SPRING 2021 from President Angela Diciccio VOLUME 26, ISSUE 1 As We Embrace 2021 Sterilization

inSurFACTS Biomaterials In memory of

SPRING 2021 From President Angela DiCiccio VOLUME 26, ISSUE 1 As we embrace 2021 sterilization. APS highlights the with determination impacts of changes in regulations for change, the on our traditional approaches SIBF board is to Extractables and Leachables committed to analysis, truly emphasizing INSIDE THIS listening to the that a generalized approach needs of our is sometimes inappropriate, membership and often insufficient to truly ISSUE and being represent and understand mindful to evolve realistic outcomes. Biocoat in a direction that echoes the importance of PAGE 2 enables growth of value. situation specific methodologies Sterilization Effects on This year will provide an and informed decision making, Hydrophilic Coatings incredible opportunity to reset, highlighting that while some refresh, rebuild and reintroduce sterilization methods may work norms of interaction while great on some substrates, the incorporating new platforms outcomes on others could have PAGE 6 and discoveries. Our intention wildly different impacts. Agonies and Thrills of Es and Ls. is to cultivate the principles After reading this episode of Part 2: Solvent Effects on which this Foundation was originally seeded and continue SurFACTs, I challenge you to stimulate an arena for non- to reflect on areas in your traditional opportunities to field where the “generalized” PAGE 10 discover, collaborate, mentor and approach might be overused Thanks to our Members! explore across interdisciplinary and a more mindful and informed stakeholders in the biomedical method would drive value and surfaces fields. and more authentic insight. If you have a great example, I Articles within this issue encourage you to submit for specifically touch upon our our next episode of SurFACTs need as a society to reevaluate or to tell us about it during this previous norms and challenge year’s Surfaces in Biomaterials the generalized approach Workshop and Symposium. Most to solving certain problems importantly, think about leaders like chemical analysis and in this field and make sure to

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1 From President Angela DiCiccio continued from page 1 nominate them for this year’s Surface new platforms for collaboration, regards to your experiences! Most in Excellence Award before the March communication, and involvement. importantly, take a deep breath and 31 deadline! Reach out if you are motivated to jump enjoy learning and growing together. in to accelerate our efforts, provide Stay tuned for announcements about perspective, or have feedback in

Sterilization Effects on Hydrophilic Coatings Bob Hergenrother, Ph.D. and Betsy Morgan, Biocoat, Inc.

Almost all medical devices on the market go through of Biological Indicators placed in the sterile load. One the process of sterilization. The sterilization of medical potential effect of ETO sterilization on medical devices devices can affect the materials and hydrophilic coatings is that the heat and humidity of the sterilization process of those devices due to the nature of methods that are can affect the materials. Another is that the ETO grafting used. The three known sterilization methods are Ethylene onto the functional groups of the surface can change the Oxide (ETO), Gamma Radiation and E-beam Radiation. properties of that surface. With data, we are able to see how these three sterilization methods can affect hydrophilic coating performance Gamma sterilization accounts for about 40% of medical 60 measured by the testing of lubricity, durability and device sterilizations in the US. This process uses Co as particulates. a radioactive isotype, which when undergoing nuclear decay produces gamma radiation to sterilize the device. ETO sterilization, the widest used method, has recently The device is exposed to the radiation which affects the seen closures to their facilities due to ETO emissions, kill by creating free radicals that result in DNA scission. prompting the FDA to issue a statement of concern about Instead of using biological indicators, microbial kill is the necessity of maintaining ETO processing capabilities. demonstrated by the probability of Sterility Assurance This in turn has prompted companies to consider of 10-6. The potential effect of using gamma sterilization qualifying other types of sterilization methods such as is that it can have chain scission on the polymers that Gamma Radiation and E-beam Radiation, for their devices. are used in the device. For example, polypropylene is However, gamma radiation poses its own availability susceptible to this degradation. concern due to the handling and security considerations of 60Co. As a hydrophilic coatings supplier, we need to The e-beam method of sterilization only accounts for understand how various sterilization methods affect the about 4% of medical device sterilizations in the US. In this materials we use and allow for potential changes to the process, electrons are accelerated in a 1 to 5 diameter sterilization choices. beam and scanned over the device to be sterilized. Microbial kill is accomplished essentially the same way as ETO, or Ethylene Oxide Sterilization, accounts for 50% gamma radiation, which is by forming DNA scissions from of all medical device sterilizations in the US. To perform free radicals that are formed, demonstrated by the Sterility this method, parts are placed in a chamber filled with Assurance level of 10-6. This potential effect results again ETO gas with a certain amount of humidity. The parts in having chain scission on the polymers. However, this are kept in the chamber for a certain amount of time at tends to be less severe than gamma radiation because an elevated temperature. They must be packaged in the depth of the electron penetration is less than the something permeable to allow the gas and water vapor gamma particles. to permeate into the device and sterilize the surface. The parts are then vacuum-purged to reduce ETO levels after At Biocoat, we evaluated our HYDAK® Thermal cure it is done. Microbial kill is accomplished by alkylation of and UV cure hydrophilic coatings using these three amine groups of the DNA, as demonstrated by the use different sterilization methods and measured post-

