Identification of Unknowns in LC-MS Using a Holistic Approach

From material knowledge to confirmed hits

6th European Forum on Extractables, Leachables, Dr. Roberto Menzel Medical Devices and Food Contact Materials Testing Sartorius Stedim Biotech GmbH Agenda Short Introduction into Sartorius Extractables and Leachables Screening in LC-HRMS measurements Chemistry of Extractables Case Study Global player with around 60 sales and production sites

Netherlands, Belgium, Ireland, Israel, India, Thailand, USA, Canada, UK, France, Spain, Sweden, Finland, Singapore, Vietnam, Puerto Rico, Denmark, Germany, Poland, Austria, China, Malaysia, Mexico, Brazil,

EMEA Hungary, Switzerland, Italy, Russia, South Korea, Japan, Argentina Americas Tunisia, South Africa Australia Asia | Pacific | Asia

25th Sept. 2019 Sales Production and Page 4 Production/Sales Main location in Göttingen, Germany

25th Sept. 2019 Page 5 Roots of both divisions reach far back

1870 1927

University mechanician | Company founder Wilhelm Sartorius founds joint venture with Nobel Florenz Sartorius invents short-beam analytical laureate and creator of membrane filtration balance Richard Zsigmondy1 Weighing Technology Filtration Core of Lab Division Core of Bioprocess Division

1) Zsigmondy25th Sept. 2019, R. and Bachmann, W. “Über Neue Filter” Z. anorg. allg. Chem., 103(1), 119–28, (1918) Page 6 25th Sept. 2019 Page 7 25th Sept. 2019 Page 8 Pioneer in single-use manufacturing technologies

Past Present

High initial cost + CAPEX reduction over entire lifecycle Considerable cleaning effort + Lower water and energy consumption Risk of cross-contamination + Higher flexibility

25th Sept. 2019 Page 9 Well positioned as a total solution provider across the biopharma process chain

Process Development Production

Cell Culture Media Fermentation Cell Harvesting Buffer| PreparationPurification

Services

25th Sept. 2019 Page 10 Extractables and Leachables – Do Not Get Confused!

The terms Extractables and Leachables are clearly defined

The impact of a single use product or container closure systems • Extractables = potential impact • Leachables = actual impact

Leads to two objectives of testing scenarios • Extractables = test the material SUS/component Suppliers responsibility • Leachables = test the final product Extractables Guides Biopharma responsibility Sartorius supports customer via ® 25th Sept. 2019 CONFIDENCE Validation Services Page 12 Extractables – Regulatory Status for SU Equipment

• There are as yet no single specific standards or guidance for SU equipment for Extractables (and Leachables) testing • USP DRAF <665> and BPOG protocol (2014) provide practical information on “what, when and how” to perform an Extractables Study (Time, Temp., Shaking, ) • Sartorius developed an Extractables Approach for material characterization and safety assessment for single-use equipment 1

Implementing new equipment into a pharmaceutical process

Design and Operational Performance Installation Qualification Qualification Qualification

Extractables data “Simulation” data Leachables data Required to identify potential leachables

1) Pahl, I. et al. Development of a Standardized Extractables Approach for Single-Use Components - General Considerations and Practical Aspects.25th Sept. Bioprocess2019 Int. 16, (2018). Page 13 Extractables & Sartorius

• Sartorius provides comprehensive Extractables information since early 1995 1 • Extractables data to support customers in pharmaceutical & biopharmaceutical industry in the DQ / IQ phase • Laboratory at Sartorius since 2015 • Defined and standardized internal Extractables testing • Data provided as Extractables guides (EG) • Since 2018 LC-HRMS screening results included in EG • Active in research 2-5

1 Reif, O. W. et al. PDA J. Pharm. Sci. Technol. 50, 399–410 (1996) Flexboy Extractables Guide 2014 Most current Extractables Guide 2019 2 Dorey, S. et al. Ind. Eng. Chem. Res. 57, 7077–7089 (2018) 3 Pahl, I. et al. Analysis and evaluation of single-use bag extractables for validation in biopharmaceutical applications PDA J. Pharm. Sci. Technol. 68, 456–71 (2014) 4 Hauk,25th A., Sept.Jurkiewicz, 2019 E., Pahl, I., Loewe, T. & Menzel, R. Filtration membranes - Scavengers for leachables ? Eur. J. Pharm. Sci. 120, 191–198 (2018) Page 14 5 Menzel, et al. Comparative Extractables Study of Autoclavable Polyethersulfone Filter Cartridges for Sterile Filtration. PDA J. Pharm. Sci. Technol. 72, 298-316 (2018) Extractables & Sartorius – Equipment LC-HRMS

