change processes and gaining regulatory acceptance for the adoption of the need to obtain global regulatory acceptance. The such methods is clearly warranted. For ATMPs, it additional barriers to change, even for a drug that has is obvious that there is no other solution. ATMPs not received regulatory approval, are categorized as may potentially have small batch sizes, short shelf technical, cost, regulatory and behavioral concerns. lives and/or be produced in a non-current good The former three categories also reflect what manufacturing practice (cGMP) facility. In addition, should be considered as key attributes prior to the benefits of continuous bioprocessing can only implementation of new detection assays, and ways to be realized if the current time required to test for mitigate these barriers are described. Any new assay adventitious agents can be reduced. In the survey, should avoid excessive manipulation, not require regulatory acceptance was cited as the main reason excessive technical expertise or analysis, minimize the for impeding implementation of new methods for potential for false positive results and must be possible the detection of adventitious agents. Meanwhile, the to validate, with the results being easy to interpret regulatory agencies have encouraged adoption of without subjective, analyst-based interpretation of the new technologies to facilitate bioprocessing and the experimental results. Additionally, consideration must channels that can be used to support end-users are be given to the complexities of comparing methods described within this document. that may have a different output or sensitivities. Ultimately, it appears that, for the biopharmaceutical Ultimately, the objective must be to demonstrate industry to achieve broad adoption of new through appropriate scientific testing and rationale technologies for the rapid detection of bacteria, fungi that the intended method is not inferior to current and viruses, the barrier does not lie with technical compendial test methods. Further, the alternative solutions. Rather, to overcome the regulatory method must enable an unequivocal decision to be hurdles, whether perceived or real, it would benefit made as to whether compliance with the standards regulatory and industry stakeholders to align on a of the monographs would be achieved if the official potential validation strategy that could cross different methods were used1. product types and production sites, such that a However, to facilitate resolution of current drug single consolidated validation package is possible. shortages or to assure patient safety for rapidly The outcome of this white paper will set forth the emerging fields in biotechnology (such as advanced foundation for this next step.

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 7 2.0

Introduction

Biological therapeutics used for the prevention The ICH Q5A guideline serves as the primary regulatory and treatment of human disease may be produced guideline for viral safety in biologic manufacturing from a variety of cells of eukaryotic or prokaryotic processes based on animal and human cell cultures. The origin, including human, non-human mammalian safety assurance is achieved through the application of a (e.g. hamster), avian, insect, fungi, yeast and robust and effective virus testing program, which adopts a bacteria². Adventitious agent safety is a three-tiered approach8: major concern during the manufacturing of • Selecting and testing of cell lines and biopharmaceuticals. In the Guidance for Industry other raw materials Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production • Assessing capacity for viral clearance and of Viral Vaccines for Infectious Disease Indications, inactivation by a manufacturing process the FDA defines an ‘adventitious agent’ as: • In-process and/or final product virus testing. “A microorganism (including bacteria, fungi, mycoplasma/spiroplasma, mycobacteria, rickettsia, A viral safety test panel for contaminants in biologics viruses, protozoa, parasites, transmissible currently includes electron microscopy (EM), assays for spongiform encephalopathy (TSE) agent) that is retroviruses, and detection of virus in cell culture and inadvertently introduced into the production of a animal host systems. In vitro virus detection has been biological product.” accomplished using multiple cell lines to which the sample An adventitious agent contamination event can arise from is applied and subsequently observed for cytopathic effect a contaminated cell line source or during production (e.g. (CPE), hemagglutination (HA) or hemadsorption (HAD) cell substrates, raw materials, equipment, facilities and/ during the 14–28 days of incubation. In vivo assays involve or operators). Although contamination of bioreactors and inoculation of specific animal species and subsequent biologic therapeutics is rare, several manufacturers have observation for mortality, and the testing of tissue for reported such incidents in biotechnology manufacturing the presence of hemagglutinins. These assays have processes or final products in the past decades, and the been considered necessary to detect new or emerging consequences can be catastrophic3, 4. This paper will focus viruses and to complement narrowly focused virus- on adventitious agent detection with special attention to specific assays such as polymerase chain reaction (PCR) virus and bacteria (including mycoplasma) agents that are that came into routine use later (e.g. Rodent Parvovirus, of concern in all biological manufacturing bioprocesses. MMV or Calicivirus 2117 detection). The use of multiple and distinct cell lines and animal hosts stems from the Several examples of bioprocess contamination by knowledge that viruses can have a limited host range (i.e. adventitious agents have been documented over infect certain species but not others), or exhibit tropisms the years, implicating Bacillus cereus, Paenibacillus for specific cell types or tissues (e.g. proliferate in kidney , Mouse Minute Virus curdlanolyticus, Leptospira licerasiae epithelial cells but not in fibroblasts). Certain host systems (MMV), Epizootic Hemorrhagic Disease Virus (EHDV), may be more permissive for virus replication and therefore Reovirus 2 (REO-2), Cache Valley Virus (CVV), and more likely to show CPE in vitro or mortality in vivo. Animal Calicivirus 21175, 6, 7. Therefore, regulatory agencies hosts were included to detect agents that are difficult to worldwide mandate testing for adventitious agents in propagate in vitro, or are non-cytopathic or cause immune- each lot/batch of material in adherence with cGMP. mediated disease (e.g. Lymphocytic Choriomeningitis Virus (LCMV))9.

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 8 Similarly, there are mandatory quality control (QC) Therefore, this paper has been written to summarize the methods that must be incorporated to assure the following key areas for consideration by the end-user for cell banks, cell cultures, cell culture harvest and final adoption of new methods: product is devoid of bacteria (including mycoplasma). 1. Key barriers to implementing alternative RMM in The methods for determination of sterility of the final the biopharmaceutical industry product are described in United States Pharmacopeia To gauge industry opinion, participants from (USP) <71> and European Pharmacopoeia (Ph. Eur.) 11 biomanufacturing organizations (representing 2.6.1, while testing for mycoplasma in cell banks, cell quality assurance (QA), QC, manufacturing and cultures and harvests is reported in USP <63> and Ph. process development) completed a focused Eur. 2.6.7. These are based on conventional growth- survey to understand current practices used for based techniques (with the exception of the indicator adventitious agent and sterility testing, as well as cell-based mycoplasma detection method10, 11, 12). Both identify the roadblocks to implementation. the sterility and mycoplasma methods are described in The key barriers identified from the survey can detail in section 3 (Current practices) of this paper. These be broadly classified into four main categories: methods assume that any contaminating microorganisms technical, cost, regulatory and behavioral. The are able to grow under the test conditions specified, with top-three specific concerns cited were regulatory the incubation period ranging from 14 days for sterility acceptance of the new method, filing a regulatory testing to 28 days for mycoplasma testing. It is noted that change control and the time to validate a new non- both the USP and Ph. Eur. now include provisions for the compendial method; use of alternative RMM13,14. 2. Regulatory perspective and acceptance for adoption However, it should be considered that regulatory of alternative rapid microbiology methods guidelines typically delineate principles to consider (in the case of ICH Q5A8 when assessing viral safety), so BioPhorum members met regulatory experts the burden is on biotechnology manufacturers to select from the FDA in October 2018 and from a selection and implement the most up-to-date solutions—both of European national regulatory agencies in June technologies and mitigation approaches—that are 2019 to discuss both technical and regulatory best suited to their current specific processes15. The considerations for adoption of alternative RMM, and industry is advancing at an unprecedented rate as novel share both industry and regulatory perspectives. manufacturing processes are being used to generate All agreed that continued engagement is necessary biological therapeutics in greater yields, over shorter going forward; production cycles, with demonstrated therapeutic 3. Roadmap to facilitate adoption of rapid methods benefits. Further, it is again emphasized that with the for real-time product release ATMP market growing rapidly, there is an urgent need for This paper includes a review of current methods vs implementation of alternative RMM. future desired technology attributes and provides As the manufacturing industry evolves, so should the an insight into the next phase of work, which will tests that are used to confirm a product’s identity, purity be focused on the sharing of best practices and and suitability. Both process and test improvements potential validation strategies that would meet should run in parallel, considering that the data regulatory expectations. generated from tests will provide the basis for the risk– The survey indicated that most participants agreed benefit decisions and the acceptability of the biological that alternative RMM will be used for lot release product for licensure. testing within the next 10 years. It is anticipated that this white paper will facilitate the biopharmaceutical industry on this journey.

