Molecular Breath Print of COPD Patients With Exacerbations Despite Triple Inhalational Therapy (TripleEX)
Conditions: COPD Exacerbation
Sponsor: Malcolm Kohler

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MOLECULAR BREATH PRINT OF COPD PATIENTS 
WITH EXACERBATIONS DESPITE TRIPLE 
INHALATIONAL THERAPY  
(TRIPLEEX) 
Research legislation: 
Study Type: 
Ordinance on human research with the exception of Clinical trials 
[HRO]  
Observational study 
Study Categorisation: 
Risk category A according to HRO Art. 7 
Study Registration BASEC: 
2020-01954 
Study Identifier: 
Sponsor-Investigator and 
Principal Investigator: 
Protocol Version and Date: 
NCT04638920
Prof. Dr. med. Malcolm Kohler 
Chair Respiratory Medicine 
Clinical Director, Pulmonary Division 
University Hospital Zurich 
Rämistrasse 100, CH-8091 Zurich, Switzerland 
Phone: +41 44 255 38 28 
E-Mail: malcolm.kohler@usz.ch
V 1.1, 02.09.2020
CONFIDENTIAL 
The information contained in this document is confidential and the property of the Sponsor. 
The information may not - in full or in part - be transmitted, reproduced, published, or 
disclosed to others than the applicable Competent Ethics Committee(s) and Regulatory 
Authority(ies) without prior written authorisation from the sponsor except to the extent 
necessary to obtain informed consent from those who will participate in the study.  

PNE_TripleEX_12522_research plan_v1.1 
 
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TABLE OF CONTENTS 
TABLE OF CONTENTS .......................................................................................................................... 3 
GLOSSARY OF ABBREVIATIONS ....................................................................................................... 5 
STUDY SYNOPSIS ................................................................................................................................. 6 
1. 
GENERAL INFORMATION & STUDY ADMINISTRATIVE STRUCTURE ..................................... 9 
1.1 
Sponsor, Sponsor-Investigator (Principal-Investigator) .............................................................. 9 
1.2 
Investigators ............................................................................................................................. 9 
1.3 
Study Site Team ....................................................................................................................... 9 
1.4 
Collaborators ............................................................................................................................ 9 
2. 
BACKGROUND AND RATIONALE .............................................................................................. 10 
2.1 
The role of exhaled breath analysis ......................................................................................... 10 
2.2 
Exhaled breath analysis by mass spectrometry ....................................................................... 11 
2.3 
Chronic Obstructive Pulmonary Disease (COPD) .................................................................... 12 
2.4 
Exacerbation ........................................................................................................................... 12 
2.5 
Exhaled breath analysis in COPD............................................................................................ 13 
2.6 
Rationale for the current study ................................................................................................. 14 
2.7 
Triple Inhalational Therapy in COPD ....................................................................................... 15 
2.8 
Role of eosinophils and targeting IL-5 in COPD ....................................................................... 15 
3. 
STUDY OBJECTIVES AND DESIGN ........................................................................................... 16 
3.1 
Hypothesis and primary objective ............................................................................................ 16 
3.1.1 Hypothesis ...................................................................................................................... 16 
3.1.2 Objective ......................................................................................................................... 16 
3.2 
Study Outcome ....................................................................................................................... 16 
3.2.1 Primary Outcome ............................................................................................................ 16 
3.2.2 Secondary Outcome Measures ....................................................................................... 16 
3.3 
Study Design .......................................................................................................................... 16 
4. 
STUDY POPULATION AND STUDY PROCEDURES .................................................................. 17 
4.1 
Project population and eligibility criteria ................................................................................... 17 
4.2 
Inclusion criteria ...................................................................................................................... 17 
4.3 
Exclusion criteria ..................................................................................................................... 17 
4.4 
Recruitment, screening and informed consent procedure ........................................................ 18 
4.5 
Study procedures .................................................................................................................... 18 
4.5.1 General study procedures ............................................................................................... 18 
4.5.2 Medical history ................................................................................................................ 19 
4.5.3 Vital parameters .............................................................................................................. 19 
4.5.4 Inflammatory markers ...................................................................................................... 19 
4.5.5 Spirometry ...................................................................................................................... 19 
4.5.6 Causative agents ............................................................................................................ 19 
4.5.7 Online breath analysis ..................................................................................................... 19 
4.5.8 Questionnaires ................................................................................................................ 20 
4.5.9 Study visits ...................................................................................................................... 21 
4.5.10 Study duration ................................................................................................................. 21 
4.6 
Withdrawal and discontinuation ............................................................................................... 21 
5. 
STATISTICS AND METHODOLOGY ............................................................................................ 21 
5.1 
Statistical analysis plan ........................................................................................................... 21 

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5.2 
Sample Size calculation .......................................................................................................... 22 
5.3 
Handling of missing data ......................................................................................................... 22 
6. 
REGULATORY ASPECTS AND SAFETY .................................................................................... 22 
6.1 
Registration and Categorisation .............................................................................................. 22 
6.2 
Local regulations/Declaration of Helsinki ................................................................................. 22 
6.3 
Participant privacy and confidentiality ...................................................................................... 23 
6.4 
Notification of safety and protective measures (HRO Art. 20) ................................................... 23 
6.5 
Serious events (HRO Art. 21) .................................................................................................. 23 
6.5.1 Definition of Seriouse Events ........................................................................................... 23 
6.5.2 Assessment of SEs ......................................................................................................... 24 
6.6 
Early termination of the study .................................................................................................. 24 
6.7 
Protocol amendments ............................................................................................................. 24 
6.8 
End of project .......................................................................................................................... 24 
6.9 
Insurance ................................................................................................................................ 25 
7. 
FURTHER ASPECTS .................................................................................................................... 25 
7.1 
Overall ethical considerations .................................................................................................. 25 
7.2 
Ethical Conduct of the project .................................................................................................. 25 
7.3 
Participant information and informed consent .......................................................................... 26 
7.4 
Risk / Benefit Assessment ....................................................................................................... 26 
7.5 
Compensation offered to participants ...................................................................................... 26 
8. 
QUALITY CONTROLE AND DATA PROTECTION...................................................................... 27 
8.1 
Quality measures .................................................................................................................... 27 
8.2 
Data management/ Data recording and Source data ............................................................... 27 
8.3 
Data handling and record keeping / archiving .......................................................................... 28 
8.3.1 Case Report Forms ......................................................................................................... 28 
8.3.2 Record keeping / archiving .............................................................................................. 28 
8.4 
Details of the Secure Handling of Data connected to DBI ........................................................ 29 
8.5 
Audits and Inspections ............................................................................................................ 29 
9. 
FUNDING /DECLARATION OF INTEREST .................................................................................. 30 
9.1 
Funding & Support .................................................................................................................. 30 
9.2 
Declaration of interest ............................................................................................................. 30 
10. PUBLICATION AND DISSEMINATION POLICY.......................................................................... 30 
11. REFERENCES ............................................................................................................................... 31 
12. APPENDIX ..................................................................................................................................... 35 
 
 
 

