Sterilization of Polymeric Implants: Challenges and Opportunities

Abstract

Degradable and environmentally responsive polymers have been actively developed for drug delivery and regenerative medicine applications, yet inadequate consideration of their compatibility with terminal sterilization presents notable barriers to clinical translation. This review discusses industry-established terminal sterilization methods and aseptic processing and contrasts them with innovative approaches aimed at preserving the integrity of polymeric implants. Regulatory guidelines, fiscal considerations, and potential pitfalls are discussed to encourage early integration of sterility regulatory considerations in material designs.

Graphical Abstract

1. INTRODUCTION

Polymer science has been an avenue of healthcare innovation for the past half century¹. Common applications include drug delivery,^2–5 orthopedic prostheses,^2,3,6,7 scaffold-assisted tissue regeneration,^8–11 cardiac implants,^2,12 and many more.^{2,3,8,9,13–15} A notable trend in recent decades is that polymeric implants have evolved from predominantly bioinert non-degradable materials intended for long-term structural support to biodegradable polymers for drug delivery and environmentally responsive polymers (e.g., shape memory polymers,^9,16–18 enzyme-cleavable hydrogels¹⁹) intended for scaffold assisted tissue regeneration. For safe in vivo uses, the sterility of all polymeric implants needs to be ensured. The US Food and Drug Administration (FDA) acknowledged four main categories of sterilization methods applicable to polymers: Class A, Class B, novel, and aseptic approaches.^2,20,21 Aseptic processing relies on the combination of sterile components in a controlled environment, while Class A, B, and novel methods utilize terminal sterilization such that the sterilization modality is applied to the completed product (e.g., in its final packaging).²¹ To achieve the necessary low bioburden, however, the minimally effective terminal sterilization conditions employed by different methods impose varying levels of risk for causing unintended changes in chemical, structural, and mechanical properties of the polymeric product of interest.

Taking orthopedic implants as an example, earlier clinical applications of polymers were dominated by non-degradable, weight-bearing implants. Ultra-high molecular weight polyethylene (UHMWPE) has been and continues to be a critical structural component of total joint arthroplasty prostheses.^22,23 Polyetheretherketone (PEEK) cages have also been commonly used in the treatment of spinal degeneration, instability, pseudoarthrosis, deformation, and other conditions that benefit from interbody fusion.^24–26 What makes UHMWPE and PEEK attractive for their respective applications are their chemical stability (resistance to in vivo degradation), biomechanical integrity (e.g., wear resistance, high strength), and biocompatibility.^22,23 However, decades of research on UHMWPE wear and biomechanical changes following sterilizations by gamma irradiation and ethylene oxide (EtO) gas sterilization demonstrate that clinically relevant deleterious changes may ensue. For instance, studies reported increased wear and premature failure of implants associated with sterilizations that induce high levels of oxidative damage due to the cleavage of C-C bonds and the resulting free radicals forming peroxide species with oxygen.^22,27–29 Accordingly, avoiding excessive high-energy irradiation and carrying out sterilization and subsequent packaging in an environment that mitigates oxidation (e.g., under vacuum or inert gas protection, or with the addition of free radical scavengers) have been explored with varying successes.^28,30–32 However, high-energy radiation even in an inert environment would still lead to oxidation due to subsequent diffusion of oxygen from the in vivo environment into the implant reacting with residual radicals.^31,32

Compared to non-degradable bioinert polymers, degradable polymers are even more sensitive to cleavage under high-energy sterilizations (e.g., polar ester linkages in polylactides are easier to cleave than C-C bonds). Reduced molecular weights due to such cleavage would significantly impact their degradation profiles,^33,34 thereby altering in vivo efficacies of their intended applications from drug release kinetics to polymeric scaffold resorption and neotissue integration. Furthermore, the preservation of dimensional integrity and structural features of porous polymeric scaffolds (e.g., porosity, pore geometry), fabricated by rapid prototyping or 3D printing, for instance, may present additional challenges compared to dense polymers using conventional sterilization modalities. Lastly, stimuli-responsive polymers designed to undergo conformational or dimensional changes in response to temperature, moisture, or pressure perturbations may experience unintended irreversible transformations during terminal sterilization.³³ For example, a macroporous biodegradable shape memory polymer (poly(lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(lactic-co-glycolic acid), or PELGA) and hydroxyapatite (HA, 25 wt%) composite-based synthetic bone graft substitutes have been designed and fabricated to precisely fit within a rat femoral segmental bone defect (Fig. 1A).⁹ Said 3D printed HA-PELGA grafts were readily deformed into a minimally invasive temporary shape to facilitate surgical placement within the defect, and subsequently triggered by a safe temperature to recover to a preprogrammed shape that stably fit within the defect (Fig. 1B).⁹ The unique hydration induced stiffening property of the amphiphilic polymer component¹⁸ further improved the graft fixation within the defect (Fig. 1C). The combination of suitable scaffold porosity and degradability then facilitated osteointegration, resulting in functional regeneration of new bone and timely resorption of the synthetic bone graft substitute.^8,9 Any sterilization conditions that may alter the permanent shape, collapse scaffold porosity, or excessively accelerate polymer degradation of such degradable shape memory polymer scaffolds could potentially compromise their surgical delivery, fixation and in vivo performances. It should be noted that the 70% ethanol and UV irradiation utilized in the published experimental study for sterilizing such synthetic bone graft substitutes⁹ are not FDA established Class A or B sterilization methods.

