D2.8 Database on structure-process and structure-activity relationship

RADIATION HARVESTING OF BIOACTIVE PEPTIDES FROM EGG PROTEINS AND THEIR INTEGRATION IN ADVANCED FUNCTIONAL PRODUCTS INNOVATION ACTION OF HORIZON EUROPE EURATOM RESEARCH AND TRAINING PROGRAMME GA NO 101061694 DELIVERABLE REPORT DELIVERABLE REPORT DELIVERABLE: D2.8 Database on structure-process and structure-activity relationship DELIVERABLE: D2.8 Document identifier: RADOV-Del-D2.8 Due date of deliverable: End of month 24 (August 2024) Report release date: 03/10/2025 Justification for delay: WP2 duration was extended to collect more data. Moreover, data presented in this report needed to be aligned with the bioactivity results (D2.5). Work package: Lead beneficiary: KTH Document status: Final WP2 [Development of laboratory scale production of irradiated egg products and peptides] 1 | Page Deliverable D2.8 Date: 03/10/2025 Database on structure-process and structure-activity relationship Grant Agreement 101061694 PUBLIC This project has received funding from the European Union’s Horizon Europe Research and Training programme under Grant Agreement No 101061694. RADOV began in September 2022 and will run for 4 years. Delivery Slip Name Partner Date Authored by Mats Jonsson KTH 28/02/2025 Edited by Åsa Emmer KTH 28/02/2025 Reviewed by Steering Committee All Partners 10/10/2025 Approved by Dagmara Chmielewska-Śmietanko INCT 10/10/2025 2 | Page Deliverable D2.8 Date: 03/10/2025 Database on structure-process and structure-activity relationship Grant Agreement 101061694 PUBLIC TABLE OF CONTENTS 1. Introduction 3 2. Results 3 2.1 Protein structure 3 2.2 Irradiation conditions 6 2.3 Conversion yields 6 2.4 Characterization of irradiated products. 8 2.5 Bioactivity 11 3. Database 13 4. Structure-process and structure-activity relationships 13 5. References 14 6. Database on different irradiated hen egg products 14 3 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC Executive summary 1. Introduction To conclude the work performed in WP2, a deliverable presenting a database and structure-process and structure-activity relationships based on the data in the database was planned. The initial plan was based on the production and characterization of peptides isolated from proteins irradiated under various conditions. The assumption was that peptides formed upon radiation-induced scission of egg proteins would display bioactivity beyond the egg proteins themselves. However, the work within WP2 has shown that irradiation of egg proteins does not lead to the formation of significant fractions of peptides. In some cases, the formation of fragments can be observed but never to the extent required to be able to isolate a given fraction. For this reason, the focus of this deliverable has been shifted from well-defined peptides to less defined products. Even though it was not possible to produce peptide fractions, egg proteins exposed to ionizing radiation under certain conditions display increased bioactivity. In this report, the structure of proteins and products formed upon irradiation of proteins will be related to irradiation conditions and bioactivity. 2. Results 2.1 Protein structure The amino acid composition of the three hen egg proteins used in this project are listed in tables 1-3. Lysozyme is a small-sized, globular protein consisting of 129 amino acids in total and with a molecular weight of around 14 kDa. It consists of 8 cysteine residues, forming four disulfide bridges, which gives the protein its high conformational To conclude the work performed in WP2, a deliverable presenting a database and structure-process and structure-activity relationships based on the data in the database was planned. In this report, qualitative relationships are presented for the limited cases where product structure and bioactivity are known to some extent. The systems discussed in terms of these relationship are solid-state irradiated lysozyme and ovalbumin as well as lysozyme irradiated in aqueous solution under reducing conditions (1 M 2-propanol purged with N2 prior to irradiation). 4 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC stability. Table 1. Amino acid composition of lysozyme, which consists of 129 amino acids in total. Amino acid Number Percentage Alanine (A) 12 9.3 % Arginine (R) 11 8.5 % Asparagine (N) 14 10.9 % Aspartic acid (D) 7 5.4% Cysteine (C) 8 6.2 % Glutamic acid (E) 2 1.6 % Glutamine (Q) 3 2.3 % Glycine (G) 12 9.3 % Histidine (H) 1 0.8 % Isoleucine (I) 6 4.7 % Leucine (L) 8 6.2 % Lysine (K) 6 4.7 % Methionine (M) 2 1.6 % Phenylalanine (F) 3 2.3 % Proline (P) 2 1.6 % Serine (S) 10 7.8 % Threonine (T) 7 5.4 % Tryptophan (W) 6 4.7 % Tyrosine (Y) 3 2.3 % Valine (V) 6 4.7 % Ovalbumin is an average-sized globular protein consisting of 386 amino acids in total and with a molecular weight of around 43 kDa. Out of the six cysteine moieties, two make up a disulfide bridge, while the remaining four provide free thiol groups. The amino acid profile can be considered balanced, and it is a quite stable protein. Table 2. Amino acid composition of ovalbumin, which consists of 386 amino acids in total. Amino acid Number Percentage Alanine (A) 35 9.1 % Arginine (R) 15 3.9 % Asparagine (N) 17 4.4 % Aspartic acid (D) 14 3.6 % Cysteine (C) 6 1.6 % Glutamic acid (E) 33 8.5 % Glutamine (Q) 15 3.9 % Glycine (G) 19 4.9 % Histidine (H) 7 1.8 % Isoleucine (I) 25 6.5 % Leucine (L) 32 8.3 % Lysine (K) 20 5.2 % 5 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC Methionine (M) 17 4.4 % Phenylalanine (F) 20 5.2 % Proline (P) 14 3.6 % Serine (S) 38 9.8 % Threonine (T) 15 3.9 % Tryptophan (W) 3 0.8 % Tyrosine (Y) 10 2.6 % Valine (V) 31 8.0 % Ovotransferrin is a relatively large, unstable, globular protein consisting of 686 amino acids and with a molecular weight of around 76 kDa. There are 30 cysteines, all participating in creating 15 disulfide bonds. Table 3. Amino acid composition of ovotransferrin, which consists of 686 amino acids in total. Amino acid Number Percentage Alanine (A) 54 7.9 % Arginine (R) 30 4.4 % Asparagine (N) 30 4.4 % Aspartic acid (D) 46 6.7 % Cysteine (C) 30 4.4 % Glutamic acid (E) 45 6.6 % Glutamine (Q) 23 3.4 % Glycine (G) 52 7.6 % Histidine (H) 12 1.7 % Isoleucine (I) 26 3.8 % Leucine (L) 49 7.1 % Lysine (K) 59 8.6 % Methionine (M) 11 1.6 % Phenylalanine (F) 26 3.8 % Proline (P) 28 4.1 % Serine (S) 48 7.0 % Threonine (T) 37 5.4 % Tryptophan (W) 10 1.5 % Tyrosine (Y) 21 3.1 % Valine (V) 49 7.1 % When we look at the amino acid composition of the three proteins, we notice that lysozyme contains the greatest percentage of amino acids with antioxidative capacity (28.8 %), followed by ovotransferrin (25.3 %), and ovalbumin (20.3 %) [1]. The fractions of amino acids with antioxidative capacity for the three proteins are listed in table 4. 