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GLOSSARY OF ABBREVIATIONS 
 
 
BASEC 
Business Administration System for Ethics Committees 
BP 
Blood Pressure 
CA 
Competent Authority (e.g. Swissmedic) 
CEC 
Competent Ethics Committee 
COPD 
Chronic obstructive pulmonary disease 
CRF 
DBI 
FeNO 
Case Report Form  
Deep Breath Initiative 
Fractional exhaled nitric oxide concentration 
FEV1 
Forced Expiratory Volume at 1 Second  
FVC 
Forced Vital Capacity 
GCP 
GOLD 
Good Clinical Practice  
Global Initiative for Chronic Obstructive Lung Disease 
H 
Hours 
HFG 
Humanforschungsgesetz (HFG = HRA) 
HFV 
Humanforschungsverordnung (HFV = HRO) 
HRA 
Federal Act on Research involving Human Beings  
HRO 
ICS 
LAMA 
LABA 
Ordinance on Human research with the exception of clinical trials 
Inhaled corticosteroids (e.g Budesonid, Fluticasonfuroat, Fluticasonpropionat) 
Long acting muscarinic antagonist (e.g. Aclidium, Umeclidium, Glycopyrronium, Tiotropium) 
Long acting β-2 agonist (e.g. Formoterol, Indacaterol, Salmeterol, Olodaterol, Vilanterol) 
MSMS 
Tandem mass spectrometry 
PI 
 
Principal Investigator 
 
PTR-MS 
Proton-transfer reaction - mass spectrometry 
SABA 
Short acting β-2 agonist (e.g. Terbutalin, Salbutamol) 
SAE 
SAMA 
SESI-HRMS 
Serious Adverse Events 
Short acting muscarinic antagonist (e.g Ipatropium) 
Secondary electro-spray ionization high resolution mass spectrometry 
SIFT-MS 
Selected ion flow tube - mass spectrometry 
SNCTP 
Swiss National Clinical Trial Portal 
SOP 
Standard Operating Procedure 
TMF 
USZ 
Trial Master File 
UniversitätsSpital Zurich  
V 
Version 
VOC 
Volatile Organic Compounds 
WHO 
World Health Organisation 
 

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STUDY SYNOPSIS  
Sponsor 
Prof. Dr. med. Malcolm Kohler 
Study Title 
MOLECULAR BREATH PRINT OF COPD PATIENTS WITH 
EXACERBATIONS DESPITE TRIPLE INHALATIONAL THERAPY 
(TripleEX) 
Study ID 
Not yet available 
Protocol Version and 
Date 
Version 1.0 of 27.05.2020 
Trial registration: 
Not yet registered (SNCTP, clinicaltrials.gov) 
Study category and 
Rationale 
Risk category A according to HFV 
Background and 
Rationale 
COPD is an inflammatory and progressive disease of the lungs and 
airways primarily characterized by irreversible airflow limitation. COPD is 
the third leading cause of death worldwide.(1) On-line breath analysis in 
COPD would allow continuous monitoring of metabolic health, disease 
progression, and medication in short time intervals. Results are obtained 
without delay which is of high importance in situations like COPD 
exacerbations.(2) The findings of a recent matched case control study in 
patients with frequent vs patients without frequent COPD exacerbations 
(3) provided data on metabolic breath-prints using untargeted real-time 
mass spectrometry in COPD. The study showed that real-time breath 
analysis by mass spectrometry allows distinguishing patients with frequent 
exacerbations from patients without frequent exacerbations on the basis 
of molecular analysis. The study provided biochemical insights in COPD 
patients at risk for exacerbations. However, because the study was 
performed in between COPD exacerbations (in a stable phase) it provided 
no information on biochemical processes during an actual COPD 
exacerbation. The identification of molecular changes during a COPD 
exacerbation could help to optimize treatment, identify new therapeutic 
targets and, is perhaps of diagnostic value.  
GOLD guidelines recommend treating symptomatic COPD patients with 
frequent exacerbations with a triple inhalational therapy i.e beta-2-
sympathicomimetics, anticholinergics and steroids (e.g. Trelegy®).(4) 
However, despite triple inhalational therapy a substantial number of 
patients continues to have exacerbations and thus are at high risk for 
disease progression, morbidity and mortality.(5) Some of these patients 
have elevated levels of eosinophils and may therefore, in theory, be 
eligible candidates for antibody treatment (e.g. with IL-5 blocking 
compounds such as Mepolizumab).(6) However, apart from eosinophil 
counts there is very little information on biochemical processes in COPD 
patients with exacerbations despite triple inhalational therapy. Thus, the 
identification of molecular changes could help to better understand 
relevant pathways and furthermoreto identify new therapeutic targets. 
Particulary those patients who are most likely to respond to an antibody 
therapy would benefit. 
Objective 
The objective of this study is to determine specific molecular breath 
patterns by secondary electrospray ionisation mass spectrometry (SESI-
HRMS) during and 8 weeks after a COPD exacerbation. Furthermore, 
breath patterns will be assessed for correlation and association to clinical 
outcomes and treatment. 

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Outcomes 
Primary Outcome Measure 
The primary outcome will be the change in exhaled breath metabolites 
during vs after COPD exacerbation. 
Secondary Outcome Measures 
Correlation of breath metabolites with symptoms (mMRC and CAT®), 
inflammation markers (CRP and eosinophils, exhaled NO), causative 
agents (sputum microbiology and viral swabs) and treatment (inhalational 
drugs, steroids, antibiotics). 
  
Study design 
40 patients with COPD (staged according to GOLD recommendations, 
aged ≥18 years) on adherent triple inhalational therapy are to be included 
in a longitudinal study. Patients will undergo two measurements of exhaled 
breath; at baseline during a hospitalization due to COPD exacerbation and 
8 weeks after the COPD exacerbation. Real-time breath analysis will be 
performed using SESI-HRMS coupled to a Q-exactive Orbitrap mass 
spectrometer (Thermo Fisher). 
 
Inclusion criteria 
(inclusive) for all 
participants 
• 
Informed Consent 
• 
age ≥ 18 years  
• 
staged according to GOLD recommendations 
• 
GOLD stage 2-4, risk groups A-D 
• 
hospitalization due to COPD exacerbation 
• 
subjects adherent to triple inhalational therapy (β-2-
sympathomimetics, anticholinergics, steroids) 
• 
suitable for follow-up assessment 
• 
<48 hours after initiation of antibiotic therapy or systemic steroid 
therapy 
Exclusion criteria 
(exclusive) for all 
participants 
• 
physical or intellectual impairment precluding informed consent 
or protocol adherence 
• 
known pregnancy 
• 
congenital defects with direct impact on central metabolism e.g. 
amino acid metabolism defect 
• 
uncontrolled diabetes (e.g. HbA1c >11% or Glc >20mmol/l). 
• 
acute or chronic pulmonary disease other than COPD  
• 
renal failure or renal replacement therapy (GFR < 15 mL/min) 
 
Number of 
Participants 
N = 40 
Study Duration 
01.08.2020 – 31.07.2023 

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Investigators from 
University Hospital 
Zurich 
Prof. Dr. med. Malcolm Kohler, Pulmonary Division,  
University Hospital Zurich, Raemistrasse 100, 8091 Zurich, Switzerland. 
malcolm.kohler@usz.ch 
 
Felix Schmidt, RPh, Pulmonary Division, 
University Hospital Zurich, Raemistrasse 100, 8091 Zurich, Switzerland. 
felix.schmidt@usz.ch 
 
Martin Osswald, MSc, Pulmonary Division, 
University Hospital Zurich, Raemistrasse 100, 8091 Zurich, Switzerland. 
martin.osswald@usz.ch 
 
Noriane Sievi, MSc, Pulmonary Division, 
University Hospital Zurich, Raemistrasse 100, 8091 Zurich, Switzerland. 
noriane.sievi@usz.ch 
Study Centre 
Pulmonary Division, University Hospital Zurich,  
Rämistrasse 100, 8091 Zurich, Switzerland 
Statistical 
Considerations 
To test the hypothesis if there is a significant difference in the values of 
exhaled metabolites during and after COPD exacerbation, we will 
perform a paired-sample t-test to evaluate differential concentrations of 
such metabolites. False discovery rate and q-values will be subsequently 
computed as defined by Storey.(7) Well-established data reduction 
techniques such as principal component analysis will be used for 
visualization purposes. Finally, high mass resolution/ high mass 
accuracy data obtained will allow us to query online databases (e.g., 
human metabolome database) to provide tentative identification of the 
subset of most informative exhaled compounds. The pre-processing of 
the raw mass spectra and the statistical analysis will be implemented 
using MATLAB (MathWorks, Massachusetts, US). 
GCP Statement 
This study will be conducted in compliance with the protocol, the current 
version of the Declaration of Helsinki(8), the principles of Good Clinical 
Practice and as well all national legal and regulatory requirements in 
Switzerland.  
 