• Waters Xevo® G2-XS QTof with ACQUITY UPLC ready for use since Q1 2016 • ESI and APCI ionization available with LockSprayTM (mass accuracy below 1 ppm) • Software is UNIFITM 1.8.2

PEG pattern obtained ESI pos using UNIFI software

25th Sept. 2019 Page 15 Target, Suspect, and Non-Target Screening - Internal LC-MS Method

Sartorius method details in brief Rational Flow 0.5 mL/min, Column Temp. 40 °C, Injection Conditions Volume = 1 µL • Neutral eluent for detection of acidic and Eluent A:10 mM NH4CH3COOH Water, B: Acetonitrile basic compounds 0 to 0.5 min B: 5%; 0.5 to 9 min B 99% Gradient 1 µL Injection volume (sensitivity fine, 9 to 19.5 min B 99% (extended up to 40 min) • 19.6 to 20 min B 5% best chromatography, less ion Detection @ λ = 200, 220, and 254 nm (and 3D λ = 220 UV/Vis – 510 nm) suppression) Column BEH C18 (1.7 µm, 2.1 x 100 mm) • LC method extended up to 40 min for Parameter Value ESI unknown samples Ionization polarity ESI positive and ESI negative Mass range 50 - 1500 m/z UV detection for weak ionizing Cone voltage 40 V • Analyzer Mode Sensitivity compounds Capillary voltage ESI pos and neg: 3 kV Source temperature 120 °C • Scan range up to m/z = 1500 including all Cone gas flow 50 L/h Desolvation gas flow and major additives 950 L/min, 550 °C temperature LockSpray every 30 s • Standard C18 column (10 cm) Ramp MSe Low CE: 4.00 eV, High CE: 40 eV

25th Sept. 2019 Page 16 Unknowns – Why Are They Such a “Nuisance”

• Evaluation of an Extractables profile includes a safety assessment • All available toxicological information are used (PDE, NOAEL, LD50 values) • The Threshold of Toxicological Concern (TTC) concept is applied for compounds with known structure w/o toxicological data 1 • ICH M7 defined TTC of 1.5 µg/day (lifetime > 10 years) with negligible risk for mutagenic compounds and typically applied also for Unknowns • All structural additional information are beneficial, e.g. exclusion of groups • Keep in mind – most E/L are of low toxicological concern 2

Quantity of a complete unknown compound might exceed allowable exposure because of (incorrect) lowest TTC – What then?

1) Koster, Sander et al. Application of the TTC Concept to Unknown Substances Found in Analysis of Foods. Food and Chemical Toxicology 49(8): 1643–60 (2011). 2)25thLi, Sept.K. et 2019 al. Creating a Holistic Extractables and Leachables (E&L) Program for Biotechnology Products. PDA J. Pharm. Sci. Technol. 69, Page 17 590–619 (2015). Target, Suspected-Target and Non-Target Screening in LC-HRMS

• Target Screening: Reference compounds available, calibration, known validation parameters, etc. “Target-Screening” → quantitative data

• Suspected-Target Screening: Expected analytes, established database, known (relative) retention time, ionization behaviour, response factors, fragmentation pattern, etc. → quantitative and/or qualitative data “Unknown-Screening” • Non-Target Screening: Low sample information, no information chemical classes, etc. → qualitative data

If possible, spent all you laboratory time to improve your suspected-target screening.

25th Sept. 2019 Page 18 Suspected-Target Screening – List of Suspects & Where to start?

• Suspected-target screening in LC-MS is extremely helpful in E/L studies • The size of the list of suspect is not important – it’s the quality • For plastic additives a start is Zweifels “Plastics Additives Handbook” (Plastic additives are no secret – Manufacturer use these additives.) 1 • Evolve library suspects with measurement results • Sartorius has about 150 secured suspects and 300 additional additives implemented

Not blindly include structures How to handle polymeric additives?

Tinuvin® 622 Irganox® 1425 Maybe use this for suspect EP/USP plastic additive Maybe they are a good starting point

25th Sept. 2019 Page 19 1) Most used antioxidants (Irgafos® 168, Irganox ® 1010, etc.): “Stabilizer systems of triaryl phosphites and phenols” DE 1976-2606358, 1976! Non-Target Screening – Where to start?