6 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC Table 4. Fractions of amino acids with antioxidant properties in lysozyme, ovalbumin and ovotransferrin Amino acid Lysozyme Ovalbumin Ovotransferrin Arginine (R) 8.5% 3.9% 4.4% Cysteine (C) 6.2% 1.6% 4.4% Histidine (H) 0.8% 1.8% 1.7% Lysine (K) 4.7% 5.2% 8.6% Methionine (M) 1.6% 4.4% 1.6% Tryptophan (W) 4.7% 0.8% 1.5% Tyrosine (Y) 2.3% 2.6% 3.1% 2.2 Irradiation conditions The irradiation conditions are given in the database (attached). The expected impact of changing the conditions is discussed in D2.1. Table 5 lists the different parameters that have been evaluated, resulting in over 250 unique conditions. Table 5. List of parameters in irradiation of proteins and egg products. Parameter Physical state Solid state /Aqueous solution Atmosphere Air/N2O/N2 Radiation High energy electrons/-radiation Temperature Room temperature/Dry ice Dose 1 and 10 kGy (and up to 500 kGy) Co-Solute None/Phosphate buffer/TRIS/2-propanol Protein concentration 0.05 – 10 mg/ml 2.3 Conversion yields The first step in being able to quantify process efficiency is to determine the conversion yields. In radiation chemistry, so-called radiation chemical yields or Gvalues are used. Radiation chemical yields are defined as amount of substance consumed or produced per unit of radiation energy absorbed. The SI-unit is mol/J but it is quite common to find the unit number of molecules/100 eV in the literature. In the following text we focus on the G-values for consumption of protein based on MALDI TOF analysis. 7 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC The consumption of lysozyme in aqueous solutions at 1 mg/ml under air, N2 or N2O, and in the absence or presence of phosphate buffer corresponds to radiation chemical yields for protein consumption in the order of 5-6 x 10-8 mol/J. In the systems containing 2-propanol, the solutions were purged with N2 prior to irradiation in order remove dissolved oxygen. The main radiolysis products formed under these conditions are the hydrated electron (e-aq) and the hydroxyl radical. The latter (and the hydrogen atom) reacts with 2-propanol and the major product of this reaction is the strongly reducing 2-hydroxy-2-propyl radical. Hence, the system becomes reducing. It has been shown that protein consumption is mainly (but not only) driven by the hydrated electron. Irradiations performed using 1 mg/ml lysozyme and either 2-propanol or t-butanol (a hydroxyl radical scavenger that does not produce a reducing radical) show that the 2-hydroxy-2-propyl radical contribute to some extent to the consumption of lysozyme. The estimated radiation chemical yield for protein consumption is 5.5 x 10-8 mol/J under these conditions. The G-value for the hydrated electron is 2.8 x 10-7 mol/J. Given the fact that the hydrated electron has been reported to have a very high rate constant for the reaction with lysozyme, the efficiency of this reaction would appear to be quite low. A possible rationale for this is that the molar concentration of protein molecules is quite low from the beginning. Based on initial protein conversion, the radiation chemical yield for protein alteration in the solid state is 6.3 x 10-7, 6.2 x 10-7 and 6.8 x 10-7 mol/J for lysozyme, ovalbumin and ovotransferrin, respectively. Interestingly, the radiation chemical yields are independent of protein structure. Due to the large differences in molecular weight, the doses required for full protein conversion differ significantly between the proteins. For proteins with higher molecular weight (ovotransferrin and ovalbumin), fewer alterations per unit mass are required to reach full conversion of the starting material. The G-values for protein consumption/conversion are summarized in table 6 Table 6. Radiation chemical yields for protein alteration under different irradiation conditions. Protein Irradiation conditions G-value (mol/J) Lysozyme Aqueous solution, N2/N2O/Air, with and without PBS* 5-6 x 10-8 Lysozyme Aqueous solution, 2propanol, N2 5.5 x 10-8 Lysozyme Solid-state 6.3 x 10-7 Ovalbumin Solid-state 6.2 x 10-7 Ovotransferrin Solid-state 6.8 x 10-7 *Note that these are six different conditions. 8 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC 2.4 Characterization of irradiated products Gammaand electron beam irradiation of aqueous protein solutions saturated with air or purged with N2O (or N2) were often found to produce soluble or insoluble aggregates. Based on both MALDI-TOF and SDS-page, irradiations of the proteins in solution under reducing conditions induces protein fragmentation. This is particularly obvious for lysozyme. From experiments where the reducing conditions were obtained in different ways it was concluded that protein fragmentation can mainly be attributed to reactions with the hydrated electron with a minor contribution from the 2-hydroxy-2propyl radical. However, it should be stressed that fragmentation to a detectable size fraction only accounts for a minor part of the overall protein consumption. Interestingly, the increase in the amount of detectable fraction of smaller fragments formed upon irradiation of lysozyme in aqueous solution under reducing conditions does not parallel the consumption of lysozyme. This could imply that the detectable fraction represents a reaction intermediate rather than a final product. In other words, it is possible that the observable fragments undergo further fragmentation upon extended irradiation. This is well in line with the arguments given above concerning the low radiation chemical yield for protein conversion. If this is the case, fragments of the desired size need to be harvested at an early stage. Given the fact that the fragments formed will be present in a high molar concentration compared to the protein, it is probable that the hydrated electrons will primarily react with the fragments in competition with the protein. For the solid-state irradiated proteins, fragmentation is also observed. As MALDI TOF, SDS-page and liquid chromatography did not reveal any sizeable product fractions that could be isolated for further analysis and use in the applications developed in WP3, alternative methods have been used to identify alterations induced by ionizing radiation. The techniques used are FTIR, Raman spectroscopy, MALDI-TOF MS/MS and NMR. The results from these studies are summarized below: Fourier Transform Infrared Spectroscopy (FTIR) ATR-FTIR analysis was performed on non-irradiated proteins dissolved in ultra pure water at concentrations ranging from 1 mg/mL to 5 mg/mL. The experimental setup was configured with a resolution of 4 cm-1 and 64 scans, and with the x-axis range set between 4000 and 500 cm-1. The samples exhibited low absorbance, with no significant peaks observed, likely due to insufficient concentration. Increasing concentration was not feasible, as it led to undissolved solids remaining in the solution. Attempts to dissolve the sample in acetonitrile (1 mg/mL) were 15 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC Sample: Lysozyme IRRADIATION pH DATE STORAGE ANALYSIS Place of delivery After shopiment/ before analysis Analysis Without DTT With DTT Sample ID Institute Atmosphere Solution composition Concentration Source Dose Temperature Before irradiation After irradiation of irradiation of receival of analysis Type of storage Visual observations Analysis method Analysis performed at Consumption of native protein Fragments Aggregates Consumption of native protein Fragments Aggregates INCT-1GLD1IL-RA INCT KTH Air Dry n/a EB/ILU 1 kGy RT 24.10.2022 RT MALDI-TOF KTH No No Marginally Few INCT-1GLD10IL-RA INCT KTH Air Dry n/a EB/ILU 10 kGy RT 24.10.2022 RT MALDI-TOF KTH No No Marginally Few