Figure 1.

Preparation and facile surgical fitting of 3D macroporous 25% HA-PELGA grafts. (A) Computer assisted design illustration and photographs of a 3D macroporous HA-PELGA graft fabricated by coprinting a dense HA-PELGA/polyvinyl alcohol (PVA) composite square prism, coring, and subsequent removal of sacrificial PVA material. (B) Photograph of placement of a cylindrical-compressed HA-PELGA graft into a 5-mm rat femoral segmental defect and the graft fixation driven by shape recovery, swelling, and stiffening of the graft upon 37°C saline rinse. (C) Peak forces required to pull HA-PELGA(8/1) grafts, HA-PELGA(2/1) grafts, or collagen sponges (n = 3) from a customized specimen holder simulating a 5-mm rat femoral segmental defect upon hydration as measured by a mechanical test machine (MTS Bionix 370, MTS Systems Corporation). Hydrated collagen sponges were dislodged with negligible force (not detectable, <10 mN). All specimens were prepared in a cylindrical shape (diameter, 3 mm; length, 5 mm) and were hydrated in 37°C water for 1 min before the pull-out test. Data are presented as means ± SEM. *P < 0.05. Reproduced with permission from reference 9. Copyright © 2019, the American Association for the Advancement of Science.

To address the unique demands set forth by emerging functional polymers and tissue grafts used for regenerative medicine, alternative sterilization methods have been explored.^1,18,23 A few recent reviews have provided general overviews on terminal sterilization methods considered by the FDA,² specific methods,^33–42 or have commented on how Class A sterilization modalities may compromise thermosensitive shape memory polymers³³ and PLA-based implants,³⁴ respectively. With the goal to inspire early integration of sterility and regulatory consideration in materials innovation, this review discusses conventional FDA established terminal sterilization modalities along with some recent novel sterilization methods not acknowledged by the FDA, highlighting their pros and cons for sterilizing labile functional polymers (degradable hydrogels or thermal responsive shape memory polymers as examples) when appropriate, as well as associated regulatory hurdles (Table 1). Approaches aimed at mitigating the negative impact of free radicals or unintended degradation, two key challenges shared by a number of Class A, Class B, and emerging novel terminal sterilization methods, are spotlighted. It should be noted that regardless of the FDA sterilization method classifications, industry requirements are such that a sterility assurance level (SAL) of 10⁻⁶ (e.g. a probability of no more than 1 viable microorganism in a million counts of sterilized final product) is met for devices labeled as sterile and that regulatory guidelines referenced here are those relevant to class III medical devices (those implanted in the body to achieve an intended purpose) where the lack of sterility would pose the highest risk and most stringent regulations are thus warranted.^{2,20,34,35,43} The classification of the sterilization modality, however, dictates the degree to which companies or product developers bear the burden of proving compliance. Finally, although aseptic processing may always serve as an alternative to terminal sterilizations when materials of interest are incompatible with existing sterilization techniques, its implementation is governed by expansive regulations as highlighted in this review (Table 2) and could require significant capital.^21,36

Table 1:

Overview of Sterilization Methods under Different FDA Classifications, and their respective Suitability for Polymeric Scaffolds (see references^{2,21,22,28,34–38,42,61,73,75–78,87,89–91,101} for general operating conditions and benefits)