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 9 3.0

Current practices

3.1 Sterility testing Sterility testing for any batch of drug product that has a testing, since it allows the possibility of testing the sterility requirement is carried out in the sterile-filtered whole product and allows for rinsing of inhibitory or final bulk drug substance (note: Ph. Eur., in its General preservative substances from the membrane19. Monograph Monoclonal Antibody for Human Use, uses Irrespective of the compendial method chosen for 16 “final lot” for drug substance) and final filled product sterility testing, two different media must be used 10 17 in compliance with USP <71> , Ph. Eur. 2.6.1 and/ depending on the microorganisms whose presence 18 or Japanese Pharmacopeia (JP) 4.06 . The methods for is being tested. Each of these media is incubated at sterility testing approved by regulatory agencies are based different temperatures. Fluid Thioglycollate Medium on direct inoculation or membrane filtration. (FTM) is mainly used for the culture of anaerobic As a precaution against contaminating the sample, both bacteria, although it can also support the detection of sterility methods are performed under aseptic conditions, aerobic bacteria. FTM is incubated at 30–35ºC. Soybean- but these conditions must not interfere with the detection Casein Digest Medium, also called Trypticase Soy Broth of any existing microorganisms in the sample. (TSB), is used to test for the presence of both fungi and These compendial sterility tests are based on bacterial aerobic bacteria. TSB is incubated at 20–25ºC10. growth methods with incubation times of at least 14 At regular intervals during the incubation period and days and have been harmonized with regards to the at the end point, the media must be examined for testing components such as media, growth promotion macroscopic evidence of microbial growth (turbidity). requirements, suitability tests, incubation conditions, If no evidence of microbial growth is found, the product amount of material to be tested, number of containers is deemed acceptable (i.e. is sterile). If, however, evidence required and the way in which results are observed and of microbial growth is found and the test is considered 19,20 interpreted . valid, the product does not comply with the sterility test. In the direct inoculation method, a pre-defined volume If the test was considered invalid, it must be repeated of product (the volume of the product to be tested must with the same sample size as in the original test. If the not be more than 10% of the volume of the growth material being tested renders the medium turbid so that medium) is transferred directly into the appropriate the presence or absence of microbial growth cannot be culture media and incubated for at least 14 days at the determined by visual examination at the end of the 14-day appropriate temperature10,17. incubation, portions of not less than 1mL of the sterility test containers must be transferred to fresh containers In the membrane filtration method, the product to be of the same medium and then the original as well as the tested is passed through a set of filters of appropriate subcultures must be incubated for a minimum of four porosity to retain any microorganisms present in the additional days10,17. sample. The filters are then rinsed to remove any retained product, transferred to the appropriate growth media When a new product is tested for sterility or when one or and incubated in an enrichment broth under appropriate more sterility testing parameters are changed, a method conditions for at least 14 days10,17. suitability test needs to be performed to show that the product conforms to USP <71>, Ph. Eur. 2.6.1 and JP 4.06. While direct inoculation methods provide a means of sterility testing for materials that are not easily filtered, membrane filtration is the preferred method for sterility

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 10 3.2 Mycoplasma testing Two classical methods approved by regulatory agencies have been employed to test for the presence The genus mycoplasma belongs to the order of mycoplasma: direct assay based on the culture , which is one of five orders in the Mycoplasmatales method and the indicator cell culture method. class of bacteria , with the others being Mollicutes Validated nucleic acid amplification techniques are

Acholeplasmatales, Anaeroplasmatales, Entomoplasmatales also accepted21 for mycoplasma detection, so long and . represents a group of Haloplasmatales Mollicutes as such methods are equivalent to the other two more than 120 species of the smallest self-replicating compendial methods26. These methods are performed prokaryotic organisms. have no cell wall, which Mollicutes employing aseptic techniques, suitable laboratory makes them penicillin-resistant. The cells vary in size conditions and trained personnel11. and morphology and cannot be Gram stained, however Direct, growth-based assay: culture method impressions of colonies on solid agar can be stained with methylene blue or equivalent stain. Most Mollicutes are The principle of detection by this method is based on the parasites and commensals, and some may be pathogenic to culture of mycoplasma on an agar plate after enrichment a variety of animal and plant hosts. In humans, mycoplasma in liquid media or broth. are usually surface parasites that colonize the epithelial Per current compendial requirements, the sample to be lining of the respiratory and urogenital tracts. Mycoplasma tested is inoculated in liquid medium (broth) in a tightly infection of a cell culture may persist for prolonged time closed container and incubated for 21 days. periods without apparent cell damage. However, these At defined time-points specified in the pharmacopeia11, 21, infections can affect nearly every metabolic pathway of the material in liquid medium is subcultured onto solid the cells in the culture, including their phenotype and media and incubated for a minimum of 1411, 21 days normal growth. Mycoplasma presence is also difficult (except the last subculture which requires only seven to observe, since it does not always result in increased days). After the incubation period11, 21, all inoculated turbidity in infected cultures or visible alteration of the solid media plates are examined microscopically for the 11 cells . To ensure the safety of biological products, testing presence of typical mycoplasma colonies with a specific for both cultivable and non-cultivable mycoplasma colony morphology (‘fried egg’ or finely granular colonies contamination in biological products is required by the with a berry-like appearance that penetrate the agar 11, 21, 22, 23 worldwide regulatory authorities . Cell lines used surface). The liquid media is also examined every two or for the manufacturing of human biological products three days and if any color change occurs a subculture (master cell banks and working cell banks), cell cultures, must be performed immediately11, 21. end of production cells and master and working virus seeds should also be tested for the presence of mycoplasma, as stated in the US Code of Federal Regulations (CFR) for biological products16, 24, 25. For production lots, testing for mycoplasma contamination should be performed in unprocessed bulk products8. Live virus vaccines should be tested prior to clarification or filtration and inactivated virus vaccines should be tested prior to inactivation24.