INCT-1GLD5IL-RA INCT IST-ID Air Dry n/a EB/ILU 5 kGy RT 24.10.2022 INCT-1GLD10IL-RA INCT IST-ID Air Dry n/a EB/ILU 10 kGy RT 24.10.2022 INCT-1GLD25IL-RA INCT IST-ID Air Dry n/a EB/ILU 25 kGy RT 24.10.2022 INCT-1LS-1ELRN/ INCT-1LS1EL-RNPO INCT KTH N2O H2O 1 mg/ml EB/EL 1 kGy RT 06.12.2022 Freezer, -18˚ C MALDI-TOF KTH Yes Few Few, but very low intensity INCT-1LS-1ELRA/ INCT-1LS1EL-RAPO INCT KTH Air H2O 1 mg/ml EB/EL 1 kGy RT 06.12.2022 Freezer, -18˚ C MALDI-TOF KTH Yes Few Few, but very low intensity INCT-1LS-1ELRI/ INCT-1LS1EL-RIPO INCT KTH N2 H2O 1 mg/ml EB/EL 1 kGy RT 06.12.2022 Freezer, -18˚ C MALDI-TOF KTH Yes Few Few, but very low intensity INCT-1LS-1ELRNP/ INCT1LS-1ELRNPOP INCT KTH N2O Phosphate buffer 1 mg/ml EB/EL 1 kGy RT 06.12.2022 Freezer, -18˚ C Precipitation MALDI-TOF KTH Yes Few Few, but very low intensity INCT-1LS-1ELRAP/ INCT1LS-1ELRAPOP INCT KTH Air Phosphate buffer 1 mg/ml EB/EL 1 kGy RT 06.12.2022 Freezer, -18˚ C Precipitation MALDI-TOF KTH Yes Few Few, but very low intensity INCT-1LS-1ELRIP/ INCT1LS-1ELRIPOP INCT KTH N2 Phosphate buffer 1 mg/ml EB/EL 1 kGy RT 06.12.2022 Freezer, -18˚ C Precipitation MALDI-TOF KTH Yes Few Few, but very low intensity INCT-1LS10EL-RN/ INCT-1LS10EL-RNPO INCT KTH N2O H2O 1 mg/ml EB/EL 10 kGy RT 06.12.2022 Freezer, -18˚ C MALDI-TOF KTH Yes Few Few, but very low intensity INCT-1LS10EL-RA/ INCT-1LS10EL-RAPO INCT KTH Air H2O 1 mg/ml EB/EL 10 kGy RT 06.12.2022 Freezer, -18˚ C MALDI-TOF KTH Yes Few Few, but very low intensity INCT-1LS10EL-RI/ INCT-1LS10EL-RIPO INCT KTH N2 H2O 1 mg/ml EB/EL 10 kGy RT 06.12.2022 Freezer, -18˚ C MALDI-TOF KTH Yes Few Few, but very low intensity 16 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC INCT-1LS10EL-RNP/ INCT-1LS10EL-RNPOP INCT KTH N2O Phosphate buffer 1 mg/ml EB/EL 10 kGy RT 06.12.2022 Freezer, -18˚ C Precipitation MALDI-TOF KTH Yes Few Few, but very low intensity INCT-1LS10EL-RAP/ INCT-1LS10EL-RAPOP INCT KTH Air Phosphate buffer 1 mg/ml EB/EL 10 kGy RT 06.12.2022 Freezer, -18˚ C Precipitation MALDI-TOF KTH Yes Few Few, but very low intensity INCT-1LS10EL-RIP/ INCT-1LS10EL-RIPOP INCT KTH N2 Phosphate buffer 1 mg/ml EB/EL 10 kGy RT 06.12.2022 Freezer, -18˚ C Precipitation MALDI-TOF KTH Yes Few Few, but very low intensity INCT-2LS-1ELRN/ INCT-2LS1EL-RNPO INCT EPS N2O H2O 2 mg/ml EB/EL 1 kGy RT 14.02.2023 INCT-2LS-1ELRA/ INCT-2LS1EL-RAPO INCT EPS Air H2O 2 mg/ml EB/EL 1 kGy RT 14.02.2023 INCT-2LS-1ELRI/ INCT-2LS1EL-RIPO INCT EPS N2 H2O 2 mg/ml EB/EL 1 kGy RT 14.02.2023 INCT-2LS-1ELRNP/ INCT2LS-1ELRNPOP INCT EPS N2O Phosphate buffer 2 mg/ml EB/EL 1 kGy RT 14.02.2023 INCT-2LS-1ELRAP/ INCT2LS-1ELRAPOP INCT EPS Air Phosphate buffer 2 mg/ml EB/EL 1 kGy RT 14.02.2023 INCT-2LS-1ELRIP/ INCT2LS-1ELRIPOP INCT EPS N2 Phosphate buffer 2 mg/ml EB/EL 1 kGy RT 14.02.2023 INCT-2LS10EL-RN/ INCT-2LS10EL-RNPO INCT EPS N2O H2O 2 mg/ml EB/EL 10 kGy RT 14.02.2023 INCT-2LS10EL-RA/ INCT-2LS10EL-RAPO INCT EPS Air H2O 2 mg/ml EB/EL 10 kGy RT 14.02.2023 INCT-2LS10EL-RI/ INCT-2LS10EL-RIPO INCT EPS N2 H2O 2 mg/ml EB/EL 10 kGy RT 14.02.2023 INCT-2LS10EL-RNP/ INCT-2LS10EL-RNPOP INCT EPS N2O Phosphate buffer 2 mg/ml EB/EL 10 kGy RT 14.02.2023 INCT-2LS10EL-RAP/ INCT-2LS10EL-RAPOP INCT EPS Air Phosphate buffer 2 mg/ml EB/EL 10 kGy RT 14.02.2023 INCT-2LS10EL-RIP/ INCT-2LS10EL-RIPOP INCT EPS N2 Phosphate buffer 2 mg/ml EB/EL 10 kGy RT 14.02.2023 INCT-2LS-1ELRN INCT IST-ID N2O H2O 2 mg/ml EB/EL 1 kGy RT 14.02.2023 INCT-2LS-1ELRA INCT IST-ID Air H2O 2 mg/ml EB/EL 1 kGy RT 14.02.2023 INCT-2LS-1ELRI INCT IST-ID N2 H2O 2 mg/ml EB/EL 1 kGy RT 14.02.2023 17 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC INCT-2LS-1ELRNP INCT IST-ID N2O Phosphate buffer 2 mg/ml EB/EL 1 kGy RT 14.02.2023 INCT-2LS-1ELRAP INCT IST-ID Air Phosphate buffer 2 mg/ml EB/EL 1 kGy RT 14.02.2023 INCT-2LS-1ELRIP INCT IST-ID N2 Phosphate buffer 2 mg/ml EB/EL 1 kGy RT 14.02.2023 INCT-2LS10EL-RN INCT IST-ID N2O H2O 2 mg/ml EB/EL 10 kGy RT 14.02.2023 INCT-2LS10EL-RA INCT IST-ID Air H2O 2 mg/ml EB/EL 10 kGy RT 14.02.2023 INCT-2LS10EL-RI INCT IST-ID N2 H2O 2 mg/ml EB/EL 10 kGy RT 14.02.2023 INCT-2LS10EL-RNP INCT IST-ID N2O Phosphate buffer 2 mg/ml EB/EL 10 kGy RT 14.02.2023 INCT-2LS10EL-RAP INCT IST-ID Air Phosphate buffer 2 mg/ml EB/EL 10 kGy RT 14.02.2023 INCT-2LS10EL-RIP INCT IST-ID N2 Phosphate buffer 2 mg/ml EB/EL 10 kGy RT 14.02.2023 INCT-1LS-1ELRNP INCT IST-ID N2O Phosphate buffer 1 mg/ml EB/EL 1 kGy RT 06.12.2022 INCT-1LS-1ELRAP INCT IST-ID Air Phosphate buffer 1 mg/ml EB/EL 1 kGy RT 13.12.2022 INCT-1LS-1ELRIP INCT IST-ID N2 Phosphate buffer 1 mg/ml EB/EL 1 kGy RT 06.12.2022 INCT-1LS-1ELRN INCT IST-ID N2O H2O 1 mg/ml EB/EL 1 kGy RT 13.12.2022 INCT-1LS-1ELRA INCT IST-ID Air H2O 1 mg/ml EB/EL 1 kGy RT 13.12.2022 INCT-1LS-1ELRI INCT IST-ID N2 H2O 1 mg/ml EB/EL 1 kGy RT 06.12.2022 INCT-1LS10EL-RNP INCT IST-ID N2O Phosphate buffer 1 mg/ml EB/EL 10 kGy RT 06.12.2022 INCT-1LS10EL-RAP INCT IST-ID Air Phosphate buffer 1 mg/ml EB/EL 10 kGy RT 13.12.2022 INCT-1LS10EL-RIP INCT IST-ID N2 Phosphate buffer 1 mg/ml EB/EL 10 kGy RT 06.12.2022 INCT-1LS10EL-RN INCT IST-ID N2O H2O 1 mg/ml EB/EL 10 kGy RT 06.12.2022 INCT-1LS10EL-RA INCT IST-ID Air H2O 1 mg/ml EB/EL 10 kGy RT 13.12.2022 INCT-1LS10EL-RI INCT IST-ID N2 H2O 1 mg/ml EB/EL 10 kGy RT 06.12.2022 INCT-1LS50EL-RN INCT KTH N2O H2O 1 mg/ml EB/EL 50 kGy RT 31.05.2023 Freezer, -18˚ C Precipitation MALDI-TOF KTH Yes No No INCT-1LS50EL-RA INCT KTH Air H2O 1 mg/ml EB/EL 50 kGy RT 31.05.2023 Freezer, -18˚ C MALDI-TOF KTH Yes No No INCT-1LS50EL-RI INCT KTH N2 H2O 1 mg/ml EB/EL 50 kGy RT 31.05.2023 Freezer, -18˚ C MALDI-TOF KTH Yes No No INCT-1LS50EL-RN-P INCT KTH N2O Phosphate buffer 1 mg/ml EB/EL 50 kGy RT 31.05.2023 Freezer, -18˚ C Precipitation MALDI-TOF KTH Yes No INCT-1LS50EL-RA-P INCT KTH Air Phosphate buffer 1 mg/ml EB/EL 50 kGy RT 31.05.2023 Freezer, -18˚ C Precipitation MALDI-TOF KTH Yes No INCT-1LS50EL-RI-P INCT KTH N2 Phosphate buffer 1 mg/ml EB/EL 50 kGy RT 31.05.2023 Freezer, -18˚ C Precipitation MALDI-TOF KTH Yes No INCT-1LS85EL-RN INCT KTH N2O H2O 1 mg/ml EB/EL 85 kGy RT 31.05.2023 Freezer, -18˚ C Precipitation MALDI-TOF KTH Yes No No INCT-1LS85EL-RA INCT KTH Air H2O 1 mg/ml EB/EL 85 kGy RT 31.05.2023 Freezer, -18˚ C MALDI-TOF KTH Yes No No INCT-1LS85EL-RI INCT KTH N2 H2O 1 mg/ml EB/EL 85 kGy RT 31.05.2023 Freezer, -18˚ C Precipitation MALDI-TOF KTH Yes No No INCT-1LS85EL-RN-P INCT KTH N2O Phosphate buffer 1 mg/ml EB/EL 85 kGy RT 31.05.2023 Freezer, -18˚ C Precipitation MALDI-TOF KTH Yes No INCT-1LS85EL-RA-P INCT KTH Air Phosphate buffer 1 mg/ml EB/EL 85 kGy RT 31.05.2023 Freezer, -18˚ C Precipitation MALDI-TOF KTH Yes No INCT-1LS85EL-RI-P INCT KTH N2 Phosphate buffer 1 mg/ml EB/EL 85 kGy RT 31.05.2023 Freezer, -18˚ C Precipitation MALDI-TOF KTH Yes No 18 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC INCT-1LS50EL-RN INCT IST-ID N2O H2O 1 mg/ml EB/EL 50 kGy RT 31.05.2023 INCT-1LS50EL-RA INCT IST-ID Air H2O 1 mg/ml EB/EL 50 kGy RT 31.05.2023 INCT-1LS50EL-RI INCT IST-ID N2 H2O 1 mg/ml EB/EL 50 kGy RT 31.05.2023 INCT-1LS50EL-RN-P INCT IST-ID N2O Phosphate buffer 1 mg/ml EB/EL 50 kGy RT 31.05.2023 INCT-1LS50EL-RA-P INCT IST-ID Air Phosphate buffer 1 mg/ml EB/EL 50 kGy RT 31.05.2023 INCT-1LS50EL-RI-P INCT IST-ID N2 Phosphate buffer 1 mg/ml EB/EL 50 kGy RT 31.05.2023 INCT-1LS85EL-RN INCT IST-ID