Michael Krainz is project manager for packaging at OFI Technologie & Innovation GmbH in Vienna.

Michael Krainz

Recent studies evaluated the effects that different sterilization methods have on the active ingredients present in four hydroxyethyl cellulose-based gels. Physical and chemical properties such as rheology, color formation, 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 contained two APIs, Lidocaine and Chlorhexidine; two gels contained only one of each of these APIs; while the fourth gel did not contain any active ingredients.

	Samples were sterilized using hot steam, gamma radiation, 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 additional gamma sterilization step, carried out at 5 and 25 kGy.

	Sterile medicinal products can be sterilized in two ways, either via terminal methods (i.e., on the product in its final container) or via aseptic processing (1). Whichever sterilization approach is used, the process must be validated in order to verify that it effectively and reliably kills any microorganisms that might be present in the product (2).

	Heat, radiation, gamma or electron beam, and steam sterilization 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.

	Radiation, a physical process that does not involve heating the sample, is often used to sterilize pharmaceuticals. 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) (3).

	In addition to evaluating sterilization’s possible impact on products, its effects on packaging and component materials must also be considered. Each polymer and chemical substance reacts differently to radiation, so it is important to verify that the dose being used does not affect product quality, 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 medical gels were also evaluated for comparison. 

 lists the sterilization methods that were evaluated. 

	This work did not consider the efficiency of the sterilization methods themselves, but, rather, focused on demonstrating whether and which physical and chemical changes occurred due to different methods of sterilization and radiation doses. Results are summarized in 

	Research centered on changes in rheology, viscosity, color, and odor, as well as the formation of volatile decomposition products, and measured the stability of the active ingredients Lidocaine and Chlorhexidine contained in most of the formulations that were evaluated.
	 

 Research involved performing the following procedures:

Sterilization of the samples--gamma, e-beam, steam.

To detect the influence of different sterilization methods, and to include any secondary reactions that might have occurred 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 a result of radiation.

Determination of the degradation behavior of Lidocaine and Chlorhexidine using high-performance liquid chromatography (HPLC) methods.

 An HPLC system (Thermo Fisher) and a variable wavelength detector were used to evaluate the samples. Separation was achieved on a 2.6-µm C18 100A, 150 x 4.6 mm, 00F-4462-E0 analytical column (Kinetex, Thermo Fisher). The mobile phase was 0.1 % trifluor 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 µl. The detection wavelength was 254 nm. Before analysis, samples were diluted 1:10 in 0.1% trifluoro acetic acid in 20% ACN to 80% water.

Determination of volatile and semi-volatile substances by gas chromatography/mass spectroscopy (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-GC/MS analysis. A DB-624 capillary column was used, with a capillary column of 320 µm x 1.8 µm 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 constant 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) library was used to estimate structures. An external calibration was used to quantify results, so concentrations are given as toluene-equivalents.

Method 2: Gas chromatography/Flame ionization detector/mass spectrometry (GC/FID/MS). 

GC/FID/MS analysis, using liquid injection, was performed using a GC system (7890A GC, Agilent Technologies), coupled to a mass selective detector (5975C inert XL MSD with a ZW-WAX Plus, Agilent Technologies) at 250 °C. A 30-m x 250-µm x 0.5-µm capillary column was used, with helium as the carrier gas. 

	For GC, the injector temperature was held at 270 °C. The oven temperature was held at 50 °C for 2 min, then increased gradually by 10 °C/min to 240 °C, and held constant at this temperature for 39 min.  The injection mode used was splitless. Compounds were identified via NIST library and quantification was done by an external calibration, so concentrations are given as toluene equivalents.
	

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 1 to 1000 1/s was used as a measuring target. Dynamic viscosity vs. shear rate was used to study the samples at different conditions.

Photometric measurement of color formation. 

A multi-label plate reader (Victor 3 -1420-011, Perkin Elmer) was used to determine absorbance at 405 nm for all of the samples that were tested.  