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 11 Indirect assay: indicator cell culture method Nucleic acid testing (NAT) The indicator cell culture method is based on the Nucleic acid testing methods are based on the cocultivation of the test sample with an indicator cell line detection of targeted nucleic acid sequences, which are (Vero cells or equivalent cell lines such as Mv1Lu cells) then amplified for their detection. The most common and incubated until the culture yields confluence, after at NAT assay is the PCR. Unlike the culture methods least three days. After this, subcultures are inoculated at described above, the performance of NAT assays low-density and incubated until cells reach approximately normally only take hours. These technologies offer 50% confluence (after three to five days), since complete extreme sensitivity and are capable of generating confluence would impair visualization of mycoplasma after millions of amplicons from as little as a single nucleic the staining step. The subculture is fixed to the surface acid template using oligonucleotide primers that bind using a fixing solution and then stained with a fluorescent specifically to a region or number of regions of the dye that binds to DNA. The subculture is then examined by DNA, leading to their amplification into detectable fluorescence microscopy. The presence of mycoplasma is levels. The amplification of these regions is performed characterized by their particulate or filamentous pattern along repeated amplification cycles (usually 30–45) at of fluorescence on the surface of the cocultured cells and the optimum elongation temperature using the enzyme in the intracellular areas11, 21. DNA polymerase27, 28. There are many different versions of PCR (see appendix), but all PCR consists of three main steps: 1. Denaturation of the double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA) at high temperatures 2. Annealing of primers to specific ssDNA sequences 3. Extension or elongation of the annealed primers by the DNA polymerase enzyme, using the ssDNA the primers are bound to as the template27. For the detection of mycoplasma, real-time PCR for real-time visualization of the DNA after each amplification cycle is commonly used. Quantitive PCR (qPCR) provides a method for the quantitation of starting DNA in the sample. The amplified DNA is detected by fluorescence and the intensity corresponds proportionally to the amplification of the target sequence, which can be measured as the amplification progresses. Three main fluorescence detection systems are used: DNA-binding agents such as SYBR green, which binds all dsDNA following each PCR cycle, fluorescent primers and fluorescent probes27.

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 12 3.3 Virus testing To ensure adequate sensitivity and specificity, the assay requires specific controls8. The virus production cell line All biotechnology products derived from cell lines share can be used as an indicator to identify cell lines capable a common risk for viral contamination, which could have of propagating the virus and using them in the assay as a serious consequences. Virus testing must therefore be positive control9. The cells used in the assay should include performed during cell line qualification (master cell bank, a human and/or a non-human primate cell line susceptible working cell bank and cells at the limit of cell age) in vitro to human viruses, which could potentially be present and unprocessed bulk. While testing is performed once for based on the origin or handling of the cells8. In addition to each one of the cell banks, every lot of unprocessed bulk cell infectivity assay, the PCR may be appropriate for the material needs to be tested8. detection of sequences of any specific viruses (e.g. Rodent Different virus testing methods can be performed during Parvovirus)30. Tests for retroviruses include infectivity cell bank qualification. Table 1 is taken from ICH Q5A8 assays in sensitive cell cultures and EM studies; for and outlines numerous assays that can be used for non-infectious retrovirus, where no sign of infectivity or the detection of endogenous and adventitious viruses retrovirus-like particles could be observed by EM, the next and their limitations. In vitro testing for adventitious suitable assay is reverse transcriptase (RT) PCR8. agents is carried out for both cell bank qualification and The in vivo testing is conducted during cell bank unprocessed bulk8, in compliance with the 2010 FDA qualification, in compliance with 21 CFR Part 630.1831, 16, Ph. Eur. 2.6.1629 (with special Guidance for Industry 21 CFR Part 630.5532 and 21 CFR Part 58 Good focus on viral vaccines for human use) and ICH Q5A8. Laboratory Practices33, and involves the inoculation of The biggest difficulty in quantitative virus assays is that specific animal species (e.g. suckling and adult mice, and the ability to detect low viral concentrations depends embryonated eggs) to reveal viruses that cannot grow in (for statistical reasons) on the size of the sample. For this cell cultures and testing the tissues for the presence of reason, no single approach will necessarily establish the hemagglutinins8. These assays are considered necessary safety of a product and, to gain confidence that infectious to detect new or emerging viruses and to complement virus is absent from the final product, a combination of narrowly focused virus-specific assays such as PCR9. direct testing and demonstration that the purification regimen is capable of removing and/or inactivating the Numerous publications show that for almost every virus viruses is required8. studied, the in vitro assay is more sensitive than the in vivo ones. A study performed to evaluate the selectivity Currently, virus detection by is performed using in vitro and sensitivity of the routine methods described above multiple susceptible indicator cell cultures capable of using 16 different viral stocks reported that the in vitro detecting a wide range of human and relevant animal systems used for routine test of bulk harvest viruses and inducing cytopathic effect, hemagglutination (e.g. MRC-5, Vero, HeLa cell line) could reliably or hemadsorption9. The sample to be tested is inoculated detect the viruses tested, whereas the in vivo systems onto the cell cultures, which are examined at different time were unable to detect about 50% of these viruses9. points over a 14-day period. The use of multiple cell lines Nevertheless, the sensitivity of the virus detection can and animal hosts derives from the knowledge that viruses be improved using PCR-based detection assays34. can have a limited host range or exhibit tropisms for specific cell types or tissues. Certain host systems may be more permissive for virus replication and therefore more likely to show CPE in vitro or mortality in vivo16.

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 13 Table 1: Use and limitation of adventitious virus testing (adapted from ICH Q5A)

Test Test article Detection capability Detection limitation Estimated assay time (days) Typically used for

Antibody production Lysate of cells Specific viral antigens Viral antigens do not induce a 49–63 (depending on animal Cell bank and their culture response in test system species) qualification medium

In vivo virus screen Lysate of cells Broad range of viruses Viral agents failing to replicate 49 Cell bank and their culture or produce signal in the animal qualification medium test system endpoints

In vitro virus screen Lysate of cells Broad range of viruses Viral agents failing to replicate 14–28 (lot release test) Lot release test for: and their or produce signal in cell and Cell bank culture medium; culture test system 28 (cell bank test) Cell bank characterization intact cells for qualification Production screen cocultivation Unprocessed bulk harvest or lysate of cells and their cell culture medium from the production reactor

Transmission Viable cells Virus and virus-like Qualitative assay with 35 Cell bank electron microscopy particles assessment of identity qualification (TEM) on: Cell-free culture Cell substrate supernatant Cell culture supernatant

Reverse transcriptase Cell-free culture Retroviruses and Only detects enzymes 14 Cell bank (RT) supernatant expressed retroviral RT with optimal activity under qualification preferred conditions. (only performed Interpretation may be difficult if infectivity is due to presence of cellular not detected and enzymes; background with no retrovirus or some concentrated samples retrovirus-like particles have been observed by EM)

Retrovirus (RV) Cell-free culture Infectious retroviruses RV failing to replicate or form 49 Cell bank infectivity supernatant discrete foci or plaques in the qualification chosen test system

Cocultivation Viable cells Infectious retroviruses RV failing to replicate 49 Cell bank 1. Infectivity 1. See above under RV qualification endpoint infectivity 2. TEM endpoint 2. See above under TEM 3. RT endpoint 3. See above under RT

Polymerase chain Cells, culture Specific virus Primer sequences must be 1–2 Cell bank reaction (PCR) fluid and other sequences present. Does not indicate qualification and/or materials whether virus is infectious lot release