Continued on page 3 2 Sterilization Effects on Hydrophilic Coatings ... continued from page 2 sterile performance via lubricity, durability, particulates and For the pinch test, there is no change in friction for the HA visualization of the coating. ETO sterilization was done at only coating (#1) using ETO sterilization over 30 cycles. 50 degrees Celsius, 30% RH (relative humidity). Gamma However, there is some change in reduction of friction and e-beam were both done at a nominal 45 kgy (kilogray when gamma and e-beam methods are applied. When sterilization dose). crosslinkers are added (#2), this stabilizes the coating and again with the pre-sterile and ETO the friction stays about For this study, we used five different coatings from our the same. With gamma, there is some degradation over HYDAK Thermal cure process. These included: time (although it is better overall than the HA only coating), whereas the e-beam method actually improves here. 1. HA (HA only) The HA coatings with acrylic polymers (#3&4) stabilizes 2. HA-X (HA with Crosslinker) the coating and again not much has changed. For one of them (#3), there is a slight increase in the friction using 3. HA-P1 (HA with Acrylic Polymer) ETO, as well as pre-sterile for gamma and e-beam. For our main coating (#4) T-70, it looks good with the different 4. HA-P2 (HA with Acrylic Polymer: T-070) – this is our main sterilizations for both the lubricity and durability. Similarly, coating the synthetic polymer (#5), which is our T-18 coating, is 5. Syn (Hydrophilic Copolymer: T-018) designed to withstand radiation and has very stable results over the course of the 30 cycles for all three sterilization We also used two different coatings from our HYDAK UV methods. (Shown in Figure 1) cure process. These include: The visualization results, which are obtained through 1. UV-2/1: 2/1 ratio of Polymer A/ Polymer B

2. UV-4/1: 4/1 ratio of Polymer A/ Polymer B

(Both of these have a photoactive basecoat and topcoat)

The lubricity was evaluated using our frictional test pad, also known as a “pinch test”, of either silicone or Delrin pads. During this process, 500 grams of force is applied to the pads while the device is pulled up through each side of the pad. We then cycle this process multiple times to measure the durability. For this study, Figure 1 we did 30 cycles using Pebax 55D, 2mm OD (outer diameter) tubing. staining the coatings, mostly match the friction results. The Visualization is accomplished by staining the hydrophilic HA only coating (#1) stayed very dark blue through ETO, but coating blue. Prior to sterilization methods, the staining of then lightened considerably through gamma and e-beam, the coating appears very dark blue. suggesting that the coating had been degraded from the The HYDAK Thermal cure results using each sterilization radiation. The HA with crosslinkers (#2) improved on the method are as follows: e-beam with some reduction in color from the gamma. Continued on page 4 3 Sterilization Effects on Hydrophilic Coatings ... continued from page 3