 
 

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1. GENERAL INFORMATION & STUDY ADMINISTRATIVE STRUCTURE  
1.1 Sponsor, Sponsor-Investigator (Principal-Investigator) 
 
Prof. Dr. med. Malcolm Kohler 
Chair Respiratory Medicine  
Clinical Director Pulmonary Division  
University Hospital Zurich  
Rämistrasse 100, 8091 Zurich (Switzerland)  
1.2 Investigators 
 
Felix Schmidt, RPh 
PhD Candidate Clinical Science 
University Hospital Zurich 
Rämistrasse 100, 8091 Zurich (Switzerland) 
 
Martin Osswald, M.Sc 
PhD Candidate Clinical Science 
University Hospital Zurich 
Rämistrasse 100, 8091 Zurich (Switzerland) 
 
Noriane Sievi, M.Sc. 
Scientist Pulmonary Division 
University Hospital Zurich 
Rämistrasse 100, 8091 Zurich (Switzerland) 
 
1.3 Study Site Team 
 
Goran Krummenacher & Selvete Bajrami 
Study Nurse 
University Hospital Zurich 
Rämistrasse 100, 8091 Zurich (Switzerland) 
 
1.4 Collaborators 
 
Deep Breath Initiative (DBI) 
University of Basel Innovation Space 
Gewerbestrasse 24, 4123 Allschwil (Switzerland) 
+41 44 255 38 28  
malcolm.kohler@usz.ch 
+41 44 255 48 01 
felix.schmidt@usz.ch 
+41 44 255 49 47 
martin.osswald@usz.ch 
+41 44 255 98 15 
noriane.sievi@usz.ch 

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2. BACKGROUND AND RATIONALE  
2.1 The role of exhaled breath analysis 
The ancient Greeks already knew that using exhaled breath as an indicator of disease can be 
revealing.(9) The famous physician Socrates mentioned characteristic smells for different types of 
diseases such as a sweet smell in diabetes, a fishy smell in patients with liver problems, a urine-like 
smell in patients with kidney problems, or a putrid smell in patients with a lung abscess.(10) The primary 
opportunity of using exhaled breath as source for biomarker discovery is the ease with which breath can 
be collected for analysis (Figure 1). Patients are only required to exhale into a device or container and 
recent technological advancements have enabled a real-time analysis of exhaled breath, where data 
are available on the spot. Breath analysis offers a unique opportunity to non-invasively retrieve 
potentially unlimited sampling of relevant information on the ongoing internal biochemical processes, as 
parts of the most volatile components of blood reach the gas phase and are subsequently exhaled (e.g. 
detection of ethanol in breath).(11) Simple methods of breath analysis have made use of this, e.g. for 
the assessment of bronchial inflammation by measuring fractional exhaled nitric oxide (FeNO).(12) 
Despite these advantages of exhaled breath testing, its use in clinical practice is still limited. This is 
mostly due to a lack of thoroughly validated techniques. 

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2.2 Exhaled breath analysis by mass spectrometry 
Analysis of exhaled breath gives biochemical information about the metabolism and the 
pathophysiological state. Chemical analysis of exhaled breath with mass spectrometry identified 
numerous volatile organic compounds (VOCs) [low molecular weight-components: < 500 Dalton (Da)].  
The origin of these VOCs is diverse. VOCs can either be endogenous (arise from the respiratory tract), 
systemic (passing the blood-air barrier) or can be of exogenous origin (inhaled/exhaled environment 
air).(2) The alveoli are intimately connected with the systemic circulation through the alveolar capillaries 
and its large blood-gas-interface. It was demonstrated that exhaled human breath contains a molecular 
fingerprint (also referred to as breathprint(9)). Breath composition changes during the course of the day 
(intra-individual variations). These temporal fluctuations may reflect circadian changes (13-15). Despite 
these changes, it was shown that each individual shows a distinct breathprint stable in time (individual 
core pattern of volatile and semi-volatile metabolites)(16). The findings of preliminary studies by others 
and our group suggest that diseases of the airways such as e.g. chronic obstructive pulmonary disease 
Figure 1: Composition and origin of exhaled breath 

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(COPD)(17, 18) may be diagnosed by a specific exhaled breath pattern. 
2.3 Chronic Obstructive Pulmonary Disease (COPD) 
COPD is an inflammatory and progressive disease of the lungs and airways primarily characterized by 
irreversible airflow limitation. COPD is the third leading cause of death worldwide.(1) Its prevalence 
among heavy smokers can reach up to 50%. According to the GOLD guidelines (Global Initiative for 
Chronic Obstructive Lung Disease) the diagnosis of COPD can be established by a fixed ratio of post-
bronchodilator forced expiratory volume (FEV1) and forced vital capacity (FVC) below 0.7 measured by 
spirometry (see http://www.goldcopd.org). The severity of airflow limitation is graded according to the 
percentage of FEV1 predicted (GOLD stage I-IV). Furthermore severity of symptoms and risk of 
exacerbation is categorized by the risk groups A - D. This functional definition has been used to 
characterize the disease for many years. However, the degree of airflow limitation is only one aspect of 
the disease and COPD is being recognized as a heterogeneous disease, which requires tailored 
therapeutic approaches.(19) While the disease course involves a slow progressive deterioration in 
airflow, it is punctuated by exacerbations (acute deterioration of the patient's respiratory symptoms, i.e. 
worsening of dyspnea, cough and/or increased sputum production), many of which are serious, requiring 
hospitalization, and are associated with high mortality and extensive costs. The risk for exacerbations 
increases with higher GOLD stage.(20-22) After the second episode of severe exacerbation, the course 
of COPD is often associated with a rapid decline in health status. Furthermore, the mortality risk 
increases in the weeks following every severe exacerbation (23). Because of the complexity of COPD 
the disease management is mainly characterized in supporting prevention and maintenance therapy, 
stable COPD management and exacerbation management.   
2.4 Exacerbation 
An exacerbation of COPD is defined as an acute worsening of respiratory symptoms that results 
in additional therapy.(24, 25) COPD exacerbations are complex events usually associated with increase 
in airway inflammation, increased mucus production and marked gas trapping. These changes 
contribute to increased dyspnea which is the key symptom of an exacerbation.(4) 
Exacerbations are classified as: 
• 
Mild (treated with short acting bronchodilators only, SABDs) 
• 
Moderate (treated with SABDs plus antibiotics and/or oral corticosteroids) 
• 
Severe (patient requires hospitalization or visits the emergency room). Severe exacerbations 