N2O H2O 1 mg/ml EB/EL 85 kGy RT 31.05.2023 INCT-1LS85EL-RA INCT IST-ID Air H2O 1 mg/ml EB/EL 85 kGy RT 31.05.2023 INCT-1LS85EL-RI INCT IST-ID N2 H2O 1 mg/ml EB/EL 85 kGy RT 31.05.2023 INCT-1LS85EL-RN-P INCT IST-ID N2O Phosphate buffer 1 mg/ml EB/EL 85 kGy RT 31.05.2023 INCT-1LS85EL-RA-P INCT IST-ID Air Phosphate buffer 1 mg/ml EB/EL 85 kGy RT 31.05.2023 INCT-1LS85EL-RI-P INCT IST-ID N2 Phosphate buffer 1 mg/ml EB/EL 85 kGy RT 31.05.2023 INCT-3.5x10^- 5LS-20EL-FITRIS INCT KTH N2 TRIS 35 uM EB/EL 20 kGy Frozen (dry ice) 23.03.2023 Freezer, -18˚ C MALDI-TOF KTH Yes No INCT-3.5x10^- 5LS-40EL-FITRIS INCT KTH N2 TRIS 35 uM EB/EL 40 kGy Frozen (dry ice) 23.03.2023 Freezer, -18˚ C MALDI-TOF KTH Yes No INCT-3.5x10^- 5LS-60EL-FITRIS INCT KTH N2 TRIS 35 uM EB/EL 60 kGy Frozen (dry ice) 23.03.2023 Freezer, -18˚ C MALDI-TOF KTH Yes No INCT-3.5x10^- 5LS-80EL-FITRIS INCT KTH N2 TRIS 35 uM EB/EL 80 kGy Frozen (dry ice) 23.03.2023 Freezer, -18˚ C MALDI-TOF KTH Yes No m/z 651, 1001, 1001.5, 1031, 1101, 1257, 1271, 1315, 1333, 1373, 1389, 1403, 1438, 1453, 1459, 1960, 1985, 2016, 2658, 3202, 3829, 4746 (S/N =>10) Concentrated with C18 tip 19 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC INCT-3.5x10^- 5LS-100EL-FITRIS INCT KTH N2 TRIS 35 uM EB/EL 100 kGy Frozen (dry ice) 23.03.2023 Freezer, -18˚ C MALDI-TOF KTH Yes No m/z 651, 1001, 1001.5, 1031, 1092, 1101, 1187, 1229, 1257, 1270, 1272, 1315, 1323, 1349, 1357, 1360, 1373, 1385, 1389, 1403, 1434, 1438, 1453, 1459, 1960, 1985, 2016, 2475, 2658, 2741, 3202, 3318, 3829, 4746, 6575, (S/N =>10) Concentrated with C18 tip INCT-3.5x10^- 5LS-20EL-FITRIS INCT IST-ID N2 TRIS 35 uM EB/EL 20 kGy Frozen (dry ice) 23.03.2023 INCT-3.5x10^- 5LS-40EL-FITRIS INCT IST-ID N2 TRIS 35 uM EB/EL 40 kGy Frozen (dry ice) 23.03.2023 INCT-3.5x10^- 5LS-60EL-FITRIS INCT IST-ID N2 TRIS 35 uM EB/EL 60 kGy Frozen (dry ice) 23.03.2023 INCT-3.5x10^- 5LS-80EL-FITRIS INCT IST-ID N2 TRIS 35 uM EB/EL 80 kGy Frozen (dry ice) 23.03.2023 INCT-3.5x10^- 5LS-100EL-FITRIS INCT IST-ID N2 TRIS 35 uM EB/EL 100 kGy Frozen (dry ice) 23.03.2023 INCT-0.1GLD50EL-RI INCT KTH N2 Dry n/a EB/EL 50 kGy RT 23.03.2023 RT INCT-0.1GLD100EL-RI INCT KTH N2 Dry n/a EB/EL 100 kGy RT 23.03.3023 RT INCT-0.1GLD200EL-RI INCT KTH N2 Dry n/a EB/EL 200 kGy RT 23.03.3023 RT INCT-0.1GLD300EL-RI INCT KTH N2 Dry n/a EB/EL 300 kGy RT 23.03.3023 RT INCT-0.1GLD50EL-RA INCT KTH Air Dry n/a EB/EL 50 kGy RT 23.03.2023 RT 20 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC INCT-0.1GLD100EL-RA INCT KTH Air Dry n/a EB/EL 100 kGy RT 23.03.3023 RT INCT-0.1GLD200EL-RA INCT KTH Air Dry n/a EB/EL 200 kGy RT 23.03.3023 RT INCT-0.1GLD300EL-RA INCT KTH Air Dry n/a EB/EL 300 kGy RT 23.03.3023 RT INCT-0.1GLD50EL-RI INCT IST-ID N2 Dry n/a EB/EL 50 kGy RT 23.03.2023 INCT-0.1GLD100EL-RI INCT IST-ID N2 Dry n/a EB/EL 100 kGy RT 23.03.3023 INCT-0.1GLD200EL-RI INCT IST-ID N2 Dry n/a EB/EL 200 kGy RT 23.03.3023 INCT-0.1GLD300EL-RI INCT IST-ID N2 Dry n/a EB/EL 300 kGy RT 23.03.3023 INCT-0.1GLD50EL-RA INCT IST-ID Air Dry n/a EB/EL 50 kGy RT 23.03.2023 INCT-0.1GLD100EL-RA INCT IST-ID Air Dry n/a EB/EL 100 kGy RT 23.03.3023 INCT-0.1GLD200EL-RA INCT IST-ID Air Dry n/a EB/EL 200 kGy RT 23.03.3023 INCT-0.1GLD300EL-RA INCT IST-ID Air Dry n/a EB/EL 300 kGy RT 23.03.3023 INCT-4LS-1ELRN-P INCT UNIPA N2O Phosphate buffer 4 mg/ml EB/EL 1 kGy RT 23.03.2023 06-04-23 11-04-23 Turbid, Mold, Vial not properly closed - INCT-4LS-1ELRA-P INCT UNIPA Air Phosphate buffer 4 mg/ml EB/EL 1 kGy RT 23.03.2023 06-04-23 11-04-23 Empty vial - INCT-4LS-1ELRI-P INCT UNIPA N2 Phosphate buffer 4 mg/ml EB/EL 1 kGy RT 23.03.2023 06-04-23 11-04-23 Mold DLS UNIPA INCT-4LS-1ELRN INCT UNIPA N2O H2O 4 mg/ml EB/EL 1 kGy RT 23.03.2023 06-04-23 11-04-23 Vial not properly closed DLS UNIPA INCT-4LS-1ELRA INCT UNIPA Air H2O 4 mg/ml EB/EL 1 kGy RT 23.03.2023 06-04-23 11-04-23 - DLS UNIPA INCT-4LS-1ELRI INCT UNIPA N2 H2O 4 mg/ml EB/EL 1 kGy RT 23.03.2023 06-04-23 11-04-23 - DLS UNIPA INCT-4LS10EL-RN-P INCT UNIPA N2O Phosphate buffer 4 mg/ml EB/EL 10 kGy RT 23.03.2023 06-14-23 11-04-23 - DLS UNIPA INCT-4LS10EL-RA-P INCT UNIPA Air Phosphate buffer 4 mg/ml EB/EL 10 kGy RT 23.03.2023 06-04-23 11-04-23 Empty vial - INCT-4LS10EL-RI-P INCT UNIPA N2 Phosphate buffer 4 mg/ml EB/EL 10 kGy RT 23.03.2023 06-04-23 11-04-23 - DLS UNIPA INCT-4LS10EL-RN INCT UNIPA N2O H2O 4 mg/ml EB/EL 10 kGy RT 23.03.2023 06-04-23 11-04-23 - DLS UNIPA INCT-4LS10EL-RA INCT UNIPA Air H2O 4 mg/ml EB/EL 10 kGy RT 23.03.2023 06-04-23 11-04-23 - DLS UNIPA INCT-4LS10EL-RI INCT UNIPA N2 H2O 4 mg/ml EB/EL 10 kGy RT 23.03.2023 06-04-23 11-04-23 - DLS UNIPA 21 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC INCT-0.1GLD50EL-RA INCT - Air Dry n/a EB/EL 50 kGy RT 23.03.2023 RT, desiccator gel electrophoresis INCT No INCT-0.1GLD100EL-RA INCT - Air Dry n/a EB/EL 100 kGy RT 23.03.3023 RT, desiccator gel electrophoresis INCT No INCT-0.1GLD200EL-RA INCT - Air Dry n/a EB/EL 200 kGy RT 23.03.3023 RT, desiccator gel electrophoresis INCT No INCT-0.1GLD300EL-RA INCT - Air Dry n/a EB/EL 300 kGy RT 23.03.3023 RT, desiccator gel electrophoresis INCT No INCT-LD-50ELRA INCT EPS Air Dry n/a EB/EL 50 kGy RT 17.05.2023 INCT-LD100EL-RA INCT EPS Air Dry n/a EB/EL 100 kGy RT 17.05.2023 INCT-LD200EL-RA INCT EPS Air Dry n/a EB/EL 200 kGy RT 17.05.2023 INCT-LD300EL-RA INCT EPS Air Dry n/a EB/EL 300 kGy RT 17.05.2023 INCT-LD-50ELRA INCT UNIPA Air Dry n/a EB/EL 50 kGy RT 4,31 17.05.2023 01-06-23 08-06-23 - Solubility test DLS UNIPA No No Yes INCT-LD100EL-RA INCT UNIPA Air Dry n/a EB/EL 100 kGy RT 4,11 17.05.2023 01-06-23 08-06-23 - Solubility test DLS UNIPA No No Yes INCT-LD200EL-RA INCT UNIPA Air Dry n/a EB/EL 200 kGy RT 3,84 17.05.2023 01-06-23 08-06-23 - Solubility test DLS UNIPA No No Yes INCT-LD300EL-RA INCT UNIPA Air Dry n/a EB/EL 300 kGy RT 3,7 17.05.2023 01-06-23 08-06-23 - Solubility test DLS UNIPA No No No INCT-1LS20EL-FI-TRIS INCT KTH N2 TRIS 1 mg/ml EB/EL 20 kGy Frozen (dry ice) 31.05.2023 Freezer, -18˚ C MALDI-TOF KTH Marginally No INCT-1LS40EL-FI-TRIS INCT KTH N2 TRIS 1 mg/ml EB/EL 40 kGy Frozen (dry ice) 31.05.2023 Freezer, -18˚ C MALDI-TOF KTH Marginally No INCT-1LS60EL-FI-TRIS INCT KTH N2 TRIS 1 mg/ml EB/EL 60 kGy Frozen (dry ice) 31.05.2023 Freezer, -18˚ C MALDI-TOF KTH Yes No INCT-1LS80EL-FI-TRIS INCT KTH N2 TRIS 1 mg/ml EB/EL 80 kGy Frozen (dry ice) 31.05.2023 Freezer, -18˚ C MALDI-TOF KTH Yes No INCT-1LS100EL-FI-TRIS INCT KTH N2 TRIS 1 mg/ml EB/EL 100 kGy Frozen (dry ice) 31.05.2023 Freezer, -18˚ C MALDI-TOF KTH Yes No No INCT-1LS20EL-FI-TRIS INCT IST-ID N2 TRIS 1 mg/ml EB/EL 20 kGy Frozen (dry ice) 31.05.2023 INCT-1LS40EL-FI-TRIS INCT IST-ID N2 TRIS 1 mg/ml EB/EL 40 kGy Frozen (dry ice) 31.05.2023 INCT-1LS60EL-FI-TRIS INCT IST-ID N2 TRIS 1 mg/ml EB/EL 60 kGy Frozen (dry ice) 31.05.2023 INCT-1LS80EL-FI-TRIS INCT IST-ID N2 TRIS 1 mg/ml EB/EL 80 kGy Frozen (dry ice) 31.05.2023 INCT-1LS100EL-FI-TRIS INCT IST-ID N2 TRIS 1 mg/ml EB/EL 100 kGy Frozen (dry ice) 31.05.2023 KTH Air H2O 1.5 mg/ml γ-source 1.1 kGy RT Freezer, -18˚ C MALDI-TOF KTH Few KTH Air H2O 5 mg/ml γ-source 1.1 kGy RT Freezer, -18˚ C MALDI-TOF KTH Yes Few KTH Air H2O 1.5 mg/ml γ-source 11 kGy RT Freezer, -18˚ C MALDI-TOF KTH Yes Few KTH Air H2O 5 mg/ml γ-source 11 kGy RT Freezer, -18˚ C MALDI-TOF KTH Yes Few KTH Air H2O 1 mg/ml γ-source 26 kGy RT