The HA with acrylic polymers (#3 & 4) look good through ETO with some changes in color from the gamma and e-beam, but more so with the gamma. The synthetic coating, T-18 (#5) has no change in color, which is consistent with the friction performance. (Shown in Figure 2)

The HYDAK UV cure results using each sterilization method are as follows:

During the development process of our new UV cure coating, looking at the two different ratios, we found that at the pre-sterile both of these coatings have about the same results for lubricity and durability. However, after ETO sterilization, one of them had worse friction results, but seemed to look good through the gamma and e-beam methods. For the 2:1 ratio coating Figure 2 (#1) there was a difference in the ETO friction compared to the 4:1 coating. We then compared Finally, for the particulate measurements, we used the this to the T-18 synthetic polymer. (Shown in Figure 3) HYDAK UV cure coatings to see how they compare after the various sterilization methods. Nailing down the For the visualization measurement, compared to the parameters and having good comparison data is critical for synthetic T-18, the results are quite good in terms of the particulate measurements. For this particulate evaluation, color after sterilization as compared to the Thermal cure we have a clinically relevant tortuous path test pathway. We coating counterparts. were aggressive on the duration of testing for this study, performing 50 cycles back and forth in the pathway, flushing with high purity water after each 10 cycles and collecting the effluent in an HDPE container. To count the particles, we immediately tested the samples in a light obscuration chamber. The results presented include all particulates greater than 10 microns. Both UV cure coatings show similar behaviors with fairly low particulate counts. There are similar

Figure 3

Continued on page 5 4 Sterilization Effects on Hydrophilic Coatings ... continued from page 4 results with pre-sterile, ETO and e-beam with gamma having slightly higher particulate counts. (Shown in Figure 4)

In conclusion, sterilization processing does have an effect on the performance of hydrophilic coatings, however, it’s possible for HA based coatings to withstand irradiation sterilization. Regardless of the cure system, Thermal cure or UV cure, as well as the type of coating, you should evaluate the sterilization effects. Testing should include looking at all aspects of a hydrophilic coating performance which Figure 4 includes lubricity, durability and particulate generation.

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5 Agonies and Thrills of Es and Ls. Part 2: Solvent Effects Mourad Rahi, Ph.D. Sr. Director, Analytical Services and Mark Smith, Ph.D. CSO Emeritus,. American Preclinical Services, Minneapolis, MN 55433