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 14 4.0

Drivers for change

As already stated, sterility, adventitious agent and So why is there such an urgency to deploy RMM mycoplasma testing continue to be the rate-limiting for real-time adventitious agent testing now? The steps for product release. From the patient’s point answer lies largely with the changing paradigm of the of view, this may have the effect of a shortage of biopharmaceutical industry. Some of these drivers for critical drugs available for administration to those change are described below. who are critically ill. From the drug manufacturer’s The issue of drug shortages is increasingly on the radar of perspective, this may require them to have large regulatory agencies and in 2018 was regarded by some as a safety stocks of inventory to ensure continued drug 37 availability in the face of long-term demand forecasts. public health crisis . In a statement released last year, Scott Such safety stocks potentially incur warehousing Gottlieb, Managing Director of the FDA, said: “While the costs and, more importantly, are not reaching patients. causes of drug shortages vary, most shortages are due to Further, for drugs only needed in limited supply, such disruptions in supply chain availability of actively marketed as orphan drugs, delays due to pending sterility results products. Among these interruptions, manufacturing and may effectively cut off the only supply chain available quality issues are the leading causes of drug shortages.” for potentially life-saving treatments. In July 2018, the FDA launched the Drug Shortages Task Force, with the objectives of not only clearly identifying By using alternative rapid adventitious agent detection the reasons for drug shortages, but, importantly, to drive methods the business risk for biomanufacturing processes long-term solutions to mitigate this risk38. In further can be minimized, since possible mycoplasma or virus communication from the FDA describing this initiative, it contaminations can be limited to harvest equipment was stated: “Manufacturers of generic drugs, with lower without contaminating expensive downstream profit margins than their brand name counterparts, may be equipment. This therefore strengthens the ability to hesitant to invest capital to upgrade their facilities to pass ensure uninterrupted supply to patients. In addition, an inspection, expand production or improve the reliability decontamination efforts will be less extensive and will of supply.” The document further stated that through result in reduced facility shutdown. Overall, significantly the Emerging Technology Program, the FDA are actively reduced reaction times will help ensure uninterrupted encouraging manufacturers to adopt new technologies that supply to patients. can facilitate production and mitigate the risk of not being The concept of using alternative RMM in bioprocessing able to supply urgently required medicines. Although testing applications has been discussed for more than two for adventitious agents is only one of multiple steps involved decades. Although some users have successfully validated in biopharmaceutical production, for instances of drug RMM to screen raw materials or perform environmental shortages, a delay in release of an urgently required drug 35,36 monitoring , there has been a reluctance to validate pending a 28-day culture-based evaluation for mycoplasma with an aim to adopt such methods for final product is clearly not aligned with this initiative. testing. As demonstrated by the results from the survey, A change to an alternative sterility test is also believed to this reluctance can be attributed to many factors, contribute to reduced manufacturing costs. For sterility including the cost, time and effort required to validate testing, there will be a cost saving associated with reduced a new method, proof of non-inferiority to current warehouse storage costs for drugs pending final sterility compendial methods, unwillingness to make a change results prior to release. It is also important to consider to an approved drug submission and difficulties, either that alternative RMM cost savings can be realized in the perceived or real, in obtaining regulatory approval for context of a faster response to a contamination event and adoption of such methods. subsequent corrective actions, extended sterility sampling plans or reprocessing costs39.

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 15 However, an even more important driver for adoption of proposed a new general information chapter new technologies for rapid sterility and adventitious virus (Ref: <1071> Rapid Sterility Testing of Short-Life Products: testing is the new generation of personalized medicines, A Risk-Based Approach)42. By adopting a risk-based approach, usually referred to as ATMPs. For this rapidly growing field the expected outcome is adoption of alternative RMM that of products, testing for adventitious agents using traditional will accommodate the unique needs of sterile compounding, compendial methods is simply not an option. ATMPs may positron emission tomography (PET) and ATMPs. be further defined as gene therapy medicinal product, There has been increased visibility in recent years to the somatic-cell therapy medicinal product, tissue-engineered possibility of recombinant protein production from plants, product and combined advanced therapy medicinal insects or fungal cell lines43, 44, 45, 46. However, along with 40. In terms of sterility and adventitious agent testing, product the potential for increased protein yields and reduced some cellular and gene-based therapies have additional manufacturing costs, it is critical that risks to patient safety complexities to be considered, including41: be addressed. Past experience using novel cell lines has • Small batch size – one batch may be administered shown the risk for contamination by adventitious agents in its entirety to one patient, creating a conundrum such as Alphanodavirus47 and Sf-Rhabdovirus48. However, as in terms of available product for microbial safety research into novel cell lines develops, it seems inevitable testing using current compendial methods. This that use of advanced molecular methods, such as next- also means that sample retention is not possible generation sequencing, may be the only realistic option to for later testing. Further, even if very low product mitigate the risk of known viruses for which no standardized volumes are available, this may be inadequate to assay exists or to detect previously unknown viruses. detect low levels of contamination. Therefore, for There is also increasing interest in novel manufacturing such applications, any future RMM may have unique platforms such as continuous bioprocessing. One of the key requirements in terms of increased sensitivity with a regulatory concerns for continuous processing is bioburden lower sample volume; control and virus safety, and how adventitious agent safety • Short shelf lives – same-day product release may can be achieved on a real-time basis. All the potential be required in many cases. Further, potential advantages of continuous bioprocessing, including flexibility heterogeneity between products with short shelf of manufacturing and reduced manufacturing costs, are lives may impact validation of current sterility and unlikely to be fully realized if final formulated product adventitious agent screening (e.g. assay interference requires storage for at least 14 days pending the results from antimicrobial steps in manufacturing); of sterility testing. Further, with continuous bioprocesses that could extend from weeks to months, use of alternative • Production environment – in many cases, production RMM for in-line monitoring seems intuitive to mitigate the of the final medicine may be in a hospital or blood risks of a potentially catastrophic contamination event. It center to facilitate administration to the patient. is anticipated that development of alternative RMM for This may limit potential technologies for assessing confirmation of process control could then be leveraged adventitious agent safety prior to administration. towards the ultimate goal of real-time release. These novel medicines provide great promise in terms of Finally, the results from the survey indicated that patient treatment, yet present a challenge in terms of patient collaborative studies with regulatory agencies, comparability safety. How can such an encouraging new treatment that is studies between different methods and sharing of clearly warranted be assured for patient safety with current standardized protocols and validation strategies within compendial methodology? Clearly that is not possible and the biopharmaceutical industry are key factors in driving this, perhaps, is the strongest driver for the adoption of new regulatory acceptance of alternative RMM. Perhaps the alternative RMM, as described later in this paper. current plea for action is most appropriately summed Indeed, there is growing recognition of this potential up by Charles Darwin: “In the long history of humankind conflict, and guidance on implementing alternative RMM is (and animal kind too) those who learned to collaborate 13 14 provided in USP <1223> cand Ph. Eur. 5.1.6. or and improvise most effectively have prevailed.”49. For the 41, 55 Ph. Eur. 2.6.27 . Further, to specifically address sterility advancement of alternative RMM, it seems that time is now. testing of products with short shelf lives, the USP has

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 16 5.0

Overcoming barriers to change

Alternative RMM can produce faster results (hours vs weeks) and potentially offer the same, or even better, sensitivity and range than current counterparts. Despite these potential advantages, several challenges have slowed the implementation of alternative RMM in place of compendial methods. To better understand these barriers, a focused survey was conducted with biopharmaceutical end-users representing 11 companies. The survey validated that compendial methods were still the predominant method being used and, indeed, that ‘time to results’ was the overwhelming number one reason for dissatisfaction (Figure 1).

Figure 1: Areas of dissatisfaction with current GMP lot release tests

% Survey respondents 20

0 Test duration – Ease of operation Test sensitivity Other Cost of test time to results Area of dissatisfaction with current GMP release test

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 17 When asked to name the three key impediments to implementing alternative RMM, the top concerns among respondents were: 1) Regulatory acceptance, 2) Filing regulatory change control and 3) Time for test validation (Figure 2). It is interesting to note that technical concerns, such as assay sensitivity, did not rank in the top three. Rather, the top-three impediments were considerations that are agnostic to the method and predicted to be concerns for future methods as well.