• Non-Target Screening (NTS) is described in literature mainly for water or biochemical analysis 1 • Possible workflow described in literature 2 • No existing guidelines on how to actually perform NTS in E/L studies

• The best NTS or “Unknown” Screening is done by: a. Know your material, better know the additives b. Think about and know your chemistry c. Read literature  Derive your possible structures and perform suspected-target screening

For confirmation authentic reference material should be used or very good justification. In chemistry, structure elucidation done by NMR, elemental composition, IR, etc.

1 General Screening and Methods 2 Workflow Krauss, M., Singer, H. & Hollender, J. Anal. Bioanal. Chem. 397, 943–951 (2010) Little, J. L. et al., J. Am. Soc. Mass Spectrom. 22, 348–359 (2011) Milman, B. L., Trends Anal. Chem. 69, 24–33 (2015) Little, J. L. et al., J. Am. Soc. Mass Spectrom. 23, 179–185 (2012) Schymanski25th Sept. 2019, E. L. et al., Anal. Bioanal. Chem. 407, 6237–6255 (2015) Müller, A.. et al., Chemosphere 85, 1211–1219 (2011) Page 20 Non-Target Screening – Sartorius Approach

Part 1 • General: Samples signal must exceed blank by 50% • Visual comparison of the BPI of the sample vs reference blank www.chemspider.com • Find unknown peak and corresponding mass signal • Only the most intense monoisotopic peak is reported

• PDA (220 nm) is used to detect compounds not ionized well by ESI www.cas.org

• Elemental Composition tool is used to generate formula • Obtained parameters e.g. used in Discovery Tool to perform structure elucidation (ChemSpider) • If no hit is obtained molecular formula is searched in Scifinder®

25th Sept. 2019 Page 21 Non-Target Screening – Sartorius Approach

Part 2 • Does the structure make sense and are they reasonable for extraction solvent? • Does the retention time make sense? • Does the ionization behavior make sense? If yes, • Structure implemented in library, checking if peak is assigned by UNIFI • Fragmentation pattern checked for reasonability Identification level should be given according to USP <1663>*

Example of a list of hits of a LC-MS suspect and non-target screening RT MS m/z m/z UV/Vis Molecular Identification i-Fit Structural Suggestion (e. g. Name, CAS-#, origin) [min] ESI pos ESI neg signal Formula Level 3.21 792.5992 850.6013 - C42H77N7O7 100 Caprolactam Heptamer (CAS 16056-00-1) confident Ethylene glycol terephthalate (3:3) (CAS 16958-96-6), 4.27 - 593.1297 - C30H26O13 100 confident suspect, PET degradant 5.02 - 785.1715 - C38H22N14O7 35 Unknown unknown 6.63 594.1606 635.1406 medium C30H24O12 Ethylene terephthalate cyclic trimer (CAS 7441-32-9) confirmed 7.05 581.4317 579.4156 - C36H56N2O4 93 Irganox 1098 degradant (tert-Butyl split off) tentative 25th Sept. 2019 Page 22 * United State Pharmacopoeia <1663> Assessment of Extractables Associated with Pharmaceutical Packaging/Delivery Systems. 38, 7166–7180 (2015). Chemistry of Extractables/Polymers – Simplify Your Screening

General Statements • Extractables are based and are formed from existing additives or processing aids or from the polymeric materials • Extractables can be traced back to the raw material • Yes, also Extractables are subject to standard chemical principles • Reactions with the extraction media are possible (e.g. Ethanol) • Reactions between Extractables in an extraction solvent are unlikely (dilution) • Analytical “False positive” hits are possible e.g. decarboxylation in GC-MS, wrong blanks, bad analytics 1 This means, all Extractables and potential “unknown” are explainable by material/manufacturing and chemical/analytical rationales.