Abstract residuals. Isocynates and epoxide by Gas Chromatography/Mass Chemical characterization of monomers constitute the building Spectrometry (GCMS), Ultra- complex medical devices presents blocks of polyurethane and Pressure-Liquid Chromatography enormous challenges even to polyethers, and are widely used Orbitrap High-Resolution Mass- the best in the field of analytical polymers in the construction of Spectrometry UPLC/HRMS) and chemistry, chromatography and various medical devices. These Inductively Coupled Plasma spectroscopy. Extractables and monomers are very unstable Spectroscopy (ICPMS). GCMS leachables (E&L) data collection, in protic solvents, they react analysis showed no detection of interpretation and toxicological risk with a broad range of chemical any compound above the analytical (8,9) assessment are major topics of compounds and yet, they are not threshold evaluation (AET). (3) ongoing forums, conferences and classified as cohorts of concern However, LCMS operating in full webinars. This article highlights despite their potential production of scan in electrospray positive and some of the shortfalls of the process DNA/RNA mutagenic adducts. negative modes, showed detection and provides guidance to improve of small amount chloro products. Solvent compatibility with data quality and ensure device The results prompted simulated- extractables are rarely investigated safety. use extraction using saline for the and present a major challenge determination of the leaching rate. for detection and identification of LCMS analysis showed detection of Introduction reactive extractables and potential The examples presented in previous various chloro-derivatives in high reactions with co-extractables such webinars (1-2) were actual data as abundance. as epoxide (4), and isocyanates reported by various laboratories and leading to identification of reaction To confirm the source of chloro- excerpts from FDA feedback. Reviews products.(5) products, simulated-use extraction of data showed rampant compound was repeated with control gelatin mis-identifications such as: In general, reaction end-products spiked with glycidylether 100 are thermodynamically more stable, 1. Anhydrides in protic solvent standard. LCMS analysis showed less reactive and less toxic, thereby extracts matching chromatographic profiles camouflaging the real risk to and perfect response ratio of 2. Significant levels of non-polar which the patient might be directly isotopic chlorine peak areas for compounds in aqueous solvent exposed. the different chloro-substituted extracts compounds. The chloro-products Results and Discussion showed no match using search 3. Polar compounds in non-polar In order to understand the chemical library. solvent and yet, no detection compatibility of the solvent on in corresponding polar extracts chemical characterization, a Hydroxy-alkyls, halide-alkyls, contrary to the principle of “like heart valve constructed of gelatin ethers including epoxides are dissolves like.” and cross-linked with glycidyl poor candidates for detection ether 100 which failed the initial by LCMS due to poor ionization. Most laboratories performing biocompatibility testing, was However, addition of halides to E&L start with a pre-investigation submitted for E&L evaluation hydroxyl-compounds promote their to ensure proper selection of following ISO-10993 part 12 and ionization through inductive effect compatible solvents to maintain 18.(6,7) or electronegativity enhancing device integrity. Complex devices negative ionization of OH group, or are constructed of a combination The device was extracted with through their lone donor electron of polymers and components hexane, isopropyl alcohol and water pair increasing electron cloud having different physico-chemical following solvent biocompatibility density on oxygen for positive properties, and are often known testing to ensure device integrity. ionization. Otherwise, the hydrolysis to contain some level of reactive The extracts were analyzed