Figure 2: Impediments to implementing alternative rapid microbiology methods for GMP release testing

Regulatory acceptance Filing regulatory change Time for test validation Decreased test sensitivity Increased test sensitivity Difﬁculty in interpreting results Other Cost of test validation Increasing testing costs

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 18 With respect to regulatory acceptance, when asked to select the two key activities that would have most impact, respondents named comparability and collaborative studies as their top two, followed by the existence of a standard protocol used by the industry (Figure 3).

Figure 3: Activities to drive regulatory acceptance of alternative rapid microbiology methods

Comparability studies between methods

Collaboration studies with agencies

Standard protocol used by industry

Publications

Co-development of test Activity Presentations

Others

Availability of certiﬁed standards

0 10 20 30 40 50 60 70 80 % Survey respondents

Ultimately, release assays are used to ensure the safety of product for use. Implementing rapid methods for this purpose is more complex and involved than just the factors identified in the survey. It is recognized that data from surveys should not be overextended or interpreted, but rather provide a basis for identifying shared industry concerns and activities to overcome barriers, including collaborative studies. The concerns cited in the survey can be broadly classified into four main categories: technical, cost (monetary and/or time), regulatory and behavioral. Three of the four categories of impediments (technical/operational, cost and regulatory) are also indicative of key attributes that new assays should have, regardless of the platform; new assays must be technically sound and non-inferior to the traditional compendial methods, have a favorable cost–benefit ratio and be accepted by regulatory agencies.

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 19 5.1 Technical/operational attributes RMM for marketed products, such questions can derail and impediments or delay an initial filing and, even if accepted in the initial filing, may result in difficult discussions in inspections. Technical/operational evaluation is the primary key to As discussed in section 5.3 (Regulatory acceptance) on determining the suitability of a proposed new technology. regulatory pathway concerns, mechanisms are in place for Properly addressing the technical and operational addressing the acceptability of new technologies that open concerns, along with a good analysis of the cost–benefit heretofore-unaddressed issues. ratio, will significantly improve the likelihood of The third key point to address, per USP <1223> and overcoming any regulatory or behavioral impediments to Ph. Eur. 5.1.6.13, 14, is that it must not be inferior to the implementing RMM. compendial method, to ensure that patient safety is not The first key point to address in the technical evaluation of compromised and that it can be validated. The critical a new method is whether it meets the user requirements. parameters, such as sensitivity/limit of detection, The assays should be simple to perform because they specificity, accuracy and precision (for an alternative are conducted on a routine basis and are expected to qualitative method) must be adequately addressed50. produce reliable results. Excessive manipulation, expert Inherent in the validation assessment is an evaluation technical hands and extensive analysis are not desired of whether the technology meets user requirements as features for such methods. In addition, the results should validation tests and whether the technology can reliably be easy to interpret. The output should be clear and operate within the required parameters. The question of unambiguous. When results are difficult to interpret the ability to validate should be an essential gatekeeper or sensitivity is reduced, then the rapid method is function used to sort technologies to be pursued from inferior to the compendial method and is not currently a those not currently ready for adoption into manufacturing. mature candidate for adoption. Similarly, false positive Promising but not ready-for-adoption technologies should results suggest that the new method is not ready for be monitored and potentially invested in by companies and implementation. Without a clear path to overcoming regulatory stakeholders because improvements over time these limitations, the new method can be shelved, thereby may overcome the current barriers. obviating the need to consider the behavioral, financial Finally, operational concerns must be addressed. A or regulatory impediments that might result from an dependable supply chain for suppliers/materials of the attempted adoption. alternative method is essential, and ideally multiple and The second key point to address during technical redundant sources would be available. However, with new evaluation of a new method is whether the technology technologies, the supply chain might not be sufficiently opens GMP questions that cannot currently be answered. robust, which could lead to time- and money-consuming One of the technical impediments cited by survey deviations and/or inability to release product. As an respondents, increased sensitivity, raises this conundrum; example, sole-sourcing a new technology is often inherent increased sensitivity inherently appears to be better, due to the newness of many technologies and intellectual but it runs the risk of detecting objects or artifacts that property barriers to others wishing to enter the same were previously unknown, unseen and/or are difficult technological space. Additional operational assurance can to characterize and explain. For example, new visual be gained through conducting an operational equivalent methods for microbial and viral detection can potentially of a hazard analysis and developing countering strategies detect viable, but not-cultivable microorganisms, virus for each concern. Mitigation options could include capsids and sample debris that are not detected using service-level agreements with vendors, pre-positioning classical growth-based methods. This ‘novel’ information or warehousing sufficient consumables to weather a must be explained, assessed for impact and may conflict temporary outage, etc. with existing limits or specification, which are currently Once technical and operational challenges are overcome, based on e.g. colony forming units that rely on the the process of gaining agreement and/or sponsorship growth of contaminants and the generation of colonies. for the change must then navigate the social aspects of It is insufficient to simply state that the background has change management. always been present. Besides hampering changing to

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 20 5.2 Cost–benefit ratio 5.3 Regulatory acceptance Once technical concerns have been addressed, the The two primary concerns cited in the survey were the cost–benefit ratio must be considered. The new assay possible lack of regulatory acceptance and filing post- should provide significant benefits in cost reductions, time approval changes. In both cases, careful consideration savings, data integrity or increased operational efficiency. must be given to the regulatory implementation strategy These benefits can be essential for newer products, for new technologies. Both companies and regulators such as viral and cellular gene therapy or personalized have little or no prior experience with approval of these therapeutics, which have short shelf lives and cannot new technologies and, therefore, strictly speaking, afford to have lengthy lot release tests on the critical there are no regulatory hurdles, but a lack of experience path. Conversely, for batch processing of a recombinant from both industry and regulators with how to navigate protein, such benefits may not be critical but are still implementing new methods. In some cases, notably in highly desirable because a faster release time will allow the cell and gene therapy areas, a more than persuasive more capacity, less inventory time, drive productivity and, imperative for adoption by regulators is driven by the ultimately, get products to the patient faster. need for new technologies that shorten timelines to meet The benefits of the new assay must also outweigh the dire patient needs. But, in most cases, the rationale for change costs, which were cited by survey respondents as regulatory acceptance does not rise to the level of an a significant concern when considering the replacement imperative. However, activities are ongoing to facilitate of compendial methods with alternative rapid methods. industry implementation, for example at the Center for Change costs cited in the survey include time for validation Biologics Evaluation and Research (CBER) to evaluate the 51. and the monetary cost associated with implementing and performance of NGS-based virus detection methods validating the new methods. The time and cost of other As innovation is driving new methods, some regulatory activities (assay development, comparability testing, filing agencies are signaling that they welcome them. while regulatory changes, etc.) were not specifically addressed in others may be more conservative and reluctant to change. the survey, but should also be considered. Consequently, organizations that wish to apply to several countries may adopt a more conservative approach to A context-specific business case should be developed for the ensure broad acceptance and avoid the perceived risk of technology to define the cost–benefit ratio and to provide delayed filing or dual-testing scenarios. assurance that the chosen technology is appropriate for the intended application. The business case will also provide a To facilitate acceptance, the new method should be justified documented baseline for why the technology was chosen, through a risk–benefit assessment and be supported by and in combination with the technical case will facilitate the a solid validation/comparability study. Comparability or process of achieving regulatory acceptance and overcoming equivalency is a key component of this assessment; to behavioral impediments. achieve regulatory acceptance, the new method must provide non-inferior assurance of patient safety. One risk with a new method is going to the regulatory agencies in a silo effect, where each company approaches individually with the package. If possible, new methods along with the validation should be shared via peer- reviewed publication and information sharing forums to drive uptake. Additionally, both the FDA and the EMA have established formal processes for discussion of new technologies prior to submission52, 53.