1) Menzel, R. “Extractables Data from Single-Use Systems for the Biopharmaceutical Industry - Need Extractables be such a difficult and 25th Sept. 2019 Page 23 “controversial” topic?”, BPI West 2019, DOI: 10.13140/RG.2.2.15050.62403/1, https://tinyurl.com/y6c26fhn Chemistry of Extractables/Polymers – Simplify Your Screening

Common Basic Chemical Reactions in E/L

• Esterification and hydrolysis, transesterification • Oxidation reaction These are not the reactions mechanism one • Radical reactions should expect for Extractables or Leachables • Acetal formation • Other nucleophilic substitution reactions (very rare) • Nitration/Nitrosation (very rare)

Remark: Catalyst (acids) produced during gamma irradiation Total synthesis of (−)-colchicine

25th Sept. 2019 Page 25 Chemistry of Extractables - Examples Oxidation Reaction

• Degradation of polyolefins is unavoidable • Crosslinking and oxidation take place

„Bolland & Gee mechanism“ polymer autoxidation

Wanted oxidation Irganox® PS 800

Oxidation degradants of BHT

Oxidation takes place also for the additives.

Bolland,25th Sept. J L, and2019 G. Gee. 1946. “Kinetic Studies in the Chemistry of Rubber and Related Materials. II. The Kinetics of Oxidation Page 26 of Unconjugated Olefins.” Transactions of the Faraday Society 42(0), 236-43, 1946 Chemistry of Extractables/Polymers – Example Hydrolysis Ester

• Esterification, hydrolysis, or transesterification takes place in aqueous extraction solutions but also during storage • Process is acid-catalyzed • Example: Irgafos® 168 - Secondary antioxidant polyolefins

„Famous“ bDtBPP (CAS 69284-93-1) Irgafos® 168 Irgafos® 168 oxidized  Usually observed after irradiation 31570-04-4 95906-11-9 because of acid-catalyzed hydrolysis  Typically, not observed after long storage

25th Sept. 2019 Page 26 Chemistry of Extractables/Polymers – Example Acetal Hydrolysis

• Acetal formation - a reversible acid-catalyzed process • Typically observed in aqueous extracts of gamma-irradiated test items • Example: Irgaclear® DM - Clarifying agent for polypropylene 1

Irgaclear® DM 4-Methylbenzaldehyde Irgaclear® DM mono Sorbitol (54686-97-4) (104-87-0) (w/o stereo 88449-62-1) (50-70-4) detected by GC-MS (with stereo 126748-37) Not detected by GC-MS or LC-MS

1) McDonald, J. G. et al. “Identification and Quantitation of Sorbitol-Based Nuclear Clarifying Agents Extracted from Common 25th Sept. 2019 Page 28 Laboratory and Consumer Plasticware Made of Polypropylene.” Analytical Chemistry 80(14), 5532–41, 2008 Chemistry of Extractables/Polymers – Example Acetal Hydrolysis

• Irgaclear® DM and its degradant „Irgaclear® XIC ESI pos, polypropylene extract DM mono“ can be detected with LC-MS easily • Compounds are present in aqueous extracts

Irgaclear® DM Irgaclear® DM mono left: ESI pos; right: ESI neg left: ESI pos; right: ESI neg

25th Sept. 2019 Page 28 Case 1: Unknown Identification - Filter Cartridge

Extractables study of a sterilizing-grade filter Material Information Filter Cartridge Membrane: PTFE Support Fleece: Polypropylene 1 Core/cage: Polypropylene 2 End Caps: Polypropylene 2 Additive package partly known

Extraction Conditions • Surface to volume ratio: 1:1; Effective Filter Area: 7500 cm2  Extraction volume: 7500 mL. • Extraction media: WFI and ethanol • Extraction parameters: t = 24 h, T = 40 °C, shaking speed = 75 rpm

25th Sept. 2019 Page 29 Case 1: Unknown Identification - Filter Cartridge

1 2 Irgafos 168 (oxidized) Irgafos 168 ESI positive Irganox 3114

sample

blank

Unknown 1: 10.36 min Two intense signals in LC-HRMS chromatogram Unknown 2: 11.13 min All other signals identified.