	Sterilization Modality	Typical Conditions	Benefits	Limitations	Contraindications
FDA Class A	Gamma irradiation (radiation energy)	15–45 kGy (ave ~25 kGy) 20–22°C	Low temperature Highly efficient penetration No toxic chemical residues	Crosslink cleavage/Chain scissions29,33,47,48 Decreased osteoinductivity of bone grafts38,102 Altered degradation profile33,50,51 (107)Change in thermal properties33,50 Altered Molecular Weights49,50 Altered packing crystallinity49 Altered scaffold porosity34,40,50 Compromised mechanical integrity29,50,51	Degradable polymers with labile linkages, porous scaffolds, tissue grafts
	E Beam irradiation (radiation energy)	25 kGy 20–22°C	Low temperature Effective penetration No toxic chemical residues	Crosslink cleavage/Chain scissions34,54 Decreased osteoinductivity of bone grafts8,13,39 Altered scaffold porosity41,50,52 Altered degradation profile34,53,54 Changes in mechanical integrity50,54 Changes in thermal properties50,54 Less effective penetration vs gamma irradiation46	Degradable polymers with labile linkages, porous scaffolds, tissue grafts
	EtO gas (chemical sterilant)	37–63°C 450–1200 mg/L gas concentration 40–80% humidity Exposure time: 1–6 h	Mild Temperature Good gaseous scaffold penetration	Toxic residues, chemical reactivity with amino, carboxyl, thiol, and hydroxyl groups33,62 Decreased osteoinductivity of bone grafts13 Impaired cell adherence64 Varying impacts on degradation64	Polymers chemically reactive with EtO, tissue grafts
	Dry & Steam autoclave (heat energy)	Dry: 160 °C Steam: 120 °C 103–117 kPa	No toxic chemical residues Not prone to induce chain scission or radical formation	Altered physical crosslinking33,55 Accelerated hydrolysis (steam autoclave) / Varied degradation profile2,33,55–57,101 Thermosensitive behavior changes33,55 Changes in structure53,58 Changes in mechanical integrity55	Thermosensitive polymers, hydrolytically degradable polymers (steam), porous scaffolds, protein-based scaffolds and tissue grafts
FDA Class B	Hydrogen Peroxide (chemical sterilant)	Vaporized: 25–50 °C Plasma: 40–65 °C Exposure time: 1–3 h	Minimal toxic residues Mild temperature	Polymer swelling72 Altered crosslinking2,103 Changes in mechanical properties34,71,72,103 Changes in degradation behavior34,71,72,103 Suboptimal penetration2,34,35,65,66	Polymers prone to oxidative damages, porous scaffolds, cellulose
	Ozone (chemical sterilant)	30–35 °C Exposure time: ~4.5 h	No toxic residue Low temperature Moderate penetration of porous structures	Changes in degradation rate74 Altered mechanical properties74 Morphological changes74 Surface chemistry alterations73	Polymers prone to oxidative damages
Selected Novel Methods	Super Critical CO2	~ 31.0 °C 7.3773 MPa/72.8 atm	Low temperature Low toxicity Minimally reactive Biomolecule/tissue property preservation High penetration	Swelling75,76 Plasticizing of polymers75,85 Additive toxicity76,82 Morphological changes104 Mechanical property changes76 Altered glass transition temperature75,85,86 High regulatory burden	Porous scaffolds or polymer chains prone to swelling in scCO2, polymers incompatible with additives
	pH Control in Gamma Irradiation Sterilization	Acidic pH protection of alkaline driven polymer degradation	Mitigate beta-eliminative degradation during steam autoclave	Niche application101	Thermosensitive or degradable polymers unstable in acidic / basic pH
	Antioxidants in Gamma Irradiation Sterilization	Vitamin E (Free radical scavenger) doped UHMWPE	Compatible with FDA Approved Sterilization In vitro mechanical property benefits	Niche application22,30,91 Modality dependent impact on crosslinking Inconsistent in vivo improvements over control UHMWPE88,94,95,97–100	Polymers do not blend well with Vitamin E
Aseptic	Aseptic Processing	Processing in such a manner that terminal sterilization is not necessary	Preservation of polymer composite structure and behavior profile	High regulatory burden Increased financial requirements	N/A

Table 2:

Outline of Aseptic Regulations Adapted from the FDA Guidance Document for Sterile Drug Products Produced by Aseptic Processing²¹

Category of Regulation	High Level Requirement	Representative Specifics
Air Quality	Critical area Supporting clean areas Clean area separation Air filtration Flow	Clean Air Classification: Class 100, 1000, 10,000, 100,000 Monitoring air quality Location of monitoring Contamination minimization Corrective action protocols Personnel training for monitoring
Personnel Compliance	Training, qualification, monitoring	Personnel must be trained on proper procedures and means of testing knowledge is required Physical entry, interaction, and