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 21 Adoption of the new technology into the clinical 5.4 Behavioral development space as the product and process is being A recognized concern in adopting new methods that was developed builds the case for the technology through evident from the survey is the ‘lack of management buy- the early regulatory filings. It is the simplest and least in’, which is considered a behavioral barrier. Behavioral burdensome pathway, but it is only available for unlicensed barriers frequently stand in the way of making changes processes and, even here, it represents a gamble to current operating practices. Behavioral barriers are because it could hold back the new product if the RMM impediments based, not on fundamental flaws in the is not accepted. This risk can be mitigated through early planned change, but rather concerns in the minds of consultative meetings with the FDA and the EMA. management and reviewers. Among the issues raised could For new technologies to be submitted to the FDA, a meeting be financial concerns, regulatory acceptance concerns, with the US regulator’s Emerging Technology Team (ETT) questions about the readiness of the technology for routine may be requested to discuss aspects of the proposed novel use, perceived issues with assay supply chain robustness, technology or novel implementation. ETT meetings are lack of understanding of the technology and risk aversion. routinely granted and are non-binding, therefore provide These issues are classifiable as to their primary root cause, an excellent opportunity for initial feasibility assessment. and performing such an analysis is useful for understanding ETT meetings can also be a valuable first step in driving the points of view, developing strategies for enhancing probability of eventual regulatory acceptance of the new communications and resolving barriers to change. Table 2 technology and/or its application. A formal Type C meeting can be used as a tool in such analyzes. with adoptability/approvability of new technologies or applications as an agenda topic may also be requested. However, Type C meetings are binding and therefore are recommended as a follow-up to an ETT meeting, rather than as a venue for the initial discussion of the new technology. Consultative meetings with the EMA can happen either at a centralized meeting or at a meeting of the health authority of a member state with mutual recognition by the other states. The choice of the pathway is largely dictated by the choice of the filing strategy, which in turn may be dictated by the therapeutic target. The information from these meetings is not binding. Already licensed processes have a higher burden of proof, since a proven technology has already been accepted through the license. Additionally, complicating post- licensure changes (post-approval changes) is that country- by-country license amendments create a shifting roster of markets that have accepted the change and those that have not yet done so. A strategy for managing this scenario based on the particulars of the product and the markets must be developed in consultation with regulatory specialists, e.g. a so-called ‘deferred implementation’ strategy.

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 22 Table2: Analysis of potential behavioral barriers to the adoption of new technologies

Behavioral barrier Primary barrier driver/form of expression Barrier classification Means to mitigate

Financial • lack of a cost-benefit analysis • lack of knowledge/fear • perform analysis • no compelling cost–benefit ratio • inadequacy, fear of financial inadequacy • do not pursue if true

Regulatory • lack of regulatory surveillance • lack of knowledge • perform analysis acceptance • risk of delaying filing • fear • both cases: ensure a compelling • no prior filings • first adopter reason to adopt and a pathway to acceptance is outlined

Readiness of the • technology feared to be not ready • fear • compile data to provide counter technology argument

Assay supply chain • unacceptable level of risk due to assay • lack of control • develop additional suppliers, robustness supply chain (e.g. sole source, weak contractual service-level suppliers) expectations, etc.

Lack of understanding • lack of knowledge • fear • educational presentations of the technology

Risk aversion • lack of knowledge • fear • articulation of risk–benefit ratio and probability of the risks • implementation of operational assurances

Inertia • human nature • comfort with the status quo • develop a compelling narrative for why the ‘activation energy’ for the change must be invested

5.5 Summary Implementation of novel technologies into GMP processes can run afoul of numerous technical and non-technical concerns. Technical concerns must be addressed first. Novel technologies must not be difficult to perform, must not be inferior to the compendial methods, must be possible to validate and must not create novel difficult-to-answer GMP questions. Operational concerns such as supply chain management should be addressed and the new method must have a favorable cost–benefit ratio. A regulatory strategy for implementation must be in place, possibly including comparability studies, peer-reviewed publication, and meetings with the FDA and EMA, among other things. Finally, behavioral barriers may arise that could be rooted in real technical, operational, cost or regulatory concerns. Methodical technical evaluation, a compelling business case including cost–benefit and careful planning of regulatory implementation strategies can be used to create a compelling and persistent message to overcome these concerns.

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 23 6.0

Advocacy for improvement and change

The objective of this white paper was However, the biotechnology world is evolving rapidly, to identify industry barriers associated and the unprecedented growth and interest in ATMPs with adoption of alternative RMM for the is forcing a change to the status quo. According to the detection of adventitious agents. From this Alliance for Regenerative Medicine, there are currently study, we determined that the biotechnology more than 906 regenerative medicine companies sector shared common barriers and, worldwide and 1,028 clinical trials involving ATMPs subsequently, could share common solutions. are in place globally54. It has long been recognized that This paper is a step towards the ultimate testing ATMPs for adventitious agents using current vision of real-time product release using compendial methods may not be feasible and regulatory such methods and delineating strategies that guidance documents have indicated support for the use can be used to overcome the current barriers of alternative RMM in such applications41, 55, 56, 57. to implementation. Further, some end-users have publicly shared their The benefits of alternative RMM were described strategies and successes in validating alternative in this paper, including reduced warehouse storage methodologies to replace compendial methods for costs, faster responses to contamination events sterility testing with ATMP applications58, 59. and, most importantly, a reduced time to get a safe Therefore, it appears that the developers of ATMPs drug product to the patient. However, based on will pioneer the validation and implementation of rapid the team’s research, concerns over execution of microbiology testing into their bioprocessing operations. validation testing, acceptance of the methods by By sharing information on this journey, it is expected the regulatory agencies and the effort involved in that other biotechnology end-users will realize that the filing a regulatory change have prevented perceived barriers are not insurmountable and will follow end-users from moving away from current the lead in the journey towards real-time release. compendial methods, despite these benefits.

Conclusions and path forwards Alternative RMM for release testing of biologics stand to have a significant impact for the biopharmaceutical manufacturer. Scientific advances are providing new, faster methods that potentially offer the same or increased sensitivity. Novel biologics such as ATMPs are releasing product using rapid sterility and mycoplasma methods because their short shelf life drives the use of rapid methods. However, to date, these represent the exception and widespread adoption of rapid methods for product release is slow to none.

As noted from the survey, the key hurdle to adoption is regulatory acceptance. To date, companies are approaching their regulatory agency with individual rapid method validation packages. This ‘silo-ed’ approach causes variations in validation approaches seen by the agencies, thus complicating and, more importantly, delaying the uptake of the new method. Additionally, the cost and time of performing validations can be a barrier to companies as well. This body of work has identified the need to focus on removing the barriers for rapid methods implementation. Ultimately, the method needs to provide assurance of the product’s safety profile, which is paramount to patient safety. Today, there are rapid methods that are available but not used in the release testing setting. It is recognized that methods will continue to evolve, becoming more sensitive, robust and accessible. However, regardless of the method, the principles that go into a validation study are constant with some information being already available14. It would benefit both regulatory and industry stakeholders to further align on what a validation study should include and share/leverage validation data on a method across different sites and product types. By doing so, industry can save time and resources, and regulatory agencies will view a single consolidated validation package. At a BioPhorum/FDA meeting in October 2018, this concept was introduced and all parties could see the potential benefits from such an approach.