LC-HRMS BPI Chromatogram ESI + ethanol extract (top sample, bottom blank chromatogram) 25th Sept. 2019 Page 30 Case 1: Unknown Identification - Filter Cartridge 1 2 monoisotopic signal monoisotopic signal + (M+H)+ (M+H)

ESI pos mass spectra low CE ESI pos mass spectra low CE

- 100 Da - 100 Da - 17 Da - 17 Da - 40 Da - 40 Da

ESI pos fragment mass spectra high CE (40 eV) ESI pos fragment mass spectra high CE (40 eV)

Unknown 2 is a homologue of Unknown 1 (+ C2H4) - longer alkane chain Unusual fragmentation pattern 25th Sept. 2019 Page 32 Case 1: Unknown Identification - Filter Cartridge

Step 1: Find Reasonable Elemental Composition • Monoisotopic mass ESI pos H+ adduct, reasonable elemental composition generated in UNIFI Elucidation Toolset

→ Unknown 1: C27H53NO3 and Unknown 2: C29H57NO3

Step 2: Database Search - Discovery Tool • Two possible “classes” of molecules for Unknown 1 identified → A) An hindered amino light stabilizer (HALS) or degradant thereof → B) Some detergent from a cleaning solution or added as flow improver

Candidate A (not available, some references) Candidate B (mainly patents)

25th Sept. 2019 “Representative” for Candidate A (available & known) Page 33 Case 1: Unknown Identification - Filter Cartridge

Step 3: Test Identified Candidates • Purchase potential/similar candidate – confirmation or additional information (fragmentation, ionization behavior) → “Rep.” Candidate A similar mass signal, different retention time, no trace of unknown peaks

Step 4: Identify Source of the Extractables • Extraction of the different raw materials → Extract of one polypropylene (PP1) showed the same unknowns

Step 5: Identification of Additives of the Raw Material • Stearic acid, Palmitic acid, Irgafos® 168, and degradant from Tinuvin® 622 detected → On request, Tinuvin® 622 was confirmed as additive from supplier

25th Sept. 2019 Page 33 Case 1: Unknown Identification - Filter Cartridge

Step 6: Develop Possible Alternatives • Plausible structure from reactions of present additives • Reaction product (transesterification) of a fatty acid (detected) and Tinuvin® 622 (confirmed) – This would be a very rare case! • Identification of potential main reaction product – chemistry of esters

Palmitic acid - or salts Tinuvin® 622 thereof (65447-77-0, EP (57-10-3, releasing plastic additive 22, agent, ubiquitous) light stabilizer)

Reaction between both compounds takes place most likely during injection molding process (high temperature, trace water always present) and not during extraction.

25th Sept. 2019 Page 34 Case 1: Unknown Identification - Filter Cartridge

Step 7: “Synthesis” – Feasibility study • Difficult to purify, NMR showed product, purity ⋲ 70% (sufficient for estimation concentration range)

In LC-MS one peak, the unknown 1 1 2

Palmitic acid from Tinuvin® 622 Stearic acid Tinuvin 622

Unknown 1: C27H53NO3 Unknown 2: C29H57NO3 m/z 440.4098, RT 10.35 confirmed m/z 468.4411, RT 11.31 min confirmed 1-Hydroxy-2,2,6,6-tetramethyl-4-piperidinyl palmitate (HTPEP) 1-Hydroxy-2,2,6,6-tetramethyl-4-piperidinyl stearate (HTPES) No CAS available, not described in literature No CAS available, not described in literature

25th Sept. 2019 Page 35 Case 1: Bringing all together • Note: It is very common to find methyl esters • Concentration range HTPEP & HTPES low ppb level!

C32H59NO6

C29H57NO3

C34H63NO6 C27H53NO3

C42H78N2O7

25th Sept. 2019 Page 36 Summary

• LC-HRMS is the technique for unknown identification • Extractables and Leachables are explainable understanding polymer chemistry and physicochemical principles • Unknowns are best identified using material knowledge and chemistry • Suspected-target screening is the key for fast “unknown” screening • Quantitative estimations should be performed by authentic reference compounds or by using orthogonal analytical methods, e.g. UV detection • “Real” non-target screening is the most time-consuming task and can take months!

“Unknown” screening can make a lot of fun, especially if everything fits together Think outside the “even/odd number-nitrogen rule-isotopic patter-fragmentation- mclafferty rearrangement” box.

Enjoy your “Eureka” moment

25th Sept. 2019 Page 37 Contact

Roberto Menzel Head of Laboratory and Manager Extractables & Leachables [email protected] Armin Hauk Principal Scientist Extractables & Leachables [email protected]

Special thanks for data and presentation support Tanja Maier Scientist Extractables & Leachables [email protected] Extractable Group Sartorius on ResearchGate Website: www.sartorius.com https://tinyurl.com/y2a4n3rw @SartoriusGlobal @SartoriusStedimBiotech Thank you for your attention!

Question & Discussion

25th Sept. 2019 Page 39