Freezer, -18˚ C MALDI-TOF KTH Yes KTH Air H2O 10 mg/ml γ-source 26 kGy RT Freezer, -18˚ C MALDI-TOF KTH Yes KTH Air Dry n/a γ-source 26 kGy RT RT MALDI-TOF KTH No Marginally KTH Air H2O 1 mg/ml γ-source 43 kGy RT Freezer, -18˚ C Precipitation MALDI-TOF KTH Yes KTH Air H2O 10 mg/ml γ-source 43 kGy RT Freezer, -18˚ C MALDI-TOF KTH Yes KTH Air Dry n/a γ-source 43 kGy RT RT MALDI-TOF KTH No KTH Air H2O 1 mg/ml γ-source 60.5 kGy RT Freezer, -18˚ C MALDI-TOF KTH Yes 22 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC KTH Air H2O 10 mg/ml γ-source 60.5 kGy RT Freezer, -18˚ C MALDI-TOF KTH Yes KTH Air Dry n/a γ-source 60.5 kGy RT RT MALDI-TOF KTH KTH Air H2O 1 mg/ml γ-source 0.36 kGy RT Freezer, -18˚ C MALDI-TOF KTH Yes KTH Air H2O 10 mg/ml γ-source 0.36 kGy RT Freezer, -18˚ C MALDI-TOF KTH Yes KTH Air H2O 1 mg/ml γ-source 0.72 kGy RT Freezer, -18˚ C MALDI-TOF KTH Yes KTH Air H2O 10 mg/ml γ-source 0.72 kGy RT Freezer, -18˚ C MALDI-TOF KTH Yes KTH Air H2O 0.5 mg/ml γ-source 0.36 kGy RT Freezer, -18˚ C MALDI-TOF KTH KTH Air H2O 0.1 mg/ml γ-source 0.36 kGy RT Freezer, -18˚ C MALDI-TOF KTH KTH Air H2O 0.05 mg/ml γ-source 0.36 kGy RT Freezer, -18˚ C MALDI-TOF KTH KTH Air H2O 0.5 mg/ml γ-source 0.72 kGy RT Freezer, -18˚ C MALDI-TOF KTH KTH Air H2O 0.1 mg/ml γ-source 0.72 kGy RT Freezer, -18˚ C MALDI-TOF KTH KTH Air H2O 0.05 mg/ml γ-source 0.72 kGy RT Freezer, -18˚ C MALDI-TOF KTH KTH Air H2O 0.5 mg/ml γ-source 1.1 kGy RT Freezer, -18˚ C MALDI-TOF KTH KTH Air H2O 0.1 mg/ml γ-source 1.1 kGy RT Freezer, -18˚ C MALDI-TOF KTH KTH Air H2O 0.05 mg/ml γ-source 1.1 kGy RT Freezer, -18˚ C MALDI-TOF KTH KTH N2 1 M 2-propanol 1 mg/ml γ-source 0.36 kGy RT Freezer, -18˚ C MALDI-TOF KTH Yes Yes KTH N2 1 M 2-propanol 1 mg/ml γ-source 1.1 kGy RT Freezer, -18˚ C MALDI-TOF KTH Yes Yes No big difference KTH N2 1 M 2-propanol 1 mg/ml γ-source 2.2 kGy RT Freezer, -18˚ C MALDI-TOF KTH Yes Yes KTH N2 1 M 2-propanol 1 mg/ml γ-source 3.6 kGy RT Freezer, -18˚ C MALDI-TOF KTH Yes Yes KTH N2 1 M 2-propanol 1 mg/ml γ-source 11 kGy RT Freezer, -18˚ C MALDI-TOF KTH Yes Yes KTH N2 1 M 2-propanol 10 mg/ml γ-source 3.6 kGy RT Freezer, -18˚ C MALDI-TOF KTH KTH N2O 1 M 2-propanol 1 mg/ml γ-source 1.1 kGy RT 3.81 3.94 06.12.2022 - 07.12.2023 Freezer, -18˚ C MALDI-TOF KTH KTH N2O 1 M 2-propanol 1 mg/ml γ-source 2.2 kGy RT 3.81 06.12.2023 - 07.12.2024 Freezer, -18˚ C MALDI-TOF KTH KTH N2O 1 M 2-propanol 1 mg/ml γ-source 3.6 kGy RT 3.81 3.93 06.12.2023 - 07.12.2025 Freezer, -18˚ C MALDI-TOF KTH INCT-LD-25ELRA INCT IST-ID Air Dry n/a EB/EL 25 kGy RT 11.10.2023 INCT-LD-50ELRA INCT IST-ID Air Dry n/a EB/EL 50 kGy RT 11.10.2023 INCT-LD100EL-RA INCT IST-ID Air Dry n/a EB/EL 100 kGy RT 11.10.2023 INCT-LD200EL-RA INCT IST-ID Air Dry n/a EB/EL 200 kGy RT 11.10.2023 INCT-LD300EL-RA INCT IST-ID Air Dry n/a EB/EL 300 kGy RT 11.10.2023 INCT-LD400EL-RA INCT IST-ID Air Dry n/a EB/EL 400 kGy RT 11.10.2023 INCT-LD500EL-RA INCT IST-ID Air Dry n/a EB/EL 500 kGy RT 11.10.2023 INCT-1LS10EL-RN INCT IST-ID N2O H2O 1 mg/ml EB/EL 10 kGy RT 3,84 3,65 11.10.2023 INCT-2LS10EL-RN INCT IST-ID N2O H2O 2 mg/ml EB/EL 10 kGy RT 3,63 3,54 11.10.2023 23 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC INCT-2LS10EL-RA INCT IST-ID Air H2O 2 mg/ml EB/EL 10 kGy RT 3,63 3,73 11.10.2023 INCT KTH Air Dry n/a EB/EL 50 kGy RT 11.10.2023 RT MALDI-TOF KTH m/z 562, 647 (S/N =>10) Very low intensity Not detected The intensity of the LYZ peak decreases more with each dose after reduction with DTT No peaks with S/N=>10 Not detected HPLC-UV KTH Yes No No SDS-PAGE KTH Still a strong band at 14 kDa No bands below 10 kDa No Still a strong band at 14 kDa Blue smear below 10 kDa No INCT KTH Air Dry n/a EB/EL 100 kGy RT 11.10.2023 RT MALDI-TOF KTH m/z 647 (S/N =>10) Very low intensity Not detected - No peaks with S/N=>10 Not detected HPLC-UV KTH Yes No No SDS-PAGE KTH Still a strong band at 14 kDa No bands below 10 kDa No Still a strong band at 14 kDa Blue smear below 10 kDa No INCT KTH Air Dry n/a EB/EL 200 kGy RT 11.10.2023 RT MALDI-TOF KTH m/z 562, 647 (S/N =>10) Very low intensity Not detected - No peaks with S/N=>10 Not detected HPLC-UV KTH Yes No No SDS-PAGE KTH Still a strong band at 14 kDa No bands below 10 kDa Faint band around 35 kDa Still a strong band at 14 kDa Blue smear below 10 kDa, more like a band No INCT KTH Air Dry n/a EB/EL 300 kGy RT 11.10.2023 RT MALDI-TOF KTH m/z 562, 647, 1715, 1886, 2001 (S/N =>10) Very low intensity Not detected - m/z 656, 973 (S/N =>10) Very low intensity Not detected HPLC-UV KTH Yes No No Yes A few new peaks, however, very low intensity SDS-PAGE KTH Still a strong band at 14 kDa No bands below 10 kDa Faint band around 35 kDa Still a strong band at 14 kDa Blue smear below 10 kDa, more like a band No INCT KTH Air Dry n/a EB/EL 400 kGy RT 11.10.2023 RT MALDI-TOF KTH m/z 562, 647, 1886, 2001 (S/N =>10) Very low intensity Not detected - m/z 886, 931, 973, 1101, 1229, 1271, 1445 (S/N =>10) Very low intensity Not detected HPLC-UV KTH Yes A couple new peaks, however, very low intensity Yes A few new peaks, however, very low intensity 24 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC SDS-PAGE KTH Still a strong band at 14 kDa Blue smear below 10 kDa appear Faint band around 35 kDa Still a strong band at 14 kDa Blue smear below 10 kDa, more like a band No INCT KTH Air Dry n/a EB/EL 500 kGy RT 11.10.2023 RT MALDI-TOF KTH m/z 562, 647, 1715, 1886, 2001 (S/N =>10) Very low intensity Not detected - m/z 886, 929, 973 (S/N =>10) Very low intensity Not detected HPLC-UV KTH Yes A couple new peaks, however, very low intensity Yes A few new peaks, however, very low intensity SDS-PAGE KTH Still a strong band at 14 kDa, perhaps a bit lighter Blue smear below 10 kDa appear Faint band around 35 kDa Still a strong band at 14 kDa, perhaps a bit lighter Blue smear below 10 kDa, more like a band No KTH N2 1 M t-BuOH 1 mg/ml γ-source 1.1 kGy RT 12.12.2022 - 13.12.2023 Freezer, -18˚ C MALDI-TOF KTH KTH N2 1 M t-BuOH 1 mg/ml γ-source 2.2 kGy RT 12.12.2023 - 13.12.2023 Freezer, -18˚ C MALDI-TOF KTH KTH N2 1 M t-BuOH 1 mg/ml γ-source 3.6 kGy RT 12.12.2023 - 13.12.2023 Freezer, -18˚ C MALDI-TOF KTH INCT-LD-50ELRA INCT UNIPA Air Dry n/a EB/EL 50 kGy RT 02-29-24 03-12-24 RT - UNIPA No No Yes INCT-LD100EL-RA INCT UNIPA Air Dry n/a EB/EL 100 kGy RT 02-29-24 03-12-24 RT - UNIPA No No Yes INCT-LD200EL-RA INCT UNIPA Air Dry n/a EB/EL 200 kGy RT 02-29-24 03-12-24 RT - DLS UNIPA No No Yes INCT-LD300EL-RA INCT UNIPA Air Dry n/a EB/EL 300 kGy RT 02-29-24 03-12-24 RT - DLS UNIPA No No No INCT-1LS-1ELRII1M INCT KTH N2 1 M 2-propanol 1 mg/ml EB/EL 1 kGy RT 3,69 3,27 19.02.2024 MALDI-TOF KTH m/z 492, 648, 650*, 751, 766, 1009, 1060, 1150, 1352, 1359, 1480, 1531, 1593*, 1621, 1636, 1683, 1758, 1831, 1859, 1944, 1960, 2059, 2477, 2549 (S/N =>10) Not detected m/z 1150, 1359, 1944, 3357 (S/N =>10) Not detected HPLC-UV KTH SDS-PAGE KTH Yes Clear bands below 10 kDa No 31 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC INCT-2OTS1EL-RIP INCT IST-ID N2 Phosphate buffer 2 mg/ml EB/EL 1 kGy RT 14.02.2023 INCT-2OTS10EL-RN INCT IST-ID N2O H2O 2 mg/ml EB/EL 10 kGy RT 14.02.2023 INCT-2OTS10EL-RA INCT IST-ID Air H2O 2 mg/ml EB/EL 10 kGy RT 14.02.2023 INCT-2OTS10EL-RI INCT IST-ID N2 H2O 2 mg/ml EB/EL 10 kGy RT 14.02.2023 INCT-2OTS10EL-RNP INCT IST-ID N2O Phosphate