Determination of the 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. 

	While the concentrations of active ingredients decreased, the sum of drug-related degradation products increased at higher radiation doses, although the increase in levels of the degradation products investigated was not as high as the loss of Chlorhexidine and Lidocaine.  Steam sterilization caused no 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 Chlorhexidine 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 of 25 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 degradation products discussed in the European Union GMP Annex (1) because the mass balance of active ingredient and known impurities is not complete. 

	As a result, most decomposition products are not detectable under the conditions specified by HPLC -UV/VIS and, therefore, will not be visible in the mass balance. For all samples evaluated, 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.

	For the steam-sterilized samples, no increases or only small increases in the sum of volatile organic compounds were seen, compared to levels in unsterilized products. At an 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 to a 25-kGy dose, compared to the original samples.

	Using GC/MS to determine levels of volatiles and semi-volatiles 

	Using GC/FID/MS, depending on the radiation intensity and the sterilization method, only slight differences in the investigated gel patterns were found. Concentration of 1-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.

	The concentration after e-beam sterilization was three times higher, while a slight increase was detected for the higher radiation dose. An increase of 1-hydroxypropan-2-one 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 1-hydroxypropan-2-one (see 

	Analysis of degradation products (hydroxyethyl cellulose and glycerol) after radiation showed an increase in the number of low 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 characteristic odor. Odor deviation was especially perceptible after gamma sterilization.

	The viscosity of all lubricants decreases significantly at higher radiation doses. This change was particularly evident 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 all gels (1-4) as the radiation dose increased (see 

). This correlates with the increase of the detected degradation products of hydroxyethyl cellulose, suggesting degradation of the polymer chain. Length of the polymeric chains after degradation was not explicitly measured, however.
	

	Photometric measurement of color formation

	Depending on the active ingredients present in the gel, there were differences in the color intensity of the irradiated samples. 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 discoloration without the addition of Lidocaine and Chlorhexidine (see 

	According to EN ISO 11137-2:2015 (3), in the case of a bioburden of 0.1, a sterilization dose of 11 kGy is necessary to ensure a sterility confidence level of 10-6 colony forming units (CFU) (5). If a radiation dose of 25 kGy is used to sterilize the medicinal gels examined in this study, it must be stated that gamma sterilization leads to a massive chemical change, in particular in the concentration of the active ingredients. In some cases, it can also lead to odor, as well as significant physical changes, especially in terms of viscosity and color. The effects are already detectable at dosage levels of 5 kGy. 

	For medicinal lubricating gels that are based on hydroxyethyl 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 degradation reactions.

EudraLex, Current Good Manufacturing Practices (cGMPs) Annex 1: Manufacture of Sterile Medicinal Products-Revision November 2008

, EC-Enterprise and Industry Directorate General-Consumer Goods-Pharmaceuticals.
	2. A. Hammad, 

Trends in Radiation Sterilization of Health Care Products

; Chapter 8.1. Page 119 (National Center for Radiation Research and Technology [NCRRT], International Atomic Energy Agency, 2008).
	3. ISO, “EN ISO 11137-2, Sterilization of Healthcare Products - Radiation-Part 2: Establishing the Sterilization Dose,” November 2015.
	4. EMA 

The Use of Ionizing Radiation in the Manufacture of Medicinal Products

(Legislative basis Directive 75/318/EEC), 

Guidelines for Validation Radiation Sterilization


	Vol. 42, No. 4
	April 2018
	Pages: 40–46

	When referring to this article, please cite it as N. Steiner-Reischütz, 

, “Evaluating the Impact of Sterilization on Gel Formulations,” 

is head of pharma, medical devices, and hygiene; 

is project manager for packaging; all at OFI Technologie & Innovation GmbH in Vienna.

Articles

Research suggests that radiation can have a significant impact on the composition and rheology of hydroxyethyl cellulose-based medicinal gels.