Ambicin

While ethylene oxide tops headlines for its potential health hazards and EPA implications, there has been increasingly louder murmurs about the use of supercritical carbon dioxide (scCO 2 ) as an ecological alternative for medical device sterilization. To learn more about scCO 2 , MD+DI sat down with Tony Eisenhut, CEO and co-founder of NovaSterilis, to understand its sterilization properties, sterility assurance levels, and potential barriers to adoption. MD+DI: What is supercritical carbon dioxide? What is it currently most used for in medtech? Eisenhut: scCO 2 is a fluid in which carbon dioxide has liquid (density) and gas (diffusivity) like properties.  The supercritical state is achieved when the temperature and pressure are above its critical points (~31C and 1,070 psi or 73 atm). scCO 2 has been broadly used in the medtech space for decades. Early uses focused on extraction of natural compounds for medicinal purposes such as the extraction of paclitaxel (Taxol) from the bark of yew trees. More recently the extraction of cannabinoids from cannabis has become a large market. In addition to extraction, scCO 2 is being used for material cleaning and the terminal sterilization of medical devices including both synthetic and biological based devices. Emerging areas in the medtech space (regenerative medicine applications) is the use in decellularization of human and animal tissues without the use of harsh chemicals such as TritonX (use has been reduced and/or eliminated in some countries). Additionally, scCO 2 is used for manufacturing of products whether it be via impregnation of bioactives into scaffolds or the creation of scaffolds themselves as aerogels. The uses of scCO 2 are growing rapidly in the medical device, regenerative medicine, and therapeutics industries. MD+DI: How does it sterilize medical devices and products? The biggest concern manufacturers have switching from EtO to something else is that EtO is said to be the most effective sterilizer that can be used without damaging a product, so how well does scCO 2 work compared to that of EtO? Eisenhut: I would assert that all sterilization modalities impact the materials they are sterilizing. What we are all working to achieve is minimization of the impacts of sterilization such that the sterilized materials still function as intended. Although EtO is broadly compatible, it still has compatibility limitations, especially with bio-based materials. We have been working with scCO 2 as a sterilant for over 20 years at this point and we have shown the ability to terminally sterilize a broad array of materials and devices. Our scCO 2 sterilization platform has been used commercially in the tissue banking industry since 2008. There have been well over 300,000 devices implanted in humans over that period without any reported adverse reactions (relative to sterilization). When comparing scCO 2 to EtO there are a number of benefits. CO 2 is an inert, non-reactive molecule. In and of itself, it is advantaged over EtO from a reactivity standpoint. That said, to successfully sterilize with scCO 2 , you need to include an entrainer (we use a peracetic acid based additive). There is a synergistic effect of between the scCO 2 and the peracetic acid so you can use minimal amounts to achieve a sterility assurance level of 10^-6. Secondly, the conditions under which scCO 2 is created results in deep penetration of the sterilant in the device even through torturous packaging configurations. scCO 2 is the most effective sterilization modality when it comes to penetration. Similar to EtO, scCO 2 is a low temperature sterilization modality (operating temperature of 35C). Unlike EtO, scCO 2 does not require an outgassing period of tens of hours (no outgassing time is required for scCO 2 ). In 2019 the FDA created an innovation challenge grant to identify and support alternatives to EtO.  NovaSterilis was selected (along with Stryker's TSO3, Noxilizer, and Steris' vaporized hydrogen peroxide).  NovaSterilis continues to work with the FDA to broaden the use of scCO 2 as a terminal sterilization modality. There were multiple FDA clearances in 2022 that used scCO 2 as a cleaning and/or terminal sterilization process in the manufactured devices. The cleared applications range from terminal sterilization of therapeutics to additive manufactured products to tissue derived products. MD+DI: There has been talk that scCO 2 could be an ecological replacement for EtO, what are your thoughts? Eisenhut: Although I am biased, I believe this to be a true statement. NovaSterilis won the Presidential Green Chemistry Small Business award in 2007 based on the use of CO 2 as an alternative to EtO. I do not think there is a "silver bullet" for the replacement of EtO. I think there will be multiple sterilization modalities that will be viable alternatives based on the needs of the products being sterilized. I actually think that for the foreseeable future, EtO will be a part of the solution as well, albeit a smaller percentage as the regulatory pressures grow and the costs continue to rise. I do believe that the alternative sterilization solutions, including scCO 2 are more economical than EtO when you look at the total cost of processing.  NovaSterilis has shown the utility of scCO 2 as a terminal sterilization solution. To date, most of the applications are mid to low volume applications because of the equipment limitations for the medtech applications. Although scale is a perceived problem, it is not a true problem. scCO 2 is used in some very large industrial applications including extraction applications of coffee and hops. We are aware of an industrial installation with 60,000L of scCO 2 capacity. The infrastructure used for extraction is the same as that used for sterilization. The difference is related to the process not the equipment. This is a long-winded way to say that the ability to scale scCO 2 sterilization to volumes processed with EtO is not a technology or engineering hurdle, it is a finance hurdle. There needs to be a forward-thinking company, with the resources that will make the investment. MD+DI: If yes, what are the biggest barriers to implementing scCO 2 in place of EtO? Knowledge of it as an alternative? Regulatory timeline issues? Cost of changing sterilization practices? Eisenhut: The primary barrier is economic investment in the scaled-up infrastructure. The second issue is awareness of the alternative.  