buffer 2 mg/ml EB/EL 10 kGy RT 14.02.2023 INCT-2OTS10EL-RAP INCT IST-ID Air Phosphate buffer 2 mg/ml EB/EL 10 kGy RT 14.02.2023 INCT-2OTS10EL-RAP INCT IST-ID N2 Phosphate buffer 2 mg/ml EB/EL 10 kGy RT 14.02.2023 INCT-1OTS1EL-RNP INCT IST-ID N2O Phosphate buffer 1 mg/ml EB/EL 1 kGy RT 06.12.2022 INCT-1OTS1EL-RAP INCT IST-ID Air Phosphate buffer 1 mg/ml EB/EL 1 kGy RT 13.12.2022 INCT-1OTS1EL-RIP INCT IST-ID N2 Phosphate buffer 1 mg/ml EB/EL 1 kGy RT 13.12.2022 INCT-1OTS1EL-RN INCT IST-ID N2O H2O 1 mg/ml EB/EL 1 kGy RT 06.12.2022 INCT-1OTS1EL-RA INCT IST-ID Air H2O 1 mg/ml EB/EL 1 kGy RT 13.12.2022 INCT-1OTS1EL-RI INCT IST-ID N2 H2O 1 mg/ml EB/EL 1 kGy RT 06.12.2022 INCT-1OTS10EL-RNP INCT IST-ID N2O Phosphate buffer 1 mg/ml EB/EL 10 kGy RT 13.12.2022 INCT-1OTS10EL-RAP INCT IST-ID Air Phosphate buffer 1 mg/ml EB/EL 10 kGy RT 06.12.2022 INCT-10OTS10EL-RIP INCT IST-ID N2 Phosphate buffer 1 mg/ml EB/EL 10 kGy RT 06.12.2022 INCT-1OTS10EL-RN INCT IST-ID N2O H2O 1 mg/ml EB/EL 10 kGy RT 13.12.2022 INCT-1OTS10EL-RA INCT IST-ID Air H2O 1 mg/ml EB/EL 10 kGy RT 13.12.2022 INCT-1OTS10EL-RI INCT IST-ID N2 H2O 1 mg/ml EB/EL 10 kGy RT 06.12.2022 INCT-1OTS50EL-RN INCT KTH N2O H2O 1 mg/ml EB/EL 50 kGy RT 31.05.2023 Freezer, -18˚C Some precipitation MALDI-TOF KTH Yes No INCT-1OTS50EL-RA INCT KTH Air H2O 1 mg/ml EB/EL 50 kGy RT 31.05.2023 Freezer, -18˚C Some precipitation MALDI-TOF KTH Yes No INCT-1OTS50EL-RI INCT KTH N2 H2O 1 mg/ml EB/EL 50 kGy RT 31.05.2023 Freezer, -18˚C Some precipitation MALDI-TOF KTH Yes No INCT-1OTS50EL-RN-P INCT KTH N2O Phosphate buffer 1 mg/ml EB/EL 50 kGy RT 31.05.2023 Freezer, -18˚C Some precipitation MALDI-TOF KTH Yes No INCT-1OTS50EL-RA-P INCT KTH Air Phosphate buffer 1 mg/ml EB/EL 50 kGy RT 31.05.2023 Freezer, -18˚C Some precipitation MALDI-TOF KTH Yes No INCT-1OTS50EL-RI-P INCT KTH N2 Phosphate buffer 1 mg/ml EB/EL 50 kGy RT 31.05.2023 Freezer, -18˚C Some precipitation MALDI-TOF KTH Yes No INCT-1OTS85EL-RN INCT KTH N2O H2O 1 mg/ml EB/EL 85 kGy RT 31.05.2023 Freezer, -18˚C Some precipitation MALDI-TOF KTH Yes No INCT-1OTS85EL-RA INCT KTH Air H2O 1 mg/ml EB/EL 85 kGy RT 31.05.2023 Freezer, -18˚C Some precipitation MALDI-TOF KTH Yes No INCT-1OTS85EL-RI INCT KTH N2 H2O 1 mg/ml EB/EL 85 kGy RT 31.05.2023 Freezer, -18˚C Some precipitation MALDI-TOF KTH Yes No INCT-1OTS85EL-RN-P INCT KTH N2O Phosphate buffer 1 mg/ml EB/EL 85 kGy RT 31.05.2023 Freezer, -18˚C Some precipitation MALDI-TOF KTH Yes No INCT-1OTS85EL-RA-P INCT KTH Air Phosphate buffer 1 mg/ml EB/EL 85 kGy RT 31.05.2023 Freezer, -18˚C Some precipitation MALDI-TOF KTH Yes No INCT-1OTS85EL-RI-P INCT KTH N2 Phosphate buffer 1 mg/ml EB/EL 85 kGy RT 31.05.2023 Freezer, -18˚C Some precipitation MALDI-TOF KTH Yes No INCT-1OTS50EL-RN INCT IST-ID N2O H2O 1 mg/ml EB/EL 50 kGy RT 31.05.2023 INCT-1OTS50EL-RA INCT IST-ID Air H2O 1 mg/ml EB/EL 50 kGy RT 31.05.2023 32 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC INCT-1OTS50EL-RI INCT IST-ID N2 H2O 1 mg/ml EB/EL 50 kGy RT 31.05.2023 INCT-1OTS50EL-RN-P INCT IST-ID N2O Phosphate buffer 1 mg/ml EB/EL 50 kGy RT 31.05.2023 INCT-1OTS50EL-RA-P INCT IST-ID Air Phosphate buffer 1 mg/ml EB/EL 50 kGy RT 31.05.2023 INCT-1OTS50EL-RI-P INCT IST-ID N2 Phosphate buffer 1 mg/ml EB/EL 50 kGy RT 31.05.2023 INCT-1OTS85EL-RN INCT IST-ID N2O H2O 1 mg/ml EB/EL 85 kGy RT 31.05.2023 INCT-1OTS85EL-RA INCT IST-ID Air H2O 1 mg/ml EB/EL 85 kGy RT 31.05.2023 INCT-1OTS85EL-RI INCT IST-ID N2 H2O 1 mg/ml EB/EL 85 kGy RT 31.05.2023 INCT-1OTS85EL-RN-P INCT IST-ID N2O Phosphate buffer 1 mg/ml EB/EL 85 kGy RT 31.05.2023 INCT-1OTS85EL-RA-P INCT IST-ID Air Phosphate buffer 1 mg/ml EB/EL 85 kGy RT 31.05.2023 INCT-1OTS85EL-RI-P INCT IST-ID N2 Phosphate buffer 1 mg/ml EB/EL 85 kGy RT 31.05.2023 INCT-4OTS85EL-RN-P INCT UNIPA N2O Phosphate buffer 4 mg/ml EB/EL 85 kGy RT 31.05.2023 05-07-23 20/07/23 Fridge; +4°C DLS UNIPA No INCT-4OTS85EL-RA-P INCT UNIPA Air Phosphate buffer 4 mg/ml EB/EL 85 kGy RT 31.05.2023 05-07-23 20/07/23 Fridge; +4°C DLS UNIPA No INCT-4OTS85EL-RI-P INCT UNIPA N2 Phosphate buffer 4 mg/ml EB/EL 85 kGy RT 31.05.2023 05-07-23 20/07/23 Fridge; +4°C DLS UNIPA No INCT-4OTS50EL-RN-P INCT UNIPA N2O Phosphate buffer 4 mg/ml EB/EL 50 kGy RT 31.05.2023 05-07-23 20/07/23 Fridge; +4°C Turbid INCT-4OTS50EL-RA-P INCT UNIPA Air Phosphate buffer 4 mg/ml EB/EL 50 kGy RT 31.05.2023 05-07-23 20/07/23 Fridge; +4°C DLS UNIPA No INCT-4OTS50EL-RI-P INCT UNIPA N2 Phosphate buffer 4 mg/ml EB/EL 50 kGy RT 31.05.2023 05-07-23 20/07/23 Fridge; +4°C DLS UNIPA No INCT0.1GOTD50EL-RA INCT KTH Air Dry n/a EB/EL 50 kGy RT 23.03.2023 RT More and more difficult to dissolve with increasing dose MALDI-TOF KTH Yes No specific peaks with S/N => 10 could be detected. No Yes 578 (S/N => 10) No More and more difficult to filter (0.45um) with increasing dose HPLC-UV KTH Yes No No SDS-PAGE KTH Yes Blue smear below 80 kDa No Yes Blue smear below 80 kDa No INCT0.1GOTD100EL-RA INCT KTH Air Dry n/a EB/EL 100 kGy RT 23.03.2023 RT MALDI-TOF KTH Yes No specific peaks with S/N => 10 could be detected. No Yes 578 (S/N => 10) No HPLC-UV KTH Yes No No SDS-PAGE KTH Yes Blue smear below 80 kDa No Yes Blue smear below 80 kDa No 33 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC INCT0.1GOTD200EL-RA INCT KTH Air Dry n/a EB/EL 200 kGy RT 23.03.2023 RT MALDI-TOF KTH Yes No specific peaks with S/N => 10 could be detected. No Yes 578 (S/N => 10) No HPLC-UV KTH Yes No No INCT0.1GOTD300EL-RA INCT KTH Air Dry n/a EB/EL 300 kGy RT 23.03.2023 RT MALDI-TOF KTH Yes No specific peaks with S/N => 10 could be detected. No Yes 578 (S/N => 10) No HPLC-UV KTH Yes No No SDS-PAGE KTH Yes Blue smear below 80 kDa No Yes Blue smear below 80 kDa No INCT-OTD400EL-RA INCT KTH Air Dry n/a EB/EL 400 kGy RT 11.10.2023 RT MALDI-TOF KTH Yes No specific peaks with S/N => 10 could be detected. No Yes 578 (S/N => 10) No HPLC-UV KTH Yes No No SDS-PAGE KTH Yes Blue smear below 80 kDa No Yes Blue smear below 80 kDa No INCT-OTD500EL-RA INCT KTH Air Dry n/a EB/EL 500 kGy RT 11.10.2023 RT MALDI-TOF KTH Yes No specific peaks with S/N => 10 could be detected. No Yes 578 (S/N => 10) No HPLC-UV KTH Yes No No SDS-PAGE KTH Yes Blue smear below 80 kDa No Yes Blue smear below 80 kDa No INCT0.1GOTD50EL-RI INCT KTH N2 Dry n/a EB/EL 50 kGy RT 23.03.2023 RT MALDI-TOF KTH Yes INCT0.1GOTD100EL-RI INCT KTH N2 Dry n/a EB/EL 100 kGy RT 23.03.2023 RT MALDI-TOF KTH Yes Yes INCT0.1GOTD200EL-RI INCT KTH N2 Dry n/a EB/EL 200 kGy RT 23.03.2023 RT MALDI-TOF KTH Yes INCT0.1GOTD300EL-RI INCT KTH N2 Dry n/a EB/EL 300 kGy RT 23.03.2023 RT MALDI-TOF KTH Yes INCT0.1GOTD50EL-RA INCT IST-ID Air Dry n/a EB/EL 50 kGy RT 23.03.2023 INCT0.1GOTD100EL-RA INCT IST-ID Air Dry n/a EB/EL 100 kGy RT 23.03.2023 34 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC INCT0.1GOTD200EL-RA INCT IST-ID Air Dry n/a EB/EL 200 kGy RT 23.03.2023 INCT0.1GOTD300EL-RA INCT IST-ID Air Dry n/a EB/EL 300 kGy RT 23.03.2023 INCT0.1GOTD50EL-RI INCT IST-ID N2 Dry n/a EB/EL 50 kGy RT 23.03.2023 INCT0.1GOTD100EL-RI INCT IST-ID N2 Dry n/a EB/EL 100 kGy RT 23.03.2023 INCT0.1GOTD200EL-RI INCT IST-ID N2 Dry n/a EB/EL 200 kGy RT 23.03.2023 INCT0.1GOTD300EL-RI INCT IST-ID N2 Dry n/a EB/EL 300 kGy RT 23.03.2023 INCT-3.5x10^- 5OTS-20EL-FITRIS INCT KTH N2 TRIS 35 uM EB/EL 20 kGy Frozen (dry ice) 23.03.2023 Freezer, -18˚C MALDI-TOF KTH