Journal Pre-proof

Manufacturing Process of Investigational Microbiome Therapeutic, SER-109,

Reduces Risk of Bacterial Pathogen Transmission

Christopher W.J. McChalicher, Mary-Jane Lombardo, PhD, Sahil Khanna, MBBS MS,
Gregory J. McKenzie, PhD, Barbara H. McGovern, MD, John G. Auniņš, PhD, The
Seres Manufacturing Adventitious Agent Working Group
PII: S2772-5723(23)00021-3
DOI: https://doi.org/10.1016/j.gastha.2023.01.018
Reference: GASTHA 248

To appear in: Gastro Hep Advances

Received Date: 2 November 2022

Revised Date: 24 January 2023
Accepted Date: 24 January 2023

Please cite this article as: McChalicher CWJ, Lombardo MJ, Khanna S, McKenzie GJ, McGovern BH,
Auniņš JG, The Seres Manufacturing Adventitious Agent Working Group, Manufacturing Process of
Investigational Microbiome Therapeutic, SER-109, Reduces Risk of Bacterial Pathogen Transmission,
Gastro Hep Advances (2023), doi: https://doi.org/10.1016/j.gastha.2023.01.018.

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© 2023 Published by Elsevier, Inc on behalf of the AGA Institute

 1 Title:
2 Manufacturing Process of Investigational Microbiome Therapeutic, SER-109, Reduces Risk of
3 Bacterial Pathogen Transmission
4
5 Authors:
6 Christopher W. J. McChalicher1, Mary-Jane Lombardo PhD1, Sahil Khanna MBBS MS2,
7 Gregory J. McKenzie PhD1, Barbara H. McGovern MD1, John G. Auniņš PhD1 and The Seres
8 Manufacturing Adventitious Agent Working Group
9
10 Contributors:
11 Elizabeth M. Halvorsen PhD1, Sanabel Almomani MSc1, Brian Schuster1 and David S. Ege PhD1
12
13 Affiliations:

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15 1. Seres Therapeutics, Cambridge, MA, USA

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17 2. Mayo Clinic, Rochester, MN, USA

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19 Correspondence:
20 Barbara McGovern, MD
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21 Seres Therapeutics Inc

22 200 Sidney Street
23 Cambridge, Massachusetts 02139
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24 UNITED STATES
25 bmcgovern@serestherapeutics.com
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27
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28 Conflicts of interest:
29
30 These authors disclose the following: Christopher W.J. McChalicher, Mary-Jane Lombardo PhD,
31 Barbara H. McGovern MD, John G. Aunins PhD, Elizabeth Halvorsen PhD, Sanabel Almomani,
32 and David S. Ege PhD are all current employees and shareholders of Seres Therapeutics.
33 Gregory J. McKenzie PhD and Brian Schuster are former employees of Seres Therapeutics. Sahil
34 Khanna MBBS receives research support from Rebioitx / Ferring, Vedanta, Finch, Seres and
35 Pfizer and also serves as a consultant for ProbioTech, Takeda, Niche and Immuron.
36
37 Data transparency statement: Data, analytic methods, and study materials will not be
38 available.
39
40 Reporting Guidelines: Not applicable
41
42 Ethical Statement: The corresponding author, on behalf of all authors, jointly and severally,
43 certifies that their institution has approved the protocol for any investigation involving humans
44 or animals and that all experimentation was conducted in conformity with ethical and humane
45 principles of research.
46
47 Funding: This research was sponsored by Seres Therapeutics.
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49

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50
51 The pathogenesis of Clostridioides difficile infection (CDI) requires a “two-hit” process:

52 exposure to spores and antibiotic-mediated disruption of the gastrointestinal microbiome, which

53 plays a key role in preventing spore germination into toxin-producing vegetative bacteria. Toxin

54 production can cause symptoms ranging from mild diarrhea to life-threatening colitis. C.

55 difficile-targeted antibiotics resolve symptoms through rapid killing of vegetative bacteria but

56 have no effect on spores which germinate within a low-diversity microbiome, facilitating

57 recurrent CDI (rCDI). Fecal microbiota transplantation (FMT) is used to prevent rCDI, as

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58 microbiome repair is an essential therapeutic goal when antibiotics fail. Efficacy rates range from

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59 67% in controlled trials to 82% in observational case series leading to guideline

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recommendations for FMT in rCDI, based on moderate quality evidence1.
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62 Recent reports have concluded that FMT has a favorable safety profile without transmission of
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63 infectious agents2,3. However, the safety follow-up in one report from a stool bank4 ended in
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64 2018 prior to transmission events in early 2019, which prompted Food and Drug Administration
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65 safety alerts4. One alert highlighted transmissions of Shiga-toxin producing Escherichia coli to

66 five recipients leading to hospitalizations and one cardiorenal death in a patient with pre-existing

67 cardiac disease; whether STEC played a role in the patient’s demise was unclear5. In addition,

68 transmission of extended-spectrum, beta-lactamase producing Escherichia coli from a hospital-

69 based FMT program was identified with prospective adverse event (AE) monitoring within two

70 FMT trials6. One immunocompromised patient died, another was hospitalized with bacteremia

71 and five became colonized. These transmission events highlight inherent safety risks of FMT7.

