PEER REVIEW: IMPACT OF STERILIZATION Evaluating the Impact of Sterilization on Gel Formulations Nicole Stelner-Relschitz, Michael Pyerin, Chrysoula Kanakaki, Daniela Neubert, Michacl Washilttl, and Michael Krainz Research suggests that radiation ‘ecent studies evaluated the effects that different steril- can havea significant impact on the ization methods have on the active ingredients present composition and rheology of hydroxyethyl a fiele ceeemnetioneash nahin cellulose-based medicinal gels. formation of volatile low molecular weight compounds, and degradation of API were studied. This article summarizes the findings. ‘To ensure unbiased results, four samples of gels with the ‘same basic composition were sterilized. One sample con- tained two APIs, Lidocaine and Chlorhexidine; two gels contained only one ofeach ofthese APIs; while the fourth gel did not contain any active ingredients. Samples were sterilized using hot steam, gamma radia- tion, and electron beam methods (with doses measured in kiloGrays, kGy). The radiation doses were 5, 10, and 25 kGy for the gamma sterilization and 5 and 25 kGy for the e-beam_ process, Unsterilized gels of each type were used as blanks. For comparison, another four commercially available gels ‘were analyzed, sterilized by radiation, and then analyzed again after an elditional gamma sterilization step, carried outat 5 and 25 kGy. Sterile medicinal products can be sterilized in two ways, either via terminal methods (ie., on the product in its final container) o via aseptic processing (1). Whichever steriliza- tion approach is used, the process must be validated in order to verify that it effectively and reliably kills any microorgan- isms that might be present in the product (2). ‘Heat, radiation, gamma or electron beam, and steam ster- ization are most frequently used to sterilize medicines. Heat sterilization is typically preferred (1); however, successful processing isn’t merely achieving a sterility assurance level ‘of 10, Sterilization should not alter or affect the product, which could reduce its efficacy and even harm patients (2). Product changes can be signaled by changes in color or the degradation of ingredients within the formulation, In addition, radiation can also result in new compounds whose potentially toxicological impacts must be evaluated. subite: y 82072 Radiation, a physical process that does not involve heat- ‘Accepted: Feb, 8, 2018, ing the sample, is often used to sterilize pharmaceuticals. 40° PhamacetialTcboogy sont 2018 Poi Tect.com ‘TRAMAWSHUTTERSTOCK-COMPEER REVIEW: IMPACT OF STERILIZATION Reference Table: Performed sterilization tests (kGy is kiloGrays of radiation), Hot team alt Non stele x x x x ‘col2 ‘Non ete x x re x ‘cals ‘Non toile x x x x ‘cola ‘Non stole x x x x In the past, a minimum dose of 25 kGy was routinely used to sterilize many pharmaceutical products and biological tissues. Today, however, dose is determined by the product and the specific type of contaminant, as recommended by the International Organization for Standardization (ISO) @). In addition to evalusting sterilization’ possible impact on products, its effects on packaging and component materials rust also be considered. Each polymer and chemical sub- stance reacts differently to radiation, so itis important to verify that the dose being used does not affect product qual- ity, safety, or efficiency throughout its intended shelf life (4). The authors performed research to evaluate the effects of certain sterilization methods on different lubricating gels with similar compositions. The lubricants were all based on hydroxyethyl cellulose, and commercially available medi- cal gels were also evaluated for comparison. Table lists the sterilization methods that were evaluated. ‘This work did not consider the efficiency of the ster tion methods themselves, but, rather, focused on demonstrat- ing whether and which physical and chemical changes oc- curred due to diferent methods of sterilization and radiation doses. Results are summarized in Table l. Research centered on changes in rheology, viscosity color, and odor, as wel as the formation of volatile decomposition products, and measured the stability of the active ingredi- ents Lidocaine and Chlorhexidine contained in most of the formulations that were evaluated. Materials and methods Experimental procedure. Research involved performing the following procedures: + Sterilization of the samples—gamma, e-beam, steam, de- tect the influence of different sterilization methods, and to include any secondary reactions that might have oc- ‘curred because of this influence, the gel samples were stored at room temperature for one month after they had been sterilized. The goal was to achieve a decay of chemical degradation processes that occurred as. result of radiation Determination of the degradation behavior of Lidocaine and Chlorhexidine using high-performance liquid chromatography (HPLO methods. PLGUVAsibleanalysis. An HPLC system (Thermo Fisher) ‘and a variable wavelength detector were used to evaluate the samples. Separation was achieved on a 2.6-4m C18 100A, 150 x 4.6 mm, OOF-4462-E0 analytical column 42 Parmacutalecalogy sera. zor Pa (Kinetex, Thermo Fisher). The mobile phase was 0.1 % irifluor acetic acid in 20% acetonitrile (ACN) in 