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may also be associated with acute respiratory failure 
During a COPD exacerbation, the worsening of the symptoms usually lasts between 7 to 10 days, but 
some events may last longer.  It is well established that COPD exacerbations contribute to disease 
progression.(26) Disease progression is even more likely if recovery from exacerbations is slow.(27) 
Exacerbations can also cluster in time and once COPD patients experience an exacerbation, they will 
show increased susceptibility to further events.(20, 28) Depending on the frequency of exacerbations, 
COPD patients are divided into infrequent (0-1 exacerbations within the last 12 months) and frequent 
exacerbators (2 or more exacerbations within the last 12 months).  Identifying COPD patients at risk for 
exacerbations, and especially frequent exacerbations, would be highly desirable to prevent further 
deterioration of early disease stages. 
2.5 Exhaled breath analysis in COPD 
Breath analysis in COPD is attractive because breath is available in nearly unlimited quantities. The 
analysis is non-invasive, and it presents no burden to the subject being measured. On-line breath 
analysis in COPD would allow continuous monitoring of metabolic health, disease progression, and 
medication in short time intervals. Data are obtained immediately which is of high importance in 
situations like COPD exacerbations.(2) 
The diagnostic “gold standard” for COPD is based on spirometry and subjective symptom assessments, 
similar to asthma with respective COPD guidelines.(29) As it is primarily a disease of the lungs, a high 
number of breath-analysis studies are available for COPD; however, only a minority of the studies 
employed on-line methods.(30) Most studies used a case-control design with an untargeted approach 
in order to identify putative discriminative markers for the disease or disease severity. Three studies 
used ion mobility separation to identify COPD-specific biomarkers in a cohort of 119, 130, and 96 
patients, respectively.(31-33) While two studies reported a 70−79% accuracy in the diagnosis of COPD 
(based on 6−10 VOCs), the other study reported 10 biomarkers, most of which were unidentified.(31) 
None of the identified substances overlapped between the two studies or was validated in an external 
cohort. Finally, two untargeted studies (17, 18) compared the breath print of COPD patients against 
healthy controls using SESI-MS with a real-time approach. Both studies yielded a Receiver Operating 
Characteristic (ROC) curve with an area under the curve of 0.88−0.91. However, in this case, with the 
aid of MS/ MS and complementary pre-separation methods some of the discriminate biomarkers could 
be identified and attributed to be metabolites of oxidative stress processes, such as fatty acids, 

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aldehydes, and amino acids.(17, 18) Most studies tried to control for the effects of smoking and there 
was evidence that lung capacity, body mass index, and certain inflammatory blood markers seem to 
play a role in the discrepancies observed between groups.(33, 34) In conclusion, studies on this topic 
are extremely heterogeneous and data processing and statistical methods are not standardized. As a 
result, none of the reported biomarkers overlaps between the studies and none of the biomarkers has 
been validated in an external cohort yet.  
2.6 Rationale for the current study 
The findings of a recent matched case control study in patients with frequent vs infrequent COPD 
exacerbations (3) provided data on metabolic breath-prints using untargeted real-time mass 
spectrometry in COPD. The study showed that real-time breath analysis by mass spectrometry allows 
distinguishing patients with frequent exacerbations from patients with infrequent exacerbations on the 
base of molecular analysis. Metabolite levels from the ω -oxidation pathway, namely ω -hydroxy, ω -
oxo, and dicarboxylic acids, were consistently decreased in frequent exacerbators. Additionally, several 
new nitro-aromatic metabolites, which were significantly increased in frequent exacerbators, were 
identified. Thus, the study provided insights about ongoing biochemical processes in patients with 
COPD at risk for exacerbations. However, because the study was performed in between COPD 
exacerbations (in a stable phase) it provided no information on biochemical processes during an actual 
COPD exacerbation. The identification of molecular changes during a COPD exacerbation could help 
to optimize treatment, identify new therapeutic targets and, is perhaps of diagnostic value.  
GOLD guidelines recommend treating symptomatic COPD patients with frequent exacerbations with a 
triple inhalational therapy i.e beta-2-sympathicomimetics, anticholinergics and steroids (e.g. 
Trelegy®).(4) However, despite triple inhalational therapy a substantial number of patients continues to 
have exacerbations and thus are at high risk for disease progression, morbidity and mortality. Some of 
these patients have elevated levels of eosinophils and may therefore, in theory, be eligible candidates 
for antibody treatment (e.g. with IL-5 blocking compounds such as mepolizumab).(6) However, apart 
from eosinophil counts there is very little information on biochemical processes in COPD patients with 
exacerbations despite triple inhalational therapy. Thus, the identification of molecular changes could 
help to better understand relevant pathways and furthermore help to identify new therapeutic targets. 
Particularly those patients who are most likely to respond to e.g. an antibody therapy would benefit from 
a deeper understanding of molecular mechanisms. 

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This will be the first study to investigate the acute effects of COPD exacerbations on molecular exhaled 
breath patterns assessed by SESI-MS. By identifying a distinctive breath pattern it may be possible to 
define biochemical processes specific of a COPD exacerbation and correlate it with causative agents, 
treatment and outcome. 
2.7 Triple Inhalational Therapy in COPD 
Triple therapy is most likely indicated in patients with COPD who are severely symptomatic, have 
moderate to very severe airflow obstruction and a history of frequent and/or severe exacerbations.(4) 
The step up in inhaled treatment to LABA plus LAMA plus ICS (triple therapy) can occur by various 
approaches.(35) This may improve lung function, patient reported outcomes and prevent exacerbations. 
Adding a LAMA to existing LABA/ICS improves lung function and patient reported outcomes, in 
particular exacerbation risk.(36-40) Double blind RCTs have reported benefits of single-inhaler triple 
therapy compared with LABA/LAMA combination therapy.(41, 42) 
  
2.8 Role of eosinophils and targeting IL-5 in COPD 
Novel approaches to COPD treatment focus on understanding and targeting molecular mechanisms of 
airway inflammation, airway obstruction, remodelling and lung destruction. The most promising biologic 
treatments at an advanced stage of development are agents blocking interleukin (IL)-5 or its 
receptor.(43) The inflammatory phenotype of COPD can be described as: 1) neutrophilic, 2) eosinophilic, 
3) mixed eosinophilic and neutrophilic and 4) paucigranulocytic nature.(44) Neutrophilic airway 
inflammation is the most predominant type seen in COPD, but eosinophilic inflammation is present in 
up to 40% of COPD patients.(45) In particular eosinophils increase the risk of exacerbations and IL-5 
expression.(45, 46) IL-5 expression is a key player in inflammatory processes of COPD. Therefore 
options such as targeting IL-5 with Mepolizumab/ Reslizumab or IL-5Rα with Benralizumab are 
encouraging. However, results of phase III clinical trials e.g. METREX(47), METRO(47), 
GALATHEA(48) or TERRANOVA(49) in eosinophilic COPD patients have not shown a breakthrough in 
exacerbation prevention. Breath analysis could lead to a deeper understanding of pathophysiological 
mechanisms of COPD exacerbations in context with IL-5 and thus help to find more suitable biomarkers 
and select those COPD patients who are most likely to benefit from IL-5 blocking therapy.  