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 25 To facilitate the strategy, this team of interested stakeholders plans to develop a Rapid viral and mycoplasma method validation framework guidebook. In this document, user requirement specifications for alternative RMM will be defined and parameters outlined for a multi-site and collaborative validation approach suitable for regulatory submission. A template validation protocol for a multi-site validation study will be included and its use illustrated with case studies using current methods, such as next-generation sequencing. The guidebook can serve as a discussion framework for industry and regulatory stakeholders to drive the uptake of rapid release methods for mycoplasma and viruses. The implementation of alternative RMM will serve to de-risk biotechnological operations, increase market supply while maintaining patient safety and will, furthermore, allow for in/at/on-line testing resulting in improved effectivity of the production line and with a potential cost reduction per test.

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Term Definition Advanced therapy medicines for human use that are based on genes, tissues or cells medicinal products Adventitious agents bacteria, yeast, mold, mycoplasma or viruses that can potentially contaminate prokaryote or eukaryote cells used in production. Potential sources of adventitious organisms include the serum used in cell culture media, persistently or latently infected cells, or the environment Alternative rapid assay methodologies to serve as alternatives to compendial microbiological methods microbiology methods Annealing recombine (DNA) in the double-stranded form following separation by heat At-line monitoring measurement where the sample is removed, isolated from and analyzed near to the process stream Bioreactor a vessel in which the central reactions of a biotechnological process takes place. Typically, the vessel contains cells grown under controlled conditions of temperature, aeration, mixing, acidity and sterility Bulk drug product a finished dosage form, for example, a tablet, capsule or solution that contains an active pharmaceutical ingredient, generally, but not necessarily, in association with inactive ingredients Bulk drug substance any substance that is represented for use in a drug and that, when used in the manufacturing, processing or packaging of a drug becomes an active ingredient or a finished dosage form of the drug. But the term does not include intermediates used in the synthesis of such substances Cell bank vials of cells of uniform composition (although not necessarily clonal) derived from a single tissue or cell, aliquoted into appropriate storage containers and stored under appropriate conditions, such as the vapor phase of liquid nitrogen Cell lines cells that have been propagated in culture since establishment of a primary culture and survival through crisis and senescence. Such surviving cells are immortal and will not senesce. Diploid cell strains have been established from primary cultures and expanded into cell banks but have not passed through crisis and are not immortal Colonization the presence of bacteria on a body surface (e.g. skin, mouth, intestines or airway) without causing disease in the person. Infection is the invasion of a host organism's bodily tissues by disease-causing organisms Colony forming unit refers to individual colonies of bacteria, yeast or mold. Used to estimate the number of viable bacteria or fungal cells in a sample Commensal microorganisms that are present on body surfaces covered by epithelial cells and are exposed to the external environment (gastrointestinal and respiratory tract, vagina, skin, etc.) Complementary DNA DNA that is complementary to messenger RNA; used for cloning or as a probe in DNA hybridization studies Continuous A process composed of all continuous unit operations running concurrently (physically bioprocessing connected) via direct connection or small-volume surge vessels Current good regulations enforced by the FDA. cGMPs provide for systems that assure proper design, manufacturing practice monitoring and control of manufacturing processes and facilities (cGMP)

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 30 Definitions /glossary (continued)

Term Definition Cytopathic effect morphological alterations of cell lines produced when cells are infected with a virus. Examples of cytopathic effects include cell rounding and clumping, fusion of cell membranes, enlargement or elongation of cells, or lysis of cells Cache Valley Virus a mosquito-borne, teratogenic Bunyavirus in the Orthobunyavirus genus of the Bunyamwera group, affecting mainly sheep Denaturation unfolding of a protein molecule into a generally bioinactive form. Also, the disruption of DNA duplex into two separate strands Deoxyribonucleic acid the basic biochemical component of the chromosomes and the support of heredity. DNA contains the sugar deoxyribose and is the nucleic acid in which genetic information is stored (apart from some viruses) Direct assay detection is based on the culture of mycoplasma on an agar plate and liquid media or broth to ensure its growth at small numbers Double-stranded DNA the basic biochemical component of the chromosomes and the support of heredity. DNA contains the sugar deoxyribose and is the nucleic acid in which genetic information is stored (apart from some viruses) Epizootic Hemorrhagic Vector-borne viral disease affecting both wild and domestic ruminants. It is transmitted Disease Virus principally by biting midges of the genus Culicoides and, although it causes disease mostly in deer, the virus can spill over into cattle during an epizootic European Medicines Government agency whose mission is to foster scientific excellence in the evaluation and Agency supervision of medicines, for the benefit of public and animal health in the European Union (EU) European Europe’s legal and scientific benchmark for pharmacopoeial standards that contribute to Pharmacopoeia delivering high-quality medicines in Europe and beyond Extension elongation of a replicate DNA strand by addition of DNA nucleotides to a growing chain that initiated from a primer and templates off a DNA complement. Also known as elongation FDA Emerging FDA team organized to promote the adoption of innovative approaches to pharmaceutical Technology Team (ETT) product design and manufacturing. Through the program, industry representatives can meet with ETT members to discuss, identify and resolve potential technical and regulatory issues regarding the development and implementation of a novel technology prior to filing a regulatory submission US Food and Drug responsible for protecting the public health by assuring the safety, efficacy and security of Administration (FDA) human and veterinary drugs, biological products, medical devices, our nation's food supply, cosmetics and products that emit radiation. The FDA also provides accurate, science-based health information to the public Fluid Thioglycollate used for sterility testing by membrane filtration or direct inoculation. It is primarily intended Medium for the detection of anaerobic bacteria Fluorescent probe fluorescently-labeled, target-specific probe designed to measure DNA amplification at each cycle of a PCR Genome all the genes carried by an organism

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 31 Definitions /glossary (continued)

Term Definition Gram stain bacterial staining method designed to differentiate types of bacteria, based on chemical and physical properties of the cell wall. Usually used as an initial classification step (Gram positive or Gram negative) Guidance document describes FDA’s interpretation of our policy on a regulatory issue (21 CFR 10.115(b)). These documents usually discuss more specific products or issues that relate to the design, production, labelling, promotion, manufacturing and testing of regulated products. Guidance documents may also relate to the processing, content and evaluation or approval of submissions as well as to inspection and enforcement policies Hemadsorption selective attachment of red blood cells to the surface of a monolayer of tissue culture cells containing a hemagglutinin-producing virus with added erythrocytes Hemagglutination binding together of red blood cells usually through an intermediary such as some viruses Indicator cell culture detection is based on the co-cultivation of the test sample with an indicator cell line and procedure incubated until the culture yields confluence Infection invasion of a host organism's bodily tissues by disease-causing organisms In-line monitoring measurement where the sample is not removed from the process stream and can be invasive or non-invasive International organization formed in 1990 for bringing together the regulatory authorities and Conference on pharmaceutical industry to discuss scientific and technical aspects of drug registration Harmonisation Japanese Pharmacopeia an official document that defines the specifications, criteria and standard test methods necessary to properly assure the quality of medicines in Japan Master cell bank (MCB) a bank of a cell substrate from which all subsequent cell banks used for vaccine production will be derived. The MCB represents a characterized collection of cells derived from a single tissue or cell Mouse Minute Virus parvovirus typically used as a representative virus particle for viral safety testing in (formerly referred to as biopharmaceuticals MVM) Microbial suitability test qualification designed to assess the antimicrobial activity of a product Mollicutes a class of wall-less bacteria descended from low G+C% Gram-positive bacteria Morphology refers to general visual characteristics of microorganisms, including size, shape and arrangement Nucleic acid testing based on the detection of targeted nucleic acid sequences Next generation term used to describe a number of different modern sequencing technologies. These sequencing technologies allow for sequencing of DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing and, as such, revolutionized the study of genomics and molecular biology Off-line monitoring the sample is tested in a conventional quality control lab outside of the production area On-line monitoring measurement where the sample is diverted from the manufacturing process and may be returned to the process stream