80% ‘water for the first 2 min, Then, the concentration of ACN, ‘was gradually increased to 90% ACN and 10% water at 20 min. Each analysis was run for 25 min. The flow rate was 1.2 mL/min., and injection volume was 10 i. ‘The detection wavelength was 254 nm, Before analysis, samples were diluted 1:10 in 0.1% trifluoro acetic acid im 20% ACN to 80% water. + Determination of volatile and semi-volatile substances by gas chromatography/mass spectroscopy (GUMS). Method 1: Headspace GC/MS, A GC system (7890A GC, Agilent Technologies), coupled to a mass selective detector (5975C inert XL MSD, Agilent Technologies), was used to perform headspace HS-GCIMS analysis. A DB-624 capillary column was used, with a capillary column of 320 im x 18 um and helium as the carrier base. Analysis was run at 250 °C. Initially, the oven temperature was kept at 40°C for 2 min and then increased by 5 °C/min. to 220 °C and held at con stant temperature for 2 min, The injection mode used was split, with ratio 20:1 of sample entering the column. The National Institute of Standards and Technology (NIST) li brary was used to estimate structures. An external calibra- tion was used to quantify results, so concentrations are given as toluene-equivalents, ‘Method 2: Gas chromatography/Flame ionization detectorimass spectrometry (GC/FIDINS). GC/FID/MS analysis, using liquid in- jection, was performed using a GC system (7890A GC, Agilent “Technologies), coupled toa mass selective detector (6975C inert XLMSD with a ZW-WAX Plas, Agilent Technologies) at 250 °C. A30-mx250-ym x0.5-pm capillary column was used, with, helium as the carrier gas. For GG, the injector temperature was held at 270°C, The Coven temperature was held at 50°C for 2 min, then increased gradually by 10 °Cimin to 240 °C, and held constant at thi temperature for 39 min, The injection mode used was split- less. Compounds were identified via NIST library and quan- tification was done by an external calibration, so concentra- tions are given as toluene equivalents Analysis of the rheological behavior. Sample rheology was analyzed by a universal rheometer (MCR 500, Anton-Paar Physica) via a rotating plate system in rotation at 23°C. A shear rate in the range of | to 1000 1/s was used as a mea- suring target. Dynamic viscosity vs. shear rate was used to study the samples at different conditions.PEER REVIEW: IMPACT OF STERILIZATION Table I Degradation of Lidocaine and Chlorhexidine in first sample (KGy is kiloGrays of radiation), Br i 2546) Udocdne | 20000 ‘9120 20703 974 902 21197 ‘9787 ehiomeian | 500 mara naeen ees aa | |e feet een ‘mass spectrometry (kGy is kloGrays ofr “(ald sanegoae eon crs eee ra lI HUT ee Comics te HIE (Mh. HE bien inning Photometrcmeasurement of color formation. A multi-label plate reader (Victor 3 -1420-011, Perkin Elmer) was used to determine absorbance at 405 nim for all of the samples, that were tested. Results and discussion Determination ofthe degradation behavior of Lidocaine and Chlorhexidine by HPLC. An increase of radiation dose was shown, to decrease the amount of active ingredients Lidocaine and Chlorhexidine in all samples that contained either one or both actives. The decrease in levels of Chlorhexidine was particularly evident, whereas Lidocaine seemed to be more resistant against the effects of radiation, 44 PammacutelTecbalegy sere zo Phar While the concentrations of active ingredients decreased, the sum of drug-related degradation products increased at hhigher radiation doses, although the increase in levels ofthe gradation products investigated was not ashigh asthe loss of Chlorhexidine and Lidocaine. Steam sterilization caused zo significant degradation of Lidocaine or Chlorhexidine in this study. Comparing the results of testing samples 1-3 using steam. sterilization and radiation showed that products were only ‘marginally affected by steam sterilization, while radiation induced significant changes. After gamma sterilization, for example, Lidocaine was found to degrade by up to 10% at 25 kGy, while Chlorhexi ine was subject to a strong degradation of up to 77% at 25 kGy, Similar results were found for e-beam sterilization, with active ingredient loss proportional to radiation intensity, Using a dose rate of25 kGy, Chlorhexidine loss was over 55%. ‘At 5 kGy, loss ranged between 7% and 33%. During radiation, degradation products of Lidocaine and Chlorhexidine arise, which do not correspond to the deg- radation products discussed in the European Union GMP ‘Annex (1) because the mass balance of active ingredient and known impurities is not complete. ‘Asa result, most decomposition products are not detect- able under the conditions specified by HPLC -UV/VIS and, therefore, will not be visible in the mass balance, For all samples evahiated, an increase in radiation dose resulted in an increase in the sum and concentration of volatile organic ‘compounds detected. This is due to chemical degradation. Forthe steam-sterilized samples, no increases or only small increases in the sum of volatile organic compounds were seen, compared to levels in unsterilized products. Atan irradiation intensity of 5 kGy, however, an increase in number as well as the concentration of volatile compounds was clearly seen in all gel samples. This increase was much higher after exposure toa 25-kGy dose, compared to the original samples. Using GC/MS to determine levels. of volatiles and semi-volatiles ‘Using GC/FIDIMS, depending on the radiation intensity and the sterilization method, only slight differences in the investigated gel patterns were found. Concentration of I-hydroxypropan-2-one and 3-methoxypropane-1,2-diol increased with higher radiation dose of e-beam treatment. 