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3.
STUDY OBJECTIVES AND DESIGN
3.1 Hypothesis and primary objective 
3.1.1 
Hypothesis 
Molecular breath patterns acquired by secondary electrospray ionization mass spectrometry (SESI-MS) 
are distinctively altered during COPD exacerbation. 
3.1.2 
Objective 
The objective is to determine specific molecular breath patterns by secondary electro ionisation-mass 
spectrometry (SESI-MS) during and 8 weeks after a severe exacerbation requiring hospitalization. 
Furthermore, molecular breath patterns will be assessed for correlation and association to clinical 
outcomes and treatment. 
3.2 Study Outcome 
3.2.1 
Primary Outcome 
The primary outcome will be the change in exhaled breath metabolites during vs after COPD 
exacerbation. 
3.2.2 
Secondary Outcome Measures 
Correlation of breath metabolites with symptoms (mMRC and CAT®), inflammation markers (CRP and 
eosinophils, exhaled NO), causative agents (sputum microbiology and viral swabs) and treatment 
(inhalational drugs, steroids, antibiotics). 
3.3 Study Design 
We will conduct a prospective, longitudinal, observational study including 40 patients with proven COPD 
staged according to GOLD recommendations and under adherent triple inhalational therapy. The 
treating physician will check during the general assessment the compliance to triple therapy treatment. 
A study flow chart is presented in Figure 2 

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Figure 2: Study flow chart. COPD = Chronic Obstructive Pulmonary Disease; BA = Breath Analysis; GOLD 
= Global Initiative for Chronic Obstructive Lung Disease; * Breath analysis by SESI_MS; +steroids & 
antibiotics 
4.
STUDY POPULATION AND STUDY PROCEDURES
4.1 Project population and eligibility criteria
A population of subjects with COPD staged according to GOLD recommendations with exacerbation 
requiring hospitalization will be screened. Furthermore, only subjects adherent to triple inhalational 
therapy i.e beta-2-sympathicomimetics, anticholinergics and steroids are eligible. An exacerbation of 
COPD is defined as an acute worsening of respiratory symptoms that results 
in additional therapy.(4) 
4.2 Inclusion criteria 
•
Informed Consent
•
Age ≥ 18 years
•
GOLD stage 2-4, risk groups A-D
•
hospitalization due to COPD exacerbation
•
Defined exacerbation according to GOLD guidelines
•
Subjects adherent to triple inhalational therapy (β-2-sympathomimetics, anticholinergics,
steroids)
•
Suitable for follow-up assessment
•
<48 hours after initiation of antibiotic or steroid impulse therapy
4.3 Exclusion criteria 
•
Physical or intellectual impairment precluding informed consent or protocol adherence
•
Known pregnancy
•
congenital defects with direct impact on central metabolism e.g. amino acid metabolism defect
Correction <48h

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• 
uncontrolled diabetes (e.g. HbA1c >11% or Glc >20mmol/l). 
• 
acute or chronic pulmonary disease other than COPD  
• 
renal failure or renal replacement therapy (GFR < 15 mL/min) 
4.4 Recruitment, screening and informed consent procedure 
Recruitment 
COPD patients will be consecutively recruited from the wards at the University Hospital Zurich. The 
treating physician will approach patients via personal communication. If interested, the treating physician 
will inform one of the investigators who then will contact the patient.  
Participants will not be offered any material compensation for their voluntary agreement to take part in 
this study (Art. 14 HFG). Whilst the scientific benefit from participation will be explained to participants, 
no other incentives will be offered. 
Patient Information and Informed Consent 
The investigators will explain to each participant the nature of the study, its purpose, the procedures 
involved, the expected duration, the potential risks and benefits and any discomfort it may entail. The 
right of the participant to refuse to participate without giving reasons will be respected, and withdrawal 
of consent will not affect the participant’s subsequent medical assistance and treatment. In the event of 
withdrawal of the participant’s consent, data collected up to the date of the withdrawal will remain coded 
in the study database. 
The participant must be informed that his/her medical records may be examined by authorised 
individuals other than their treating physician. The date and time of the consent will be recorded by the 
investigator (or his designee) and it will be retained as part of the study records. 
The participant information sheet and the consent form will be submitted with the protocol for review 
and approval for the study by the CEC. The formal consent of a participant, using the approved consent 
form, must be obtained before that participant is submitted to any study procedure.   
The participant should read and consider the statement before signing and dating the informed consent 
form, and should be given a copy of the signed document. The consent form must also be signed and 
dated by the investigator (or his designee) and it will be retained as part of the study records. 
4.5 Study procedures 
4.5.1 
General study procedures 
Upon participation in the study, participants will be requested to answer general questions on their 

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medical history. The participant will be asked to fill in questionnaires and vital parameters will be 
determined. Subsequent on-line breath analysis will be performed. 
4.5.2 
Medical history 
General information: age, gender, weight, height, eating habits, activity habits, exacerbation history 
medication, co-morbidities, smoking history, disease specific images and clinical chemistry test results. 
4.5.3 
Vital parameters 
Vital parameters are measured to reflect the basic function of the human body. We focus on blood 
pressure and heart rate, which are measured in sitting position after 3 min of rest. Furthermore, 
temperature and oxygen saturation will be measured. 
4.5.4 
Inflammatory markers 
During the hospitalization of patients with COPD exacerbation blood sampling is routinely performed. 
CRP level and eosinophil level will be documented for this study during visit 1 and 2. 
4.5.5 
Spirometry 
A spirometry will be performed if the physical sate of the patient allows at visit 1 and visit 2. Spirometry 
results (forced expiratory volume at 1 second [FEV1] and forced vital capacity [FVC]) will be expressed 
in percentage of predicted values according to the European reference equations.(50) Furthermore a 
FeNO Test will be used to investigate inflammatory characteristics more in detail. A FeNO Test is 
routinely performed in asthma patients. The patients should exhale into a to certified sensitive instrument 
where the NO concetration in the exhaled breath gives conclusion about inflammatyr celllines. 
4.5.6 
Causative agents 
Sputum samples and viral smear is collected routinely during the clinical assessment during 
hospitalization (visit 1). 
4.5.7 
Online breath analysis 
Real-time breath analysis will be performed with SESI-HRMS. SESI-HRMS is a new developed real 
time mass spectrometric method besides PTR-MS (51) and SIFT-MS. (52) The big advantage of the 
SESI method is that the ionisation takes place at atmosphere pressure. This characteristic brings the 
possibility to connect the SESI ionization source with high resolution mass analyser like the Orbitrap 