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 32 Definitions /glossary (continued)

Term Definition Pathogenic causing disease Phenotype the set of observable characteristics of an individual resulting from the interaction of its genotype with the environment Polymerase chain laboratory technique used to make multiple copies of a segment of DNA reaction (PCR) Primers short sequences of DNA (oligonucleotides), usually designed to a specific region of DNA. Primers anneal to template DNA and allow for extension of the new DNA strand Quality assurance part of quality management focused on providing confidence that quality requirements will be fulfilled Quality control part of quality management focused on fulfilling quality requirements Qualitative assay used to assess the general microbial quality of compendial articles. This category includes assays that are intended to demonstrate the absence of microorganisms in a compendial article Quantitative assay yield a numerical (enumerative) result in terms of the microbial content of a compendial article Quantitative a type of real-time PCR (using primers and fluorescent probes) used to track resulting DNA polymerase chain amplification in real time and, in conjunction with a known standard DNA control dilution reaction of known concentration, provides accurate initial starting concentration of unknown DNA template Retrovirus virus that uses RNA as its genome and, upon successful infection, usurps the host cells functions to reverse transcribe insert itself into the host genome Real-time PCR a type of PCR that allows for real-time analysis of PCR amplification by employing and detecting either the hydrolysis of a fluorescently labeled probe or incorporation of a dsDNA-specific Hoechst dye (SYBR Green). It is used for many qualitative and quantitative applications, including gene expression analysis, microRNA analysis for identification of cancer biomarkers, single nucleotide polymorphism (SNP) genotyping, copy number variation (CNV) analysis and even protein analysis Real-time release electronic systems, process analytical technology (PAT) and advanced process control testing strategies working in concert to enable raw material and product release contemporaneously with drug substance/drug product manufacture, thereby reducing reaction time to demand signals, lower inventory and fewer process deviations. This aspirational real-time release testing goal is to encourage the development and regulatory acceptance of rapid methods and technology platforms to replace long lead-time assays and to reduce the quality assurance review time for methods by converting them to validated in-line, on-line or at-line tests Reverse transcriptase enzyme used to convert RNA into DNA Ribonucleic acid (RNA) basic biochemical component of the chromosome that is found mainly in the nucleolus and ribosomes. Messenger RNA transfers genetic information from the nucleus to the ribosomes in the cytoplasm and also acts as a template for the synthesis of polypeptides. Transfer RNA transfers activated amino acids from the cytoplasm to messenger RNA Sterile free from microorganisms

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 33 Definitions /glossary (continued)

Term Definition Transmission electron microscopy technique used to observe structures that are difficult to resolve using light or microscopy phase contrast microscopy. A beam of electrons is passed through a fixed and stained ultra- thin specimen. The micro-structures are resolved based on the various densities, which allow the electrons to pass at different rates Trypticase Soy Digest general purpose liquid enrichment medium used in qualitative procedures for the sterility test Broth and for the enrichment and cultivation of aerobic microorganisms that are not excessively fastidious US Code of Federal an annual codification of the general and permanent rules published in the Federal Register by Regulations the executive departments and agencies of the federal government United States US agency organized to improve global health through public standards and related programs Pharmacopeia that help ensure the quality, safety and benefit of medicines and foods Validation confirmation using objective evidence, obtained from the execution of preapproved protocols, that the particular requirements for a specific intended use can be consistently fulfilled, e.g. process, system or method validations

Acronym/abbreviation Definition ATMP Advanced therapy medicinal products CFR US Code of Federal Regulations for biological products CFU Colony forming unit cGMP Current good manufacturing practice CPE Cytopathic effect CVV Cache Valley Virus ds-DNA Double-stranded DNA EDQM European Directorate for the Quality of Medicines EHDV Epizootic Hemorrhagic Disease Virus EM Electron microscopy EMA European Medicines Agency ETT FDA Emerging Technology Team FDA US Food and Drug Administration FTM Fluid Thioglycollate Medium GMP Good manufacturing practice HA Hemagglutination HAD Hemadsorption JP Japanese Pharmacopeia LCMV Lymphocytic Choriomeningitis Virus MMV Mouse Minute Virus NAT Nucleic acid testing NGS Next-generation sequencing PCR Polymerase chain reaction PET Positron emission tomography Ph. Eur European Pharmacopoeia RV Retrovirus QA Quality assurance QC Quality control qPCR Quantitative polymerase chain reaction REO-2 Reovirus 2 RMM Rapid microbiology methods RT Reverse transcriptase ssDNA Single-stranded DNA TEM Transmission electron microscopy TSB Trypticase Soy Broth USP United States Pharmacopeia

©BioPhorum Operations Group Ltd | October 2019 Rapid detection of bacteria and viruses 35 Appendix Different types of PCR

There have been many iterations of PCR since its inception • Real-time PCR using a Hoechst dye (such as in 1983, some of these include (but are not limited to): SyberGreen®). The Hoechst dye is a double- stranded DNA binding molecule that fluoresces • Touchdown PCR is used to increase PCR specificity when attached to double-stranded DNA (e.g. at the by starting with an initial annealing temperature end of an elongation cycle) and is non-fluorescent above the projected melting temperature (Tm) of the primers being used and then reducing the when it dissociates when the DNA becomes single- annealing temperature by one or two degrees stranded again (e.g. at the denaturation step). This detection involves instruments that excite (laser) with each cycle. Any difference in Tm between correct and incorrect annealing will produce and detect (CCD camera) available fluorescent an exponential advantage of twofold per cycle. signal after each amplification cycle; This allows the template-primer hybridizations • Reverse-transcriptase PCR uses RNA as a starting with the highest fidelity to outmatch those that template. The RNA is first transcribed into DNA have less fidelity. This assay usually relies on gel using reverse transcriptase and then subjected to electrophoresis for detection of the product; another PCR technique as a complementary DNA • Real-time PCR using a probe in which the PCR (cDNA) template; reaction includes a sequence-specific probe labeled • Quantitative PCR (qPCR) is a type of real-time with a reporter dye at the 5’ end and a quencher PCR that includes a DNA standard curve, which is molecule at the 3’ end. Real-time PCR uses an a dilution series of known quantities of template engineered Taq polymerase such as TaqMan®, DNA. The amount of fluorescence from an these polymerases possess a 5’ to 3’ exonuclease unknown sample can be compared to the standard activity that cleaves (one base at a time) any curve fluorescence to derive a starting quantity of DNA (in this case the probe) it encounters during template DNA; polymerization. When the reporter molecule is • Unidirectional PCR uses one primer to amplify cleaved off it becomes free of the steric quenching one strand of template DNA. This provides a linear effect of the quencher molecule and can now amplification of the template and is used in DNA fluoresce and be detected when excited at the sequencing applications15. appropriate wavelength. The sequence-specific probe also adds another level of specificity to the assay. This detection involves instruments that excite (laser) and detect (CCD camera) available fluorescent signal during each amplification cycle; • Multiplex real-time PCR uses two different primer probe sets to detect different target sequences. The reporter molecules on the 5’ end of the probes will need to excite and be detected at different wavelengths to avoid interference with each other. This method is usually used to detect internal control spikes;

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