Microbiological Attributes, Specifications, and Risk Assessment of Culture-Based Therapeutic Products

Abstract

A major challenge with emerging heterogeneous, live culture-based products is setting consistent, risk-based microbial specifications to protect the recipients of these product from potential microbial infection. A review of the literature and the published regulatory requirements demonstrates a lack of consensus as to donor and/or product infectious disease screening that may inevitably harm patients along with increased costs and delayed product availability. This review article addresses the microbiological attributes, specifications, screening methods, and risk assessment of these unique products and makes recommendations as to the path forward.

Introduction

Currently our microbiological standards largely address pharmaceutical drug products, botanicals and dietary supplements that are either sterile or have a moderate to low microbiological content using traditional culture-based methods. Emerging products, which may have health claims that contain live microbiological cultures, include probiotics, fecal microbiota transplantations, fecal-derived consortium cultures and therapeutic bacteriophage products, although sharing some common attributes, they lack comprehensive microbial standards. These live biotherapeutic products challenge standard-setting organizations, regulators, and microbiologists alike. Fundamental questions that must be answered include what is the microbial contamination risk associated with these products that target different at-risk patient populations, what would be their acceptable unintended bioburden level, and what microorganisms would be objectionable in these products, and how do we assess their formulated microbiological purity?

Description of These Emerging Biological Products

Probiotics

The definition of probiotics found in the Food and Agriculture Organization/World Health Organization (FAO/WHO) guidelines is “Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host” The U. S. Food and Drug Administration (FDA) has defined probiotics as live microbial food supplements which beneficially affect the host animal by improving its intestinal microbial balance. If a probiotic is marketed in the U.S.A. to prevent, treat or cure a disease then the FDA would consider the product as a live biotherapeutic agent and would require regulatory review and approval by the FDA Center for Biologics Research and Evaluation (CBER).

According to USP <64> Probiotic Tests probiotics are live microorganisms that, when administered in adequate amounts, may confer health benefits to the recipient. Probiotics are typically identified at the strain level as their characteristics and benefits are considered strainspecific. The USP chapter applies to probiotics produced in specialized fermenters under strict hygiene conditions for dietary supplements or pharmaceutical applications. Fermentation media are formulated to the specific growth requirements of the microbial species or strain and typically contain nutrients such as proteins, carbohydrates, vitamins, and minerals. After the microbial cells are grown, they are harvested, usually by centrifugation. Suitable protectants may be added to the concentrated probiotic biomass, and the biomass is freeze-dried or spray-dried to a powdered form. The dried biomass then undergoes formulation, which may involve blending one or more strains with suitable excipients. Formulated probiotic ingredients can be further processed into a range of dosage forms, e.g., compressed tablets, powder-filled capsules, softgels, powders, or gels.

Typically, dietary supplements are either fermented dairy products like yogurt containing live bacterial cultures, milk, juices or desserts fortified with live cultures or capsulated probiotics or suspensions marketed as dietary supplements. Probiotics have been mainly selected from the genera Lactobacillus and Bifidobacterium, because of their long history of safe use in fermented milk by the dairy industry and their natural presence in the human intestinal tract. Probiotics are promising but their efficacy is largely unproven in clinical trials (Su et al, 2020).

Regulatory and compendial expectations may be found in the FAO/WHO Guidelines, U.S. Code of Federal Regulations 21 CFR 170.3(i) Subchapter B Food for Human Consumption, USP <64> Probiotic Tests and the Food Chemical Codex Appendix XV: Microbial Food Cultures Including Probiotics.

Fecal Microbiota Transplantations

The publication of a randomized clinical trial (van Nood et al, 2013) showing that fecal transplantation was superior to vancomycin treatment of chronic, recurrent Clostridium difficile infection jumpstarted this field. Since this publication, the use of fecal microbiota transplantation (FMT) to treat chronic C. difficile (C. diff.) infection not responding to standard antibiotic therapies has become a recognized and established treatment option. As described by Carlson (2020), at a 2013 FDA-stakeholder workshop, the FDA noted that the use of FMT and any clinical studies to evaluate its safety and effectiveness to treat or prevent C. diff. infection are subject to regulation by the FDA. Following this workshop, CBER issued a draft guidance document for industry for immediate implementation. This outlined a policy to exercise enforcement discretion regarding the requirements for Investigational New Drug (IND) applications in the use of FMT when used to treat C. diff. infections not responsive to standard therapies.