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spectrometer by Thermo Fisher. This results in sensitive and selective, yet real-time, analysis of trace 
vapour species.(53) Our SESI-HRMS is situated in a separate, lockable room at the University 
Hospital Zurich. Access to the designated room is only permitted to members of the staff. Participants 
will be asked to refrain from eating, drinking, chewing gum, alcohol, tobacco, caffeine use or brushing 
their teeth at least 1 hour before the measurements will be performed. Room temperature and lighting 
will be set at the same level for all measurements. Participants will exhale through a disposable 
mouthpiece, into a heated tube (stainless steel, Silconert coated) (50 cm long, 4 mm in diameter) 
connected to the curtain AUX gas port of a Orbitrap spectrometer (Thermo Fisher, Waltham, 
Massachussetts, USA) that is provided by DBI. The sampling tube is surrounded with an isolated 
heating tape at 130 °C to prevent water condensation and to minimize losses of exhaled compounds 
onto the walls of the tube. While performing full exhalations, the subjects will keep the volume through 
the sampling line at 8L/min monitored by a digital flow meter. A nano electrospray plume (FIT, Madrid, 
Spain), provided by DBI, will be used, where compounds of exhaled breath get ionized and 
subsequently are mass analysed. The technique, by which breath is analysed in real time by injecting 
exhaled breath into electrospray plumes of pure solvent and thus causing ionization of relevant 
exhaled compounds(54-56), has been referred to as secondary electrospray ionization (SESI)(57). 
Breathprints will be collected in real-time recording six replicates through the mouthpiece. The raw 
mass spectra will be exported as mzXML files for further processing using MATLAB (Mathworks, Inc). 
The spectra of exhaled breath will be averaged. In this process, only the last few seconds (typically 
around 6 sec.) of each exhalation will be considered, thus the first part of the exhalation, which reflects 
mostly the dead space in the upper respiratory tract, will be excluded from the analysis. SESI-MS 
allows for real-time breath-printing by detection of both volatile and non-volatile trace components in 
breath without any sample pre-treatment. The de-identified mass spectrometric breath signatures of 
patients are subsequently analysed by DBI with AI algorithms for spectroscopic profiling. 
4.5.8 
Questionnaires 
4.5.8.1 
COPD Assessment Test (CAT) & midified Medical Research Council (mMRC) 
For assessing symptoms, GOLD guidelines primarily recommend the use of the COPD Assessment 
Test (CAT) or the modified Medical Research Council (mMRC) dyspnea score. The CAT score is a 
patient-completed instrument to assess and quantify health-related quality of life and symptom burden 
in COPD patients. It comprises 8 questions, each is presented as a semantic 6-point (0-5) differential 

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scale, providing a total score out of 40. Scores of 0-10, 11-20, 21-30, 31-40 represent mild, moderate, 
severe or very severe clinical impact, respectively. The mMRC dyspnea score is a 5-point (0-4) scale 
based on the severity of dyspnea.(58) 
4.5.8.2 
QUIT 
QUIT is a Questionnaire to assess the probability of therapy adherence, especially in inhaled therapies. 
The Test was developed by the healthcare research unit of the Federal Association of Pneumologists 
in Germany. The Questionnaire was initially designed with focus on asthma patients. In COPD the 
adherence to inhalational therapy is much higher due to the consistency of symptoms during the disease 
progression. We use an adapted version with focus on COPD therapy.(59) 
4.5.9 
Study visits 
Participation in the study requires two on-site study visits. Visit 1 will be performed while the participant 
is hospitalized. This visit will have a duration of approximately 45 minutes. After approximately 8 weeks 
patients are invited to the hospital to perform visit 2. This visit will take again approximately 45 minutes. 
Further information in Appendix. 
4.5.10 Study duration 
Individual participation time in this study will be approximately 8 weeks. Overall recruitment of patients 
will last until the targeted sample size is reached. 
4.6 Withdrawal and discontinuation 
Consent withdrawal of the patient will lead to an exclusion of the study. Data obtained up to the 
withdrawal will be included for the analysis. 
5. STATISTICS AND METHODOLOGY 
5.1 Statistical analysis plan 
For data processing, we will follow the same procedure as described in our previous studies [Gaugg, 
2016]: raw mass spectra will be transformed to *.mzXML format using the online tool msConvert. The 
*.mzXML files will be imported into Matlab. Each mass spectrum is then interpolated (shape-preserving 
piecewise cubic interpolation method), mass calibrated using an iterative grid search algorithm, and the 
resulting spectra are centroided. To test the hypothesis that there is a significant difference in the values 
of exhaled metabolites during and after the exacerbation, we will perform a paired-sample t-test to 

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evaluate differential concentrations of such metabolites. False discovery rate and q-values will be 
subsequently computed as defined by Storey.(60) To account for instrumental drift (e.g., detector 
sensitivity), the mass spectra are normalized using standard methods such as interquartile range, total 
ion current. Well-established data reduction techniques such as principal component analysis will be 
used for visualization purposes. Finally, high mass resolution/high mass accuracy data obtained will 
allow us to query online databases (e.g., human metabolome database) to provide tentative 
identification of the subset of most informative exhaled compounds. The preprocessing of the raw mass 
spectra and the statistical analysis will be implemented using MATLAB (MathWorks, Massachusetts, 
US). 
For correlation analysis, the Pearson’s linear correlation coefficient and 95% bootstrap confidence 
intervals will be applied. Continuous data will be summarized by means (SD) or medians (quartiles) as 
appropriate. A two-sided p value of <0.05 will be considered to be statistically significant for all tests.  
 
5.2 Sample Size calculation 
As this is an exploratory study it is not possible to perform a conventional sample size calculation. Based 
on the results of our published previous study including a control group(18), a group size of 40 patients 
has been chosen for this longitudinal study.Overall recruitment of patients will last until the targeted 
sample size is reached and full data sets of 40 subjects are acquired (two visits with breath analysis).  
 
5.3 Handling of missing data 
Subjects with missing data are excluded if the data is considered relevant to the specific analysis. Drop-
outs will be replaced.  
6. REGULATORY ASPECTS AND SAFETY  
6.1 Registration and Categorisation 
The study will be registered in the study registry ClinicalTrials.gov (61). The study is in line with risk 
category A according to HFV(62) and entails only minimal risks and burdens. 
6.2 Local regulations/Declaration of Helsinki 
This research project will be conducted in accordance with the protocol, the Declaration of Helsinki(8), 
the principles of Good Clinical Practice, the Human Research Act (HRA) and the Human Research 

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Ordinance (HRO) as well as other locally relevant regulations.(62) The Principle Investigator 
acknowledges his responsibilities as both the Principle Investigator and the Sponsor.  
6.3 Participant privacy and confidentiality  
The investigator affirms and upholds the principle of the participant's right to privacy and that they shall 
comply with applicable privacy laws. Especially, anonymity of the participants shall be guaranteed when 
presenting the data at scientific meetings or publishing them in scientific journals.  
Individual subject medical information obtained as a result of this study is considered confidential and 
disclosure to third parties is prohibited. Subject confidentiality will be further ensured by utilising subject 
identification code numbers to correspond to treatment data in the computer files. 
For data verification purposes, authorised representatives of the Sponsor (-Investigator), a competent 
authority (CA) (e.g. Swissmedic), or an ethics committee may require direct access to parts of the 
medical records relevant to the study, including participants’ medical history. 
6.4 Notification of safety and protective measures (HRO Art. 20) 
The project leader is promptly notified (within 24 hours) if immediate safety and protective measures 
have to be taken during the conduct of the research project. The Ethics Committee will be notified via 
BASEC of these measures and of the circumstances necessitating them within 7 days. 
6.5 Serious events (HRO Art. 21) 
If a serious event occurs, the research project will be interrupted and the Ethics Committee notified on 
the circumstances via BASEC within 7 days according to HRO Art. 211.  
Due to the health state of our subjects and their life expectancy, we expect the subjects to have non-
related serious events during the study time. Subject will be instructed to report serious events to the 
study team. Serious events else than “related” will not be reported to the Ethics Committee. 
6.5.1 
Definition of Seriouse Events 
A serious event (SE) is any unfavourable event for which a causal relationship to sampling of biological 
material or the collection of health related personal data cannot be ruled out, and which:   
• 
requires hospitalization or prolongation of  an inpatients’ hospitalization, 
                                                     
1 A serious event is defined as any adverse event where it cannot be excluded, that the event is attributable to the 
sampling 
of 
biological 
material 
or 
the 
collection 
of 
health-related 
personal 
data. 
 