The regulatory timeline is not any longer than other sterilization modalities including EtO. We had multiple partners receive clearance in 2022 and the timelines were what one would normally expect. One of the barriers is professional risk. The willingness to try a "newer" — I put it in quotes because scCO 2 has been used in the food industry for ~50 years — sterilization modality is tempered by decision makers fear of what it means for their carrier should it fail, even if the risk of failure is no greater than the other sterilization solutions. MD+DI: How is the process of sterilization changed when using CO2, or CO2 with additives like trifluoroacetic acid, peracetic acid, hydrogen peroxide, nisin, and NovaKill? Eisenhut: CO 2 alone will not allow you to achieve a sterility assurance level 10^-6.  Bacterial endospores are not able to be fully inactivated with CO 2 alone. An entrainer (additive) is necessary to achieve terminal sterilization. We have explored the use of several additives. Most of the oxidative agents (trifluoroacetic acid, H2O2, and peracetic acid) can achieve success. NovaKill is our proprietary formulation, but it is a peracetic acid-based solution as well. MD+DI: As I am new to the conversation surrounding scCO 2 , please highlight anything that you think our readers would be interested to know or that is a common misconception of using the sterilization method. Eisenhut: It is often believed that it is only cost effective on high value, low volume products. That is not true. The cost of operation is lower than that of EtO and often radiation when you evaluate the total cost of sterilization.

Researchers have developed modified alginate hydrogels that can endure the growth of bacteria, allowing them to synthesize important enzymes. Encapsulating cells -- both prokaryotic and eukaryotic -- allows researchers to carry out experiments in hydrated environments over prolonged periods of time. However, cell growth under these conditions can exert a lot of pressure on the encapsulating shells, resulting in cell leakage. In a new study, researchers at the University of Illinois Urbana-Champaign have developed modified alginate hydrogels that can endure the growth of bacteria, allowing them to synthesize important enzymes. Hydrogels are polymers that are reinforced by different chemical bonds, and are capable of absorbing water and swelling without breaking down. As a result, biotechnology researchers have often turned to these structures to provide stability and structural support for their cell cultures. "Hydrogel capsules have been utilized for over 50 years. There are many different types that can be made by combining different kinds of cells in different hydrogel environments," said Yoon Jeong, a graduate student in the Irudayaraj (CGD/EIRH) lab. "The problem with combining microorganisms with hydrogel capsules is that they leak out." To address this problem, Jeong decided to focus on alginate, a naturally occurring, edible compound found in brown algae. Although it has been previously looked at, using it to encapsulate bacteria has been challenging. "My strategy was to make a hydrogel membrane on the surface of the hydrogel structure," Jeong said. Although the change may seem small, it works well. Jeong tested his system with genetically-modified Lactococcus lactis and saw that without the layer, the bacteria leaked out and were unable to form biofilms -- a collection of microorganisms that stick to each other. On the other hand, L. lactis colonies inside the modified hydrogels were able to grow for over 10 days; the hydrogels provided a stable platform that did not rupture. Jeong also looked genetically-modified Escherichia coli, which can synthesize a host of different molecules, only when they are able to achieve a high cell density. He looked at E. coli cells that can produce green fluorescent protein, which emits a green signal when the cells are subjected to ultraviolet light. "Although growing GFP-producing E. coli is simple, they quickly die," Jeong said. "I showed that inside the hydrogel they form colonies, which continuously increase in size, produce GFP, and do not leak out." He achieved the same results when he used E. coli cells that are bioluminescent. These bacteria encode the lux genes that result in blue cells that glow in the dark. The researchers saw that once the bacteria reached a certain cell density, the luminescence continued to increase for the next 3 days. The main purpose of making these hydrogels is to develop bioreactors that can support the growth of bacteria while they make important compounds. To test whether the modified hydrogels were capable of sustaining such processes, Jeong also tested the ability of L. lactis to make nisin, a peptide that is used as a food preservative. In agreement with their previous results, the bacteria were able to grow well in the modified hydrogels and were able to produce the compound. "Although at a glance it can seem simple to make these hydrogel structures, it is actually difficult. You have to control their size, thickness, and prevent agglomeration since these capsules stick together," Jeong said. "Researchers who have a different scientific background have found this process difficult. We plan to publish a detailed protocol soon so that people can use this cheap and easy technique." The researchers are also interested in continuing their tests in human and cancer cells, with the hope that the hydrogels will be able to provide a reliable platform for a wide range of applications. The work was partially funded by the National Institute of Biomedical Imaging and Bioengineering of the NIH.