These materials can be administered as suspensions in the upper or lower gastrointestinal tract or as encapsulated materials administrated orally. The relative infection risk of these modes of administration is largely unknown.

Fecal-Derived Microbial Consortium Products

An innovation to fecal microbiota transplantation is the isolation of individual species from the intestinal microbiota, their production in anaerobic pure culture from well characterized master and working cell banks, and manufacture as pharmaceutical dosage forms to treat patients with intestinal microbiota malfunctions including chronic C. diff. infections (Petrof et al., 2013). Multiple companies currently have these products in randomized, double-blinded clinical trials.

Bacteriophage Therapeutic Products

Viruses termed bacteriophages that infect and replicate in bacterial cultures, are of interest to microbiologists because of the their role in the contamination of starter cultures in cheese and other dairy product manufacturing, probiotics, bacterial cell cultures used for the production of biopharmaceuticals, and the increased potential usage as therapeutic phage preparations. Felix D’Hérelle working at the Pasteur Institute described bacteriophage, first seen in 1917, as spots on the culture plates of the dysentery bacillus Shigella dysenteriae and recognized their therapeutic value in treating dysentery. Interest in their therapeutic value was largely eclipsed by the discovery and development of antibiotics but lately due the prevalence of multidrug resistance organisms, e.g., methicillin-resistant Staphylococcus aureus, interest in therapeutic bacteriophage has been renewed.

Phage therapy uses obligate lytic phages that selectively kill their antibiotic-resistant bacterial hosts. Major advantages are their host specificity, lack of effect on mammalian cells and other human microbiota outside their host range and safety for parenteral, topical, inhalation and oral administration (Furfaro et al, 2018; Fernandez et al, 2019).

The quality and safety requirements of a therapeutic bacteriophage product will depend on the dosage form and will include a quantitative determination of the active ingredient, i.e., the bacteriophage in plaque-forming units per weight or volume using the target bacterium, a genomic identity test, the host range on a panel of target organisms, residual nucleic acid and other cellular components, sterility (sterile dosage forms), bacterial endotoxin content (parenteral products), and absence of potential pathogens (non-sterile dosage forms).

Setting Microbial Requirements for Live Culture Therapeutic Products

Requirements of the Different Therapeutic Products

A comparison of the salient features and microbiological testing requirements of live culture products is found in Table 1.

As a starting point to evaluate microbiological specifi cations, we can use the USP/Ph. Eur./J. P. pharmacopeial requirements for non-sterile drug products (Table 2).

In addition, there is a U.S. Federal Good Manufacturing Practices (GMP) requirement as found in 21 CFR 211.113 Control of microbiological contamination to exclude objectionable microorganisms from non-sterile drug products. The reader is referred to the 2014 PDA Technical Report No. 67 Exclusion of Objectionable Microorganisms from Non-sterile Pharmaceutical and OTC Drug Products, Medical Devices and Cosmetics.

Other USP guidance for microbiological specification setting may be found in USP <64> Probiotics Tests.

Challenges Associated with Microbiological Testing

Identity Testing

A consensus is developing that identity testing based on the genotype as advocated in USP <64> Probiotics Tests represents the best approach. Type stains can be recognized by PCR methods with specific primers for the specific strain used in the product. In the future, if the equipment and reagent costs become affordable, whole genome sequencing (WGS) may replace 16s rRNA base sequencing as the method of choice.

Microbial Content

As potency has always been a prerequisite to setting an efficacious and safe dosage, the author believes that the number of viable microorganisms in the product must be known and may be part of the labeling requirements. A product, like a probiotic, containing large numbers of viable organisms, e.g. billions per g, must be diluted into a countable range and enumerated on a selective culture medium using the appropriate incubation conditions. Products containing multiple microorganisms that are closely related will present unique challenges to microbial enumeration. Carlson (2020) discussed the difficulties in establishing FMT potency for release and stability testing by enumerating an anaerobic microbiota through microbial count, viable staining or a qPCR approach.

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Tests for Specified Microorganisms

Cultural isolation of contaminating microorganisms from the high background of the product is a challenge and strategies include exploiting physiological requirements such as media selection, incubation temperature, and presence or absence of oxygen (aerobic, anaerobic or microaerophilic conditions) and CO2 supplemented atmosphere. Overgrowth of the culture can be suppressed using antibiotics, media formulation, thermal shock, filtration, and bacteriophages (Lagler et al, 2015). For robust, live culture therapeutic products based on purified strains grown in cell culture such as microbial ecosystem and bacteriophage therapies, the specified microorganisms for each dosage form contained in USP <1111> should be sufficient. For example, it would be hard to justify screening lyophilized pure cultures of intestinal-derived anaerobic bacteria delivered orally in a capsule for the absence of S. aureus, P. aeruginosa, C. albicans and A. niger. Screening for the absence of E. coli and C. sporogenes may be justified.

Contaminating Microorganisms

Recent USP dietary supplement monographs for probiotics have requirements for an absence of Listeria spp. in 25 g. The author questions the justification for this requirement.

The FDA Bad Bug Book states: “Many foods have been associated with L. monocytogenes. Examples include raw milk, inadequately pasteurized milk, chocolate milk, cheeses (particularly soft cheeses), ice cream, raw vegetables, raw poultry and meats (all types), fermented raw-meat sausages, hot dogs and deli meats, and raw and smoked fish and other seafood. L. monocytogenes can grow in refrigerated temperatures, which makes this microorganism a particular problem for the food industry.”

A recent review of probiotic manufacturing emphasizes that ultrahigh temperature sterilization is used for the culture media, purified cultures are used as the source of inocula, the cultures are harvested by centrifugation, frozen by liquid nitrogen and lyophilized so the risk of listeria contamination is slight (Fenster et al, 2019).

With a heterogeneous therapeutic product like FMT the fecal material of the donor may contain intestinal pathogens without presenting symptoms of recognizable illness. As with other donations of human tissues, both the donor and stool samples may be screened to known pathogens.

In Tables 4, 5 and 6 the pathogen screening recommended by the Australian Therapeutic Goods Authority (TGA), Health Canada, the FDA, and a European Consensus Conference are summarized. The reader should be aware that, although there is not a strong consensus, screening requirements are evolving, and these summaries may not represent current regulatory thinking.

The source documents are the Australian TGA Draft Standards for Fecal Microbiota Transplant dated November 2019, the Health Canada website, Carlson (2020) for the FDA position and Cammarota et al (2017) for the European consensus.

This is a very extensive list for pathogens screening, which may be arguably unnecessary with healthy donors who exhibit no signs of intestinal infection. According to Bakken et al (2011) this can be achieved by PCR screening for C. diff. toxin B, routine screening for enteric bacterial pathogens, and screening for fecal Giardia and Cryptosporidium antigens.

One attractive solution is to use multiplex qPCR technologies that are increasingly used in a clinical setting. For example, the BIOFIRE® FILMARRAY® Gastrointestinal (GI) Panel tests for the 22 most common gastrointestinal pathogens (see Table 7) including viruses, bacteria and parasites that causes infectious diarrhea and other gastrointestinal symptoms in clinical specimens (Buss et al 2015).

On June 13, 2019, the FDA informed health care providers and patients of the potential risk of serious or life-threatening infections with the use of fecal microbiota for transplantation (FMT). Bacterial infections caused by multi-drug resistant organisms (MDROs) have occurred due to transmission of a MDRO from use of investigational FMT, resulting in the death of one individual.

According to the FDA, FMT donor stool testing must now include MDRO testing to exclude use of material that tests positive for MDROs. As E. coli is a major component of the intestinal microbiota, screening of the absence of E. coli would not be an effective strategy, as isolates would need to be screen for their antibiotic resistance. The MDRO tests should at minimum include extended spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, vancomycin-resistant enterococci (VRE), carbapenem-resistant Enterobacteriaceae (CRE), and methicillinresistant Staphylococcus aureus (MRSA). Culture of nasal or peri-rectal swabs is an acceptable alternative to stool testing for MRSA only.

On April 7, 2020 the FDA issued another safety alert warning health care providers of the potential risk of life-threatening infections due enteropathogenic E. coli (EPEC) and shigatoxin-producing E. coli (STEC) following the investigative us of fecal microbiota for transplantation (FMT). Details of the circumstances of the FMT related infections were recently published (DeFilipp et al, 2020)

The screening options are: 1) conducting dilution and disk diffusion antibiotic susceptibility screening of potential enteric bacterial pathogens, 2) using selective or chromogenic solid media for specific enteric pathogens, 3) use toxin EIA screening methods, 4) RT-PCR gene screening for antibiotic resistance, and 5) Whole Genomic Sequencing (WGS) methods.

Risk Assessment and Mitigation

Risk Analysis

In terms of relative risk of microbial infection to the recipients, fecal matter transplantations because of their heterogeneous nature and the uncertainty of the health status of the donor represent the greatest risk and bacteriophages because they are not human pathogens represent the lowest risk to the recipient. Probiotics derived from dairy cultures that have a long history of safe use, but have unproven medical benefits, have a low risk. The risk associated with intestinal-derived stool repopulating products needs more scientific or regulatory definition. Strict and facultative anaerobes are isolated from fecal microbiota as pure cultures, identified, characterized, and stored frozen as master and working cell banks. Working cell banks are used to inoculate sterile anaerobic culture broth incubated in an anaerobic isolation chamber, the cells are harvested by centrifugation, lyophilized, and encapsulated in hard shell capsules for oral administration. Using this approach, the microbial composition of this product will be known and the risk of bacterial and viral contaminants largely eliminated.

Based on the above discussion, this suggests the following order of microbial risk: fecal matter transplant >> fecal-derived stool repopulating products ≥ probiotics > bacteriophage therapeutic products.

Blaser (2019) pointed out that at least 10,000 FMT, and probably a lot more, are being performed in the U.S. annually and as a biological product heterogeneous across donors carries a real risk of transmission of infectious agents in treating patients with recurrent C. difficile infection and often other comorbidities.

To determine what pathogen should be monitored and the donor and/or stool material excluded from transplantation, we need to examine the risk to the recipient. Given the relatively recent introduction of FMT and the low numbers of annual transplantation this is a relative small body of experience. Using prevalence and severity of foodborne illness as a measure of risk, the epidemiological data suggests that the highest risk would be associated with Salmonella spp., Campylobacter spp., STEC E. coli and Yersinia enterocolitica. In contrast, foodborne Listeria monocytogenes infections, although they occur at a low frequency, are associated a high rate of hospitalization and death per 100,000 patients (See Table 8).

Specific Challenges Associated with Microbial Screening Test

Screening for Clostridium difficile

Clostridium difficile can be detected by a culture method using Clostridium difficile Selective Agar or Clostridium difficile toxin gene detection by PCR assay. As C. difficile may be a normal part of the intestinal microbiota without exhibiting symptoms, the gastroenteritis disease is best detected using the PCR assay.

Screening for Enteric Pathogens

Methods for screening for enteric bacterial pathogens may be obtained from many sources including the FDA Bacteriological Analytical Manual, the USDA/FSIS Microbiological Laboratory Guidebook, CDC Guidelines, the USP and the ASM Manual of Clinical Microbiology. Screening of specific foodstuffs for pathogens is risk-based (Table 8) and clinical microbiologists respond to patient histories and symptoms while that pathway may not be available for live culture products.

As stated above, E. coli is major component of the intestinal microbiota so screening must be directed toward enteropathogenic strains. For example, Shiga Toxin-Producing E. coli O157:H7 can be conveniently detected by a culture method using either sorbitol MacConkey agar or E. coli O157:H7-specific chromogenic agar. Shiga toxin 1 and 2 by enzyme immuno-assay (EIA) from enrichment broth supernatants or detection of the genes encoding these toxins by PCR is required for diagnosis of infection due to non- O157:H7 STEC.

Screening for SARS-CoV-2 Coronavirus

On March 23, 2020 the FDA in response to literature publications issued a safety alert addressing the use of FMT and SARS-CoV-2 recommending the identification of donors currently or recently infected with the virus, testing donors and/or donor stool for SARS-CoV-2, and developing criteria for the exclusion of donors and donor stool based on screening and testing.

Screening for Other Important Attributes

To re-establish microbial populations in the gut microbiota, attributes like acid and bile tolerance (oral administration only), adherence to the intestinal wall, and the absence of genes for antibiotic resistance should be considered during product development.

Conclusions

The advent of emerging live culture products is an ongoing challenge in terms of setting microbiological requirements. A small production experience base with these products compounds this challenge. The author encourages continued research and development for these innovative products. Perhaps with information from the NIH Microbial Human genome project, the pharmaceutical industry can apply this knowledge on how to prepare therapeutic probiotics for patients in need of these products.

References

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