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• 
results in persistent or significant disability or incapacity, or 
• 
is life-threatening or results in death, 
If a serious event occurs the research project must to be set on hold. 
6.5.2 
Assessment of SEs 
The assessment by the project leader regarding the causal relationship of an event to the project-specific 
measure is done according to the following definitions:  
Unrelated 
• 
The event started in no temporal relationship to the project-specific 
measures applied and 
• 
The event can be definitely explained by underlying diseases or other 
situations. 
Related 
 
• 
The event started in plausible time relationship to the project-specific 
measures applied and 
• 
The event cannot be definitely explained by underlying diseases or other 
situations. 
 
All SEs are to be documented in the subjects´ file and on the SE report form. 
 
6.6 Early termination of the study  
The Sponsor-Investigator may discontinue the study prematurely according to certain circumstances:  
• 
ethical concerns,  
• 
insufficient participant recruitment,  
• 
alterations in accepted clinical practice that make the continuation of a clinical study unwise  
6.7 Protocol amendments 
Substantial amendments are only implemented after approval of the CEC. Under emergency 
circumstances, deviations from the protocol to protect the rights, safety and well-being of human 
participants may proceed without prior approval of the sponsor and the CEC. Such deviations shall be 
documented and reported to the sponsor and the CEC as soon as possible. 
6.8 End of project 
Upon project termination, the Ethics Committee is notified within 90 days.  

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6.9 Insurance 
Insurance is covered by “Versicherung für klinische und nicht-klinische Versuche des Kantons Zürich 
für das UniversitätsSpital Zürich (Policennummer 15.369.591)“. Any damage developed in relation to 
study participation is covered by this insurance. So as not to forfeit their insurance cover, the participants 
themselves must strictly follow the instructions of the study personnel. The investigator must also be 
informed instantly, in the event of health problems or other damages during or after the course of study 
treatment. 
The investigator will allow delegates of the insurance company to have access to the source 
data/documents as necessary to clarify a case of damage related to study participation. All involved 
parties will keep the patient data strictly confidential. A copy of the insurance certificate will be placed in 
the Investigator’s Site File. 
7. FURTHER ASPECTS 
7.1 Overall ethical considerations 
This will be the first study to investigate the acute effects of COPD exacerbations on molecular exhaled 
breath patterns assessed by SESI-HRMS. By identifying a distinctive breath pattern it may be possible 
to define biochemical processes specific of a COPD exacerbation and correlate it with causative agents, 
treatment and outcome. It may also be possible to identify those patients who need, and are most likely 
to respond to additional therapy such as e.g. antibody treatment. 
The study procedure (contact to patients and relatives) will be conducted in accordance with 
internationally accepted quality criteria and entails only minimal risks and burdens.  
7.2 Ethical Conduct of the project 
The study will be carried out in accordance with principles enunciated in the current version of the 
Declaration of Helsinki (8),  the principles of GCP, the Federal Act on Research involving Human Beings 
(HRA), the Ordinance on Human research with the exception of clinical trials (HRO)(62), and Swiss 
Academy of Medical Sciences’ ethics guidelines (63). Before this study will be conducted, the protocol, 
the proposed participant information and all other study-specific documents, which are recommended 
by swissethics(64), will be submitted to Competent Ethics Committee (CEC) of the Canton of Zurich in 
agreement with local legal requirements, for formal approval.  
The decision of the competent authority concerning the conduct of the study will be made in writing to 
the Sponsor-Investigator before commencement of this study. The study can only begin once approval 

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from all required authorities has been received. Any additional requirements imposed by the authorities 
shall be implemented. No substantial amendments are made to the protocol without prior CEC approval, 
except where necessary to eliminate apparent immediate hazards to study participants. 
The following are considered to be significant changes/substantial amendments: 
• 
changes affecting the participants’ safety and health, or their rights and obligations; 
• 
a change of research site or conducting the research project at an additional site; or 
• 
a change of the project leader or sponsor. 
The regular end of the study or premature study end or interruption is reported to the CEC within 90 
days. 
7.3 Participant information and informed consent 
Prior to the project start each participant has to give their written informed consent after he/she was 
comprehensively informed - verbally and in writing - on the nature, relevance and impact of the project. 
The content of this information should be documented on the informed consent. Each participant must 
be informed that the participation in the project is voluntary and that he/she may withdraw from the 
project at any time and that withdrawal of consent will not affect his/her subsequent medical treatment. 
The participants will be given enough time to make their decision whether they would like to participate 
or not. 
The consent to participate into the research project should be signed and dated by both, the participant 
and the project leader. A copy of the signed and dated subject information and informed consent form 
has to be handed out to the participant. 
No project procedures are to be conducted before a legally accepted written informed consent has been 
given. 
 
7.4 Risk / Benefit Assessment 
This study does not impose any potential threats or harms to the participants. Participants will undergo 
the regular diagnosis and treatment pathway for patients with COPD as it is standard in the University 
Hospital Zurich. The knowledge and insights emanating from this study will potentially advance research 
efforts focused on COPD and may ultimately lead to better diagnosis and treatment of this condition. 
7.5 Compensation offered to participants 
Participants will not be offered any material compensation for their voluntary agreement to take part in 

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this study. Whilst the scientific benefit from participation will be explained to participants, no other 
incentives will be offered.  
 
8. QUALITY CONTROLE AND DATA PROTECTION  
8.1 Quality measures 
The Sponsor-Investigator is implementing and maintaining quality assurance and quality control 
systems with written SOP and working instructions to ensure that studies are conducted and data are 
generated, documented (record), and reported in accordance with the protocol, the Declaration of 
Helsinki(8), the Human Research Act (HRA) and the Human Research Ordinance (HRO)(62) as well as 
other locally relevant regulations. Trainings will be performed including all the study staff before start of 
the study. During the study, one independent internal quality control will take place. Designated study 
team members will be responsible for data collection, confidentiality, and data management. 
For quality assurance the Ethics Committee may visit the research sites. Direct access to the source 
data and all project related files and documents must be granted on such occasions. 
8.2 Data management/ Data recording and Source data 
Designated research team members will be responsible for data collection and data management. Data 
are registered in a case report form (CRF). Paper documents and informed consent forms will be stored 
in a lock-secured cupboard in a dedicated research office at the University Hospital Zurich. Only study 
team members have access to these documents. Authorized staff of the responsible ethics committee 
can request access to these data for monitoring and auditing purposes. Electronic data will be stored in 
a password-secured folder. The files will be stored within the University Hospital Zurich intranet server. 
It can only be accessed by authorized users from registered computers at the University Hospital Zurich. 
All personal and medical information obtained for this study is confidential and disclosure to third parties 
other than those noted below is prohibited. Confidentiality of the subjects will be maintained by assigning 
subjects a study number (ID), keeping identifiers separate from the data and storing data in a locked file 
and secure computer database in line with Swiss legal requirements. Patient identifier will be assigned 
with a code of letters and numbers (the first letter for the population, a consecutive number for the 
subject, the visit and the sample or data type resp. e.g. TX0001V1). 
The USZ will handle administration of the study database. The data entry will be performed by the study 