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6 Agonies and Thrills of Es and Ls. Part 2: Solvent Effects continued from page 6 products would go undetected, the potential to affect structure require expertise in material and identification of any residual of analytes through formation science or analytical chemistry to epoxide completely overlooked. of adducts. Concentration also provide the necessary qualitative enhances reaction kinetics amongst and quantitative data that a The major advantage of HRMS is its potential co-extractants to yield risk assessor can use to assess selectivity allowing measurement of by-product(s) that would eventually medical device safety.” Yes, the exact mass and distinction in minor result in false positive identification emphasis is on expertise. In spirit, changes in chemical structure. of extractables. the guidelines are good but study However, HRMS stops short of conduct and application suffer from distinguishing between compounds Conclusion major flaws due to competition, with the same molecular formula The search for the culprit cost control, business pressure, leading to E&L search library illusion molecules responsible for failure etc. The design of experiments as and confusion. A literature search of biologically compatible extracts is generally being practiced has as an example for molecular formula to pass the routine and common become so generic as applied C H O , https://pubchem.ncbi.nlm. 22 20 9 biocompatibility tests for cytotoxicity throughout a large sector of the nih.gov/#query=C H O , shows 22 20 9 or irritation for similar devices may industry following protocol design over 200 different structural, binary have been prematurely halted had limiting solvent selection to device and ternary adducts compounds the simulated-use extraction been integrity and identification based on exhibiting different substructures performed using water or alcohol search libraries. The results often and various level of reactivity and as extraction medium. The use of fall short of expectations mainly toxicity. saline and subsequent reactions due to lack of expertise in data of Cl- with epoxides enhanced the interpretation and collaboration, Adduct formation in E&L presents detection and identification of the basic knowledge of principles of a big challenge for LCMS chemicals causing failure. chromatography and spectroscopy, identification and quantification chemical and physical properties of and these reactions can range Another issue that might have solvents and extractables. Search from simple to more complex. The emerged, had the simulated-use Libraries and exact molecular mass spectra fragmentation pattern extracts been analyzed following formula should be a tool but never is also affected by instrument target analysis as recommended the only unconfirmed tool for parameters, concentration, mobile by guidelines. Target analysis may (10) identification. phase composition interference have resulted in a release rate (11) or contaminants, rendering intra- of chloro-byproducts causing no The responsibility of any laboratory laboratory reproducibility of E&L toxicological risk, and the device conducting E&L is paramount (12) data enormously difficult. may have passed the approval because the ultimate safety process without any question assessment of the device depends In method development/validation asked. In such a case, identification on laboratory results. Toxicologists of analyte(s) in complex matrices, of the chemical structures of the and reviewers often overlook how the sample extract is often “cleaned various chloro-byproducts is not the identification of compounds are up” using various techniques to as important as identification of generated as long as a standard control matrix effect. However, E&L the parent compound to which the process is followed. At the end, extracts are by design, analyzed patient is directly exposed. it is possible that regulatory “as is” without any substantial approval could be granted based preparation. As a general rule, In fairness to guidelines being on reporting reaction by-products, the more complex the device, ambiguous, it is not possible mis-identification or wrong data the more complex the matrix to be more specific given the interpretation. effect, and it is directly related to variables that could easily affect the volume reduction/concentration outcome. However, the guidelines Finally, most laboratories’ processes which is often performed to meet 10993-18: 2020, page 29 states: as well as reviewers put more Analytical Evaluation Threshold "The successful completion of emphasis on compliance to ensure (AET) requirements. Concentration the chemical characterization the box is checked. We all believe generates a complex medium with outlined in this document can that expertise pays off. Good E&L Continued on page 8 7 Agonies and Thrills of Es and Ls. Part 2: Solvent Effects continued from page 7 data provides guidance to medical a. BioInterface Webinar. Oct 2020 8. ISO/TS 21726:2019(E). Biological device manufacturers to either b. E&L USA Webinar. Oct 2020. Evaluation of medical devices- Ap- c. American Preclinical Services plication of the threshold of toxico- revisit their manufacturing process Webinar. June 2020 logical concern (TTC) for assessing to improve the quality of their 2. Mourad Rahi. Agonies and Thrills of Es biocompatibility of medical device and Ls. BioInterface Convention, Salt constituents product or face regulatory scrutiny, Lake City, 2019 9. M7(R1) Assessment and control of risk device failure and compromise 3. Biological evaluation of medical DNA reactive (Mutagenic) Impurities patient safety. Finally, data quality devices-Application of threshold of in pharmaceutical to limit potential toxicological concern (TTC) for assess- carcinogenic risk. ICH Guidance for provides unique standing to any ing biocompatibility of medical devices Industry. 2018 organization in line with: You get constituents. ISO/TS 21726:2019(E) 10. https://www.pennstatehershey. what you pay for, and the data is 4. Leon Shechter, et al. Glycidyl Ether org/c/document_library/get_ Reactions with Amines, Ind. Eng. file?uuid=67335431-8f0b-4a77-96c0- as good as the one behind the Chem. 1956, 48, 1, 94–9. https://doi. e5625f40e119&groupId=2384708 machine. org/10.1021/ie50553a029 11. Lopes, N. P. et al. A Study of the 5. J. Bernardini et al. Exploitation of Effect of pH, Solvent System, N.B: In the next article: Part 3. Arundo donax L. Hydrolysis Residue Cone Potential, and the Addition of for the Green Synthesis of Flexible Crown Ethers on the Formation of Solvents selection and analytical Polyurethane Foams. Bioresources the Monensin Protonated Parent Ion techniques for characterization of 12(2):3630-3655A (2017) in Electrospray MassSpectrometry. reactive extractables and covalently 6. Biological evaluation of medical devic- Analyst2001,126,1630 es: Sample preparation and reference 12. Bob Przygoda. Variation in chemical bonded toxic un-extractables. materials. Part 12: 2012(E) characterization results from four 7. Biological evaluation of medical test laboratory. ISO-TC 194-WG15_N References devices: Chemical Characterization 142-Presentation_on_SOT_2018 1. Mourad Rahi. Data Collection, of medical device materials within a Collaboration and Data Interpretation. risk management processISO10993- 18:2020(E)

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