3-methoxypropane-1,2-diol could not be identified in the non-sterile sample. The substance shows nearly the same concentrations in gamma radiated and steam sterilized samples, independent of radiation dose.PEER REVIEW: IMPACT OF STERILIZATION Impact of e-beam sterilization ‘The concentration after e-beam sterilization was three times higher, while a slight increase was detected for the higher radiation dose, An increase of I-hydroxypropan-2 cone was detected with higher radiation dose, The influence is more significant for the gamma-radiated samples, than for the samples treated by e-beam, Steam sterilization did not affect the concentration of I-hydroxypropan-2-one (see Figure). ‘Analysis of degradation products (hydroxyethyl cellu Jose and glycerol) after radiation showed an increase in the number oflow molecular weight oxygenated substances, also depending on radiation dose. All compounds were detected in ppm range, but generation of these substances appeared to increase in samples with higher concentrations of API at higher doses of radiation. In particular, short-chain alcohols, aldehydes, and ketones were detected, each with a characteris ticodor. Odor deviation was especialy perceptible after gamma sterilization, Analysis of the rheological behavior ‘The viscosity of all lubricants decreases significantly at higher radiation doses. This change was particularly evi- dent by comparing samples 1-4 after gamma irradiation at 5, 10 and 25 kGy, with the non-sterile samples. The decrease in viscosity was less pronounced after e-beam radiation, Regardless of the active ingredient contained in the gels, the rheology of steam-sterilized samples 1-4 barely differed from that of untreated gels. After gamma sterilization, however, sigificant decreases in viscosity were seen in al gels (1-4) as the radiation dose increased (see Figure2). This correlates with the increase of the detected degradation products of hydroxyethyl cellulose, suggest- ing degradation of the polymer chain Length of the poly- ‘meric chains afer degradation was not explicitly measured, however 48 Pharmscuatealehalogy au. 208 Pha Vooh.c0 Photometric measurement of color formation Depending on the active ingredients present in the gel, there were differences in the color intensity of the irradiated sam- ples, Discoloration has already been detected after radiation ‘with 5 KGy. Gel matrices containing Chlorhexidine show a greater degree of yellowing than the samples containing only Lidocaine. In contrast, radiation with 25 kGy causes no dis coloration without the addition of Lidocaine and Chlothexi- dine (see Figure3) Conclusion ‘According to EN ISO 11137-2:2015 @), in the case of a bio- burden of 0.1, a sterilization dose of 11 kGy is necessary to ensure a sterility confidence level of 10° colony form- ing units (CEU) (8), Ifa radiation dose of 25 kGy is used to sterilize the medicinal gels examined in this study, it must be stated that gamma sterilization leads toa massive chemi- cal change, in particular in the concentration of the active ingredients. In some cases, itcan also lead to odor, as well as significant physical changes, especially in terms of viscos- ity and color. The effects are already detectable at dosage levels of 5 kGy. ‘Gor medicinal lubricating ges that are based on hydroxy- ethyl cellulose, steam sterilization is clearly a better option if the final product is to remain intact?This sterilization ‘method has been shown to result in relatively few degrada- tion reactions. References 1, European Commission (EC), Eudratex, Current Good Manufac- turing Practices (GMP) Annex: Manufacture of Sterile Medicinal Products-Revsion November 2008, EC-Enterprise and Industry Directorate General-Consumer Goods-Pharmaceuticals 2, A. Hammad, Trends in Radiation Sterilication of Health Care Products Chapter 8.1. Page 119 (National Cente fr Radiation Research and Technology [NCRRT], International Atomic Energy Agency, 2008) 3, 180, "EN ISO 111372, Sterlization of Heatheare Products-Radi- stion-Part 2: Establishing the Sterilization Dose" November 201, 4. EMA Guideline 3AQa, The Use of lonizing Radiation inthe Man- facture of Medicinal Products, 1982 (Legislative basi Directive 75/318/BEC), gmp-complianceorg, www.emaca.curopact, WWW mp-complianceorg/guidemge/ies3AQUAEN PDF 5. Stcrigencs, Oak Brook, ILwnensterigenics.com, Guidelines for Vl dation Radiation terization, Press Release, 2015, wewsterigenic comiservices/medical_steriizationcontrat. sterilization! guide lines for validation_radation sterilization pdf PT Nicole Steiner-Reischdtz is coordinator; Michael Pyerin is, hhead of pharma, medical devices, and hygiene; Chrysoula Kanakaki is GC chromatography analyst; Daniela Noubort Is liquid chromatography analyst; Michael Washiit! is hhead of packaging; Michael Krainz fs project manager for packaging: all at OFI Technologle & Innovation GmbH in Vienna,