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team, consisting of investigators and study nurses. Study researchers will only be given access to 
database exports and will not have direct system access. Only de-identified database exports will be 
made accessible to the project collaborators DBI.  
8.3 Data handling and record keeping / archiving  
The study will strictly follow the protocol. If any changes become necessary, they must be laid down in 
an amendment to the protocol. All amendments of the protocol must be signed by the Sponsor-
Investigator and if essential submitted to CEC. 
8.3.1 
Case Report Forms  
The investigators will use paper case report forms (CRF), one for each enrolled study participant, to be 
filled in with all relevant data pertaining to the participant during the study. All participants who either 
entered the study or were considered not-eligible or were eligible but not enrolled into the study 
additionally have to be documented on a screening log. The investigator will document the participation 
of each study participant on the Subject Master List. 
CRFs must be kept current to reflect participant status at each phase during the course of study. 
Participants must not to be identified in the CRF by name. It must be assured that any authorized person, 
who may perform data entries and changes in the CRFs, can be identified. A list with signatures and 
initials of all authorized persons will be filed in the study site file and the trial master file, respectively. 
Documented medical history and narrative statements will be additionally maintained. For further 
analysis, data will be transferred into electronic data sheets (spreadsheets) including an audit trail and 
will be stored password-secured. The paper CRF as well as electronic data will serve as source for 
study data. 
8.3.2 
Record keeping / archiving  
The study data (Trial Master File [TMF], CRF, etc.) will be archived in a dedicated research office at the 
Department of Pulmonology, University Hospital Zurich, Rämistrasse 100, CH-8091 Zürich. A 
designated research member will be responsible for data collection, confidentiality, and data 
management. The collected data will be stored in the Trial master folder, which will be stored in a 
designated research office (Room RAE C 25) in a lock-secured cupboard. Some data will be 
documented on paper sheets and may be stored as certified copies only. For further analysis, data will 
be transferred into electronic data sheets (spreadsheets) including an audit trail and will be stored 

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password-secured. The file will be stored within the Division of Pulmonology research-section of the 
University Hospital of Zurich intranet server. The file itself can be only accessed by researchers from 
registered computers at the University of Zurich. Access is electronically recorded and password-
secured. The study personnel will preserve the confidentiality of participants taking part in the study 
according to HFV art. 5(62). All study data must be archived for a minimum of 10 years after study 
termination or premature termination of the clinical trial. 
8.4 Details of the Secure Handling of Data connected to DBI 
 
Data will be collected at University Hospital Zurich premises only by using Hardware and data acquisition 
tools (software), under the medical and administrative supervision and control of USZ. Data collection, 
data storage and data analytics will be described in more details in the following: 
Medical records and de-identification of patients  
All patients data will be de-identified by providing only a patient number with corresponding labels 
(gender, age, COPD stage/healthy,..) to DBI. This file is stored at DBI. DBI will not get any additional 
medical records other than these evaluated during the study visits. 
Data collection and Data transfer 
The unique patient identifier (number) will be used for the data collection as the only label received from 
USZ, added to the corresponding data acquired. The time series of data spectra will be locally encrypted 
on Hardware, transferred via internet to a Swiss-based, secure cloud storage, stored in encrypted state 
in the cloud, and transferred and finally decrypted at DBI. DBI will report the de-identified and clustered 
output data to USZ. 
Data analytics at DBI 
At DBI, data will be subdivided into temporal series of data and merged with the relevant labels (gender, 
age, GOLD status, …) Based on the subject-matter expert input, all data will be analyzed regarding 
specific VOCs in a targeted way (using a data subset with filtered VOCs levels) or broadband in a 
untargeted way (using the full spectrum). Various Machine Learning (“ML”) and Artificial Intelligence 
(“AI”) algorithms will be tested. 
8.5 Audits and Inspections  
A quality assurance audit/inspection of this study may be conducted by the CEC. The quality assurance 
auditor/inspector will have access to all medical records, the investigator's study related files and 

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correspondence, and the informed consent documentation that is relevant to this clinical study. The 
investigator will allow the persons being responsible for the audit or the inspection to have access to the 
source data/documents and to answer any questions arising. All involved parties will keep the patient 
data strictly confidential. As part of the informed consent, participants agree that authorized members 
of staff of the responsible ethics committee can obtain access to confidential data for monitoring and 
auditing purposes under strict maintenance of participant’s confidentiality. 
9. FUNDING /DECLARATION OF INTEREST 
9.1 Funding & Support 
This investigator initiated trial (IIT) will be conducted in collaboration with the Deep Breath Initiative 
(DBI). GlaxoSmithKline plc. will provide financial support. DBI will provide the mass spectrometer as 
well as the ionisation source, technical support and assist in the data analysis process by employing 
machine learning algorithms.   
9.2 Declaration of interest  
Professor Dr. med. Kohler declares that he owns shares of DBI, other than that none of the authors has 
any conflict of interest (independence, intellectual, financial, proprietary). Professor Dr. med. M. Kohler 
is cofounder of the Deep Breath Initiative (DBI), a company that provides services in the field of breath 
analysis (www.dbi.ch). 
10. PUBLICATION AND DISSEMINATION POLICY  
After the statistical analysis of this study every endeavour to publish the data via publication in peer-
reviewed scientific media will be made. Dissemination of results will be independent of negative or 
positive findings and submission of results for publication will follow the consensus decision of all 
investigators. Authorship credit is based on 1) substantial contributions to conception and design, 
acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for 
important intellectual content; and 3) final approval of the version to be published. Each author should 
have participated sufficiently in the work to take public responsibility for appropriate portions of the 
content. 

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11. REFERENCES  
1. 
WHO. Top 10 causes of death 2018 [Access date: 28.04.2020]. Available from: 
https://www.who.int/gho/mortality_burden_disease/causes_death/top_10/en/. 
2. 
Bruderer T, Gaisl T, Gaugg MT, Nowak N, Streckenbach B, Muller S, et al. On-Line Analysis of 
Exhaled Breath Focus Review. Chem Rev. 2019;119(19):10803-28. 
3. 
Gaugg MT, Nussbaumer-Ochsner Y, Bregy L, Engler A, Stebler N, Gaisl T, et al. Real-Time 
Breath Analysis Reveals Specific Metabolic Signatures of COPD Exacerbations. Chest. 
2019;156(2):269-76. 
4. 
GOLD. 2020 Global Strategy for Prevention, Diagnosis and Management of COPD 2020 
[Access date: 28.04.2020]. Available from: https://goldcopd.org/. 
5. 
Viniol C, Vogelmeier CF. Exacerbations of COPD. Eur Respir Rev. 2018;27(147). 
6. 
Romero GAF, Beros J, Criner G. Mepolizumab for the prevention of chronic obstructive 
pulmonary disease exacerbations. Expert Rev Resp Med. 2019;13(2):125-32. 
7. 
Storey JD. A direct approach to false discovery rates. J Roy Stat Soc B. 2002;64(3):479-98. 
8. 
WMA. Declaration of Helsinki - Ethical Principles for Medical Research Involving Human 
Subjects 
2013 
[Access 
date: 
23.05.2014]. 
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12. APPENDIX 
Overview study procedures 
 
Study Procedure 
Parameter Analysis 
 
 V 1 
(day 0) 
V 2 
(8 weeks) 
ICF  
 
x 
 
Medical history 
 
age, gender, weight, height, eating 
habits, activity habits,  medication, co-
morbidities, smoking history, disease 
specific images and clinical chemistry 
test results, exacerbation history 
x 
x 
Vital parameter 
 
BP, HR, T, SpO2 
x 
x 
Inflammatory markers 
 
CRP, eosinophil level 
x 
x 
Spirometry 
FEV1, FVC 
x2 
x2 
FeNO 
 
x2 
x2 
Sputum & viral smear 
 
x1 
 
Observational data 
collection 
On-line breath analysis 
x 
x 
Questionnaire (eg. COPD 
Assessment Tool) 
CAT, mMRC, QUIT 
x 
x 
SE review 
 
x 
x 
1  collected routinely during the clinical assessment after hospitalization 
2 if applicable