Yes Not really INCT-3.5x10^- 5OTS-40EL-FITRIS INCT KTH N2 TRIS 35 uM EB/EL 40 kGy Frozen (dry ice) 23.03.2023 Freezer, -18˚C MALDI-TOF KTH Yes Not really INCT-3.5x10^- 5OTS-60EL-FITRIS INCT KTH N2 TRIS 35 uM EB/EL 60 kGy Frozen (dry ice) 23.03.2023 Freezer, -18˚C MALDI-TOF KTH Yes Not really INCT-3.5x10^- 5OTS-80EL-FITRIS INCT KTH N2 TRIS 35 uM EB/EL 80 kGy Frozen (dry ice) 23.03.2023 Freezer, -18˚C MALDI-TOF KTH Yes Not really INCT-3.5x10^- 5OTS-100ELFI-TRIS INCT KTH N2 TRIS 35 uM EB/EL 100 kGy Frozen (dry ice) 23.03.2023 Freezer, -18˚C MALDI-TOF KTH Yes Not really Unclear since control did not show peak Yes INCT-3.5x10^- 5OTS-20EL-FITRIS INCT IST-ID N2 TRIS 35 uM EB/EL 20 kGy Frozen (dry ice) 23.03.2023 INCT-3.5x10^- 5OTS-40EL-FITRIS INCT IST-ID N2 TRIS 35 uM EB/EL 40 kGy Frozen (dry ice) 23.03.2023 INCT-3.5x10^- 5OTS-60EL-FITRIS INCT IST-ID N2 TRIS 35 uM EB/EL 60 kGy Frozen (dry ice) 23.03.2023 INCT-3.5x10^- 5OTS-80EL-FITRIS INCT IST-ID N2 TRIS 35 uM EB/EL 80 kGy Frozen (dry ice) 23.03.2023 INCT-3.5x10^- 5OTS-100ELFI-TRIS INCT IST-ID N2 TRIS 35 uM EB/EL 100 kGy Frozen (dry ice) 23.03.2023 INCT-4OTS1EL-RN-P INCT UNIPA N2O Phosphate buffer 4 mg/ml EB/EL 1 kGy RT 23.03.2023 06-04-23 11-04-23 Fridge, +4°C Turbid UNIPA No INCT-4OTS1EL-RA-P INCT UNIPA Air Phosphate buffer 4 mg/ml EB/EL 1 kGy RT 23.03.2023 06-04-23 11-04-23 Fridge, +4°C Turbid UNIPA No INCT-4OTS1EL-RI-P INCT UNIPA N2 Phosphate buffer 4 mg/ml EB/EL 1 kGy RT 23.03.2023 06-04-23 11-04-23 Fridge, +4°C Turbid UNIPA No INCT-4OTS1EL-RN INCT UNIPA N2O H2O 4 mg/ml EB/EL 1 kGy RT 23.03.2023 06-04-23 11-04-23 Fridge, +4°C Turbid UNIPA No 35 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC INCT-4OTS1EL-RA INCT UNIPA SAir H2O 4 mg/ml EB/EL 1 kGy RT 23.03.2023 06-04-23 11-04-23 Fridge, +4°C Turbid UNIPA No INCT-4OTS1EL-RI INCT UNIPA N2 H2O 4 mg/ml EB/EL 1 kGy RT 23.03.2023 06-04-23 11-04-23 Fridge, +4°C DLS UNIPA No INCT-4OTS10EL-RN-P INCT UNIPA N2O Phosphate buffer 4 mg/ml EB/EL 10 kGy RT 23.03.2023 06-14-23 11-04-23 Fridge, +4°C Turbid UNIPA No INCT-4OTS10EL-RA-P INCT UNIPA Air Phosphate buffer 4 mg/ml EB/EL 10 kGy RT 23.03.2023 06-04-23 11-04-23 Fridge, +4°C DLS UNIPA No INCT-4OTS10EL-RI-P INCT UNIPA N2 Phosphate buffer 4 mg/ml EB/EL 10 kGy RT 23.03.2023 06-04-23 11-04-23 Fridge, +4°C DLS UNIPA No INCT-4OTS10EL-RN INCT UNIPA N2O H2O 4 mg/ml EB/EL 10 kGy RT 23.03.2023 06-04-23 11-04-23 Fridge, +4°C DLS UNIPA No INCT-4OTS10EL-RA INCT UNIPA Air H2O 4 mg/ml EB/EL 10 kGy RT 23.03.2023 06-04-23 11-04-23 Fridge, +4°C DLS UNIPA No INCT-4OTS10EL-RI INCT UNIPA N2 H2O 4 mg/ml EB/EL 10 kGy RT 23.03.2023 06-04-23 11-04-23 Fridge, +4°C DLS UNIPA No INCT0.1GOTD50EL-RA INCT - Air Dry n/a EB/EL 50 kGy RT 23.03.2023 RT, desiccator gel electrophoresis INCT No INCT0.1GOTD100EL-RA INCT - Air Dry n/a EB/EL 100 kGy RT 23.03.2023 RT, desiccator gel electrophoresis INCT No INCT0.1GOTD200EL-RA INCT - Air Dry n/a EB/EL 200 kGy RT 23.03.2023 RT, desiccator gel electrophoresis INCT No INCT0.1GOTD300EL-RA INCT - Air Dry n/a EB/EL 300 kGy RT 23.03.2023 RT, desiccator gel electrophoresis INCT No INCT-OTD50EL-RA INCT EPS Air Dry n/a EB/EL 50 kGy RT 17/05/202 3 INCT-OTD100EL-RA INCT EPS Air Dry n/a EB/EL 100 kGy RT 17/05/202 3 INCT-OTD200EL-RA INCT EPS Air Dry n/a EB/EL 200 kGy RT 17/05/202 3 INCT-OTD300EL-RA INCT EPS Air Dry n/a EB/EL 300 kGy RT 17/05/202 3 INCT-OTD50EL-RA INCT UNIPA Air Dry n/a EB/EL 50 kGy RT 4,8 17/05/202 3 01-06-23 08-06-23 RT - Solubility test; DLS UNIPA No No Yes INCT-OTD100EL-RA INCT UNIPA Air Dry n/a EB/EL 100 kGy RT 4,63 17/05/202 3 01-06-23 08-06-23 RT - Solubility test; DLS UNIPA No No Yes INCT-OTD200EL-RA INCT UNIPA Air Dry n/a EB/EL 200 kGy RT 4,49 17/05/202 3 01-06-23 08-06-23 RT - Solubility test; DLS UNIPA No No Yes INCT-OTD300EL-RA INCT UNIPA Air Dry n/a EB/EL 300 kGy RT 4,52 17/05/202 3 01-06-23 08-06-23 RT - Solubility test; DLS UNIPA No No Yes INCT-1OTS20EL-FI-TRIS INCT KTH N2 TRIS 1 mg/ml EB/EL 20 kGy Frozen (dry ice) 31.05.2023 Freezer, -18˚C MALDI-TOF KTH Yes Not really INCT-1OTS40EL-FI-TRIS INCT KTH N2 TRIS 1 mg/ml EB/EL 40 kGy Frozen (dry ice) 31.05.2023 Freezer, -18˚C MALDI-TOF KTH Yes Not really INCT-1OTS60EL-FI-TRIS INCT KTH N2 TRIS 1 mg/ml EB/EL 60 kGy Frozen (dry ice) 31.05.2023 Freezer, -18˚C MALDI-TOF KTH Yes Not really INCT-1OTS80EL-FI-TRIS INCT KTH N2 TRIS 1 mg/ml EB/EL 80 kGy Frozen (dry ice) 31.05.2023 Freezer, -18˚C MALDI-TOF KTH Yes Not really INCT-1OTS100EL-FI-TRIS INCT KTH N2 TRIS 1 mg/ml EB/EL 100 kGy Frozen (dry ice) 31.05.2023 Freezer, -18˚C MALDI-TOF KTH Yes Not really Unclear since control did not show peak m/z 802 (S/N =>10) 36 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC INCT-1OTS20EL-FI-TRIS INCT IST-ID N2 TRIS 1 mg/ml EB/EL 20 kGy Frozen (dry ice) 31.05.2023 INCT-1OTS40EL-FI-TRIS INCT IST-ID N2 TRIS 1 mg/ml EB/EL 40 kGy Frozen (dry ice) 31.05.2023 INCT-1OTS60EL-FI-TRIS INCT IST-ID N2 TRIS 1 mg/ml EB/EL 60 kGy Frozen (dry ice) 31.05.2023 INCT-1OTS80EL-FI-TRIS INCT IST-ID N2 TRIS 1 mg/ml EB/EL 80 kGy Frozen (dry ice) 31.05.2023 INCT-1OTS100EL-FI-TRIS INCT IST-ID N2 TRIS 1 mg/ml EB/EL 100 kGy Frozen (dry ice) 31.05.2023 INCT-4OTS10EL-RN-P INCT UNIPA N2O Phosphate buffer 4 mg/ml EB/EL 10 kGy RT 14.06.2023 05.07.23 20.07.23 RT Empty vial DLS UNIPA No No INCT-4OTS10EL-RA-P INCT UNIPA Air Phosphate buffer 4 mg/ml EB/EL 10 kGy RT 14.06.2023 05.07.23 20.07.23 RT - DLS UNIPA No No INCT-4OTS10EL-RI-P INCT UNIPA N2 Phosphate buffer 4 mg/ml EB/EL 10 kGy RT 14.06.2023 05.07.23 20.07.23 RT - DLS UNIPA No No INCT-4OTS20EL-RN-P INCT UNIPA N2O Phosphate buffer 4 mg/ml EB/EL 20 kGy RT 14.06.2023 05.07.23 20.07.23 RT Precipitation DLS UNIPA No No INCT-4OTS20EL-RA-P INCT UNIPA Air Phosphate buffer 4 mg/ml EB/EL 20 kGy RT 14.06.2023 05.07.23 20.07.23 RT Vial not properly closed DLS UNIPA No No INCT-4OTS20EL-RI-P INCT UNIPA N2 Phosphate buffer 4 mg/ml EB/EL 20 kGy RT 14.06.2023 05.07.23 20.07.23 RT Vial not properly closed DLS UNIPA No No INCT-4OTS30EL-RN-P INCT UNIPA N2O Phosphate buffer 4 mg/ml EB/EL 30 kGy RT 14.06.2023 05.07.23 20.07.23 RT Precipitation DLS UNIPA No No INCT-4OTS30EL-RA-P INCT UNIPA Air Phosphate buffer 4 mg/ml EB/EL 30 kGy RT 14.06.2023 05.07.23 20.07.23 RT - DLS UNIPA No No INCT-4OTS30EL-RI-P INCT UNIPA N2 Phosphate buffer 4 mg/ml EB/EL 30 kGy RT 14.06.2023 05.07.23 20.07.23 RT Vial not properly closed DLS UNIPA No No INCT-4OTS40EL-RN-P INCT UNIPA N2O Phosphate buffer 4 mg/ml EB/EL 40 kGy RT 14.06.2023 05.07.23 20.07.23 RT Precipitation DLS UNIPA No No INCT-4OTS40EL-RA-P INCT UNIPA Air Phosphate buffer 4 mg/ml EB/EL 40 kGy RT 14.06.2023 05.07.23 20.07.23 RT - DLS UNIPA No No INCT-4OTS40EL-RI-P INCT UNIPA N2 Phosphate buffer 4 mg/ml EB/EL 40 kGy RT 14.06.2023 05.07.23 20.07.23 RT Vial not properly closed DLS UNIPA No No KTH Air H2O 1.5 mg/ml γ-source 1.1 kGy RT Freezer, -18˚C MALDI-TOF KTH Yes KTH Air H2O 5 mg/ml γ-source 1.1 kGy RT Freezer, -18˚C MALDI-TOF KTH Yes KTH Air H2O 1.5 mg/ml γ-source 11 kGy RT Freezer, -18˚C MALDI-TOF KTH Yes KTH Air H2O 5 mg/ml γ-source 11 kGy RT Freezer, -18˚C MALDI-TOF KTH Yes KTH Air H2O 1 mg/ml γ-source 26 kGy RT Freezer, -18˚C Precipitation MALDI-TOF KTH Yes KTH Air H2O 10 mg/ml γ-source 26 kGy RT Freezer, -18˚C MALDI-TOF KTH Yes KTH Air Dry n/a γ-source 26 kGy RT RT MALDI-TOF KTH No Yes 37 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC KTH Air H2O 1 mg/ml γ-source 43 kGy RT Freezer, -18˚C Precipitation MALDI-TOF KTH Yes KTH Air H2O 10 mg/ml γ-source 43 kGy RT Freezer, -18˚C MALDI-TOF KTH Yes KTH Air Dry n/a γ-source 43 kGy RT RT MALDI-TOF KTH No KTH Air H2O 1 mg/ml γ-source 60.5 kGy RT Freezer, -18˚C MALDI-TOF KTH Yes KTH Air H2O 10 mg/ml γ-source 60.5 kGy RT Freezer, -18˚C MALDI-TOF KTH Yes KTH Air Dry n/a γ-source 60.5 kGy RT RT MALDI-TOF KTH KTH Air H2O 1 mg/ml γ-source 0.36 kGy RT Freezer, -18˚C MALDI-TOF KTH Yes KTH Air H2O 10 mg/ml γ-source 0.36 kGy RT Freezer, -18˚C MALDI-TOF KTH Yes KTH Air H2O 1 mg/ml γ-source 0.72 kGy RT Freezer, -18˚C MALDI-TOF KTH Yes KTH Air H2O 10 mg/ml γ-source 0.72 kGy RT Freezer, -18˚C MALDI-TOF KTH Yes KTH Air H2O 0.5 mg/ml γ-source 0.36 kGy RT Freezer, -18˚C MALDI-TOF KTH KTH Air H2O 0.1 mg/ml γ-source 0.36 kGy RT Freezer, -18˚C MALDI-TOF KTH KTH Air H2O 0.05 mg/ml γ-source 0.36 kGy RT Freezer, -18˚C MALDI-TOF KTH KTH Air H2O 0.5 mg/ml γ-source 0.72 kGy RT Freezer, -18˚C MALDI-TOF KTH KTH Air H2O 0.1 mg/ml γ-source 0.72 kGy RT Freezer, -18˚C MALDI-TOF KTH KTH Air H2O 0.05 mg/ml γ-source 0.72 kGy RT Freezer, -18˚C MALDI-TOF KTH KTH Air H2O 0.5 mg/ml γ-source 1.1 kGy RT Freezer, -18˚C MALDI-TOF KTH KTH Air H2O 0.1 mg/ml γ-source 1.1 kGy RT Freezer, -18˚C MALDI-TOF KTH KTH Air H2O 0.05 mg/ml γ-source 1.1 kGy RT Freezer, -18˚C MALDI-TOF KTH 38 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC KTH N2 1 M 2-propanol 1 mg/ml γ-source 0.36 kGy RT Freezer, -18˚C MALDI-TOF KTH Yes Yes KTH N2 1 M 2-propanol 1 mg/ml γ-source 1.1 kGy RT Freezer, -18˚C MALDI-TOF KTH Yes Yes - Yes KTH N2 1 M 2-propanol 1 mg/ml γ-source 2.2 kGy RT Freezer, -18˚C MALDI-TOF KTH Yes Yes KTH N2 1 M 2-propanol 1 mg/ml γ-source 3.6 kGy RT Freezer, -18˚C MALDI-TOF KTH Yes Yes KTH N2 1 M 2-propanol 1 mg/ml γ-source 11 kGy RT Freezer, -18˚C MALDI-TOF KTH Yes Yes KTH N2 1 M 2-propanol 10 mg/ml γ-source 3.6 kGy RT Freezer, -18˚C MALDI-TOF KTH INCT-OTD25EL-RA INCT IST-ID Air Dry n/a EB/EL 25 kGy RT 11.10.2023 INCT-OTD100EL-RA INCT IST-ID Air Dry n/a EB/EL 100 kGy RT 11.10.2023 INCT-OTD400EL-RA INCT IST-ID Air Dry n/a EB/EL 400 kGy RT 11.10.2023 INCT-OTD500EL-RA INCT IST-ID Air Dry n/a EB/EL 500 kGy RT 11.10.2023 39 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC Sample: Ovalbumin IRRADIATION pH DATE STORAGE ANALYSIS Place of delivery After shopiment / before analysis Analysis Without DTT With DTT Sample ID Institute Atmosphere Solution composition Concentration Source Dose Temperature Before irradiation After irradiation of irradiation of receival of analysis Type of storage Visual observations Analysis method Analysis performed at Consumption of native protein Fragments Aggregates Consumption of native protein Fragments Aggregates INCT-1GOD5IL-RA INCT IST-ID Air Dry n/a EB/ILU 5 kGy RT 24.10.2022 INCT-1GOD10IL-RA INCT IST-ID Air Dry n/a EB/ILU 10 kGy RT 24.10.2022 INCT-1GOD25IL-RA INCT IST-ID Air Dry n/a EB/ILU 25 kGy RT 24.10.2022 INCT-OL50EL-RA INCT EPS Air Dry n/a EB/EL 50 kGy RT 17.05.2023 INCT-OL100EL-RA INCT EPS Air Dry n/a EB/EL 100 kGy RT 17.05.2023 INCT-OL200EL-RA INCT EPS Air Dry n/a EB/EL 200 kGy RT 17.05.2023 INCT-OL300EL-RA INCT EPS Air Dry n/a EB/EL 300 kGy RT 17.05.2023 INCT-OL50EL-RA INCT UNIPA Air Dry n/a EB/EL 50 kGy RT 6,3 17.05.2023 01-06-23 08-06-23 RT Solubility test; DLS UNIPA No No Yes INCT-OL100EL-RA INCT UNIPA Air Dry n/a EB/EL 100 kGy RT 6,66 17.05.2023 01-06-23 08-06-23 RT Solubility test; DLS UNIPA No No Yes INCT-OL200EL-RA INCT UNIPA Air Dry n/a EB/EL 200 kGy RT 6,95 17.05.2023 01-06-23 08-06-23 RT Solubility test; DLS UNIPA No No Yes INCT-OL300EL-RA INCT UNIPA Air Dry n/a EB/EL 300 kGy RT 7,01 17.05.2023 01-06-23 08-06-23 RT Solubility test; DLS UNIPA No No No INCT-OL50EL-RA INCT IST-ID Air Dry n/a EB/EL 50 kGy RT 17.05.2023 INCT-OL100EL-RA INCT IST-ID Air Dry n/a EB/EL 100 kGy RT 17.05.2023 INCT-OL200EL-RA INCT IST-ID Air Dry n/a EB/EL 200 kGy RT 17.05.2023 INCT-OL300EL-RA INCT IST-ID Air Dry n/a EB/EL 300 kGy RT 17.05.2023 KTH Air H2O 1 mg/ml γ-source 0.36 kGy RT Freezer, -18˚C MALDI-TOF KTH KTH Air H2O 10 mg/ml γ-source 0.36 kGy RT Freezer, -18˚C MALDI-TOF KTH KTH Air Dry n/a γ-source 0.36 kGy RT RT MALDI-TOF KTH KTH Air H2O 1 mg/ml γ-source 1.1 kGy RT Freezer, -18˚C MALDI-TOF KTH KTH Air H2O 10 mg/ml γ-source 1.1 kGy RT Freezer, -18˚C MALDI-TOF KTH KTH Air Dry n/a γ-source 1.1 kGy RT RT MALDI-TOF KTH KTH Air H2O 1 mg/ml γ-source 20.2 kGy RT Freezer, -18˚C MALDI-TOF KTH KTH Air H2O 10 mg/ml γ-source 20.2 kGy RT Freezer, -18˚C MALDI-TOF KTH KTH Air Dry n/a γ-source 20.2 kGy RT RT MALDI-TOF KTH KTH N2 1 M 2-propanol 1 mg/ml γ-source 0.36 kGy RT Freezer, -18˚C MALDI-TOF KTH KTH N2 1 M 2-propanol 1 mg/ml γ-source 1.1 kGy RT Freezer, -18˚C MALDI-TOF KTH KTH N2 1 M 2-propanol 1 mg/ml γ-source 2.2 kGy RT Freezer, -18˚C MALDI-TOF KTH 40 | Page Database on structure-process and structure-activity relationship Deliverable D2.8 Date: 03/10/2025 Grant Agreement 101061694 PUBLIC KTH N2 1 M 2-propanol 1 mg/ml γ-source 3.6 kGy RT Freezer, -18˚C MALDI-TOF KTH INCT-OL25EL-RA INCT IST-ID Air Dry n/a EB/EL 25 kGY RT 11.10.2023 INCT-OL50EL-RA INCT IST-ID Air Dry n/a EB/EL 50 kGy RT 11.10.2023 INCT-OL100EL-RA INCT IST-ID Air Dry n/a EB/EL 100 kGy RT 11.10.2023 INCT-OL200EL-RA INCT IST-ID Air Dry n/a EB/EL 200 kGy RT 11.10.2023 INCT-OL300EL-RA INCT IST-ID Air Dry n/a EB/EL 300 kGy RT 11.10.2023 INCT-OL400EL-RA INCT IST-ID Air Dry n/a EB/EL 400 kGy RT 11.10.2023 INCT-OL500EL-RA INCT IST-ID Air Dry n/a EB/EL 500 kGy RT 11.10.2023 INCT-OL25EL-RA INCT KTH Air Dry n/a EB/EL 25 kGy RT 11.10.2023 RT SDS-PAGE KTH Yes Faint blue smear below 40 kDa, however, no clear bands Faint blue smear above 40 kDa, however, no clear bands Yes Faint blue smear below 40 kDa, however, no clear bands Faint blue smear above 40 kDa, however, no clear bands MALDI-TOF Yes m/z 897, 1416, 1717, 3345, 4330, 4458, 5966 (S/N =>10) however, low intensity Not detected Yes m/z 4079, 4330, 4458 (S/N =>10) however, low intensity Not detected HPLC-UV INCT-OL50EL-RA INCT KTH Air Dry n/a EB/EL 50 kGy RT 11.10.2023 RT SDS-PAGE KTH Yes Faint blue smear below 40 kDa, however, no clear bands Faint blue smear above 40 kDa, however, no clear bands Yes Faint blue smear below 40 kDa, however, no clear bands Faint blue smear above 40 kDa, however, no clear bands MALDI-TOF Yes m/z 897, 1416, 1717, 4330, 4458, 5966 (S/N =>10) however, low intensity Not detected Yes m/z 3345, 4079, 4330, 4458 (S/N =>10) however, low intensity HPLC-UV INCT-OL100EL-RA INCT KTH Air Dry n/a EB/EL 100 kGy RT 11.10.2023 RT SDS-PAGE KTH Yes Faint blue smear below 40 kDa, however, no clear bands Faint blue smear above 40 kDa, however, no clear bands Yes Faint blue smear below 40 kDa, however, no clear bands Faint blue smear above 40 kDa, however, no clear bands MALDI-TOF Yes m/z 897, 1416, 1717, 3345, 4458, 5966 (S/N =>10) however, low intensity Not detected Yes m/z 3345, 4458 (S/N =>10) however, low intensity Not detected HPLC-UV INCT-OL200EL-RA INCT KTH Air Dry n/a EB/EL 200 kGy RT 11.10.2023 RT SDS-PAGE KTH Yes Faint blue smear below 40 kDa, however, no clear bands Faint blue smear above 40 kDa, however, no clear bands Yes Faint blue smear below 40 kDa, however, no clear bands Faint blue smear above 40 kDa, however, no clear bands