72

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73 Potential risk mitigation includes continuous review for new pathogens with fecal shedding,

74 screening every donation, quarantining donations with bookend screening and comprehensive,

75 standardized donor testing. These methodologies may increase costs and safety concerns remain.

76 Most assays that assess for pathogens are validated in patients with active infection rather than in

77 asymptomatic carriers who may have lower levels of fecal shedding. Furthermore, donor

78 screening algorithms are focused on known organisms while emerging pathogens require de

79 novo assay development with inherent delays. Finally, retrospective AE reporting can preclude

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80 timely recognition of a donor as a transmission source8. A strategic therapeutic approach that

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81 provides efficacy while mitigating risk is needed.
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83 SER-109, an oral investigational microbiota therapeutic, was superior to placebo in reducing risk
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84 of rCDI in a randomized-controlled Phase 3 trial in patients with history of recurrence9. The

85 manufacturing process was designed to achieve a purified Firmicutes bacterial spore product
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86 through exposure to high ethanol concentrations to selectively inactivate non-product, vegetative

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87 bacteria, including potential undetected pathogenic bacteria. We evaluated the effect of ethanol-
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88 based inactivation operations on representative vegetative bacteria.

89
90 The inactivation step was validated using spiking studies whereby specific model organisms

91 were incorporated into manufacturing process intermediates and bacterial kill curves were

92 measured after ethanol exposure. Four species were used to model the range of pathogenic or

93 drug-resistant bacteria: Salmonella enterica for rod-shaped Gram-negative pathogens such as

94 Escherichia coli and representative of carbapenem-resistant Enterobacteraceae (CRE); Listeria

95 innocua for rod-shaped Gram-positive pathogens such as Listeria or Corynebacterium;

96 Enterococcus faecalis for Enterococcus and Streptococcus including vancomycin-resistant

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 97 Enterococci (VRE); and Staphylococcus aureus for Gram-positive clustering cocci such as

98 methicillin-resistant S. aureus (MRSA). Strain sources, strain characteristics, study execution,

99 and methods to assess relative impact to risk mitigation are described in the Supplementary

100 Materials.

101
102 Bacterial log reduction factors ranged from 6.5 log10 to 7.4 log10 (Figure 1). The full range of

103 estimates is limited by the maximum bacterial titers that could be achieved in culture and the

104 lower limits of assay detection. In all cases, lysis of the spiked organisms in the process matrix

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105 was rapid with titers reaching the assay limit of detection within 30 seconds for every replicate

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106 (Figure 1). The minimum observed inactivation rate was 13.4 log10/minute corresponding to an

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inactivation d-value (time required to achieve 1 log10 reduction) of 4.5 seconds, consistent with
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108 rapid bacterial inactivation. For perspective, the reported d-value for steam autoclaving is 20
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109 times slower at 1.5 minutes. The SER-109 manufacturing process maintains an ethanol contact
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110 for a minimum of 5 minutes, which is capable of bacterial inactivation well in excess of the total
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111 vegetative bacterial content of donor stool.

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113 These experiments demonstrate substantial and rapid inactivation of representative model

114 organisms, supporting the potential benefit of SER-109 manufacturing processes to mitigate risk

115 to patients while providing beneficial Firmicutes needed for efficacy in reducing risk of rCDI.

116 Firmicutes spores possess several inherent attributes that enable selective purification and

117 efficient drug delivery. Resistance of spores to ethanol enables selective inactivation of

118 vegetative microbes, including drug-resistant bacteria. Spore dormancy, which includes

119 resistance to aerobic conditions, enables the full processing time required for spore purification

120 from fibers and fecal solids, leading to removal of 99% of the total mass of donor materials.

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121 Spore resistance to gastric acid and oxygen enables oral delivery. Germination of spores into

122 replicating vegetative bacteria also supports a low pill burden.

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124 Although the reported risk is low, FMT-related transmissions of pathogenic bacteria are arguably

125 “predictable outcomes” and may be underreported due to few controlled trials with rigorous AE

126 reporting7. Additionally, emerging bacterial pathogens are continually being recognized and

127 cannot always be anticipated. Drug-resistant bacteria can lead to hard-to-treat infections with

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128 high rates of morbidity and mortality, highlighting the implications of undetected bacterial

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129 pathogens, which have been identified in healthy community residents without demographic risk

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factors. Finally, there remains no safety net for FMT products when screening failures occur due
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131 to false negative testing, asymptomatic carriage below the limit of assay detection or
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132 unrecognized novel bacterial pathogens. As demonstrated here, validated processes for
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133 inactivation reduce bacterial pathogen transmission risk beyond donor screening alone. In

134 addition, we reported that the SER-109 manufacturing steps inactivate fungi, parasites and
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135 viruses, including a model coronavirus9,10.

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137 In conclusion, clinical development of microbiome therapeutics needs to consider efficacy in

138 addition to risk mitigation. SER-109 manufacturing processes lead to a purified Firmicutes

139 product providing efficacy, as observed in a randomized-controlled trial, while mitigating

140 pathogen transmission risk, a highly desired outcome for vulnerable patients at risk for rCDI.

141

142
143 Figure 1. Inactivation kill curves (Titer versus Exposure Time) obtained for four model
144 organisms spiked into SER-109 manufacturing process intermediates. (A) Salmonella enterica,
145 (B) Listeria inoccua, (C) Enterococcus faecalis, and (D) Staphylococcus aureus. Each panel

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146 contains three replicate studies for each organism. Horizontal dashed lines represent limit of
147 detection (LOD, 200 CFU/mL) for the plating assay. Error bars are presented for each sample
148 with a titer above LOD and represent 1 standard deviation of the measured value based on
149 triplicate measurements.
150
151
152
153 References
154

155 1. Johnson S, Lavergne V, Skinner AM, et al. Clinical Practice Guideline by the Infectious
156 Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America
157 (SHEA): 2021 Focused Update Guidelines on Management of Clostridioides difficile Infection
158 in Adults. Clin Infect Dis 2021;73:ciab549-.

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159 2. Osman M, Budree S, Kelly CR, et al. Effectiveness and safety of fecal microbiota

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160 transplantation for Clostridioides difficile infection: Results from a 5,344 patient cohort study.
161 Gastroenterology 2022.

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3. Saha S, Mara K, Pardi DS, et al. Long-term Safety of Fecal Microbiota Transplantation for
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163 Recurrent Clostridioides difficile Infection. Gastroenterology 2021;160:1961-1969.e3.
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164 4. FDA. Important Safety Alert Regarding Use of Fecal Microbiota for Transplantation and Risk
165 of Serious Adverse Reactions Due to Transmission of Multi-Drug Resistant Organisms. 2019.
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166 https://www.fda.gov/vaccines-blood-biologics/safety-availability-biologics/important-safety-
167 alert-regarding-use-fecal-microbiota-transplantation-and-risk-serious-adverse. Accessed July 20,
168 2022.
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169 5. FDA. Safety Alert Regarding Use of Fecal Microbiota for Transplantation and Risk of Serious
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170 Adverse Events Likely Due to Transmission of Pathogenic Organisms. 2020.

171 https://www.fda.gov/vaccines-blood-biologics/safety-availability-biologics/safety-alert-
172 regarding-use-fecal-microbiota-transplantation-and-risk-serious-adverse-events-likely. Accessed
173 July 20, 2022.

174 6. DeFilipp Z, Bloom PP, Soto MT, et al. Drug-Resistant E. coli Bacteremia Transmitted by
175 Fecal Microbiota Transplant. New Engl J Med 2019;381:2043-2050.

176 7. Blaser MJ. Fecal Microbiota Transplantation for Dysbiosis — Predictable Risks. New Engl J
177 Med 2019;381:2064-2066.

178 8. Zellmer C, Sater MRA, Huntley MH, et al. Shiga Toxin–Producing Escherichia coli
179 Transmission via Fecal Microbiota Transplant. Clin Infect Dis 2020;72:e876-e880.

180 9. Feuerstadt P, Louie TJ, Lashner B, et al. SER-109, an Oral Microbiome Therapy for Recurrent
181 Clostridioides difficile Infection. New Engl J Med 2022;386:220-229.

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182 10. McChalicher C, Abdulaziz A, Zhou SS, et al. Manufacturing Process of SER-109, a Purified
183 Investigational Microbiome Therapeutic, Reduces Risk of Coronavirus Transmission From
184 Donor Stool. Open Forum Infect Dis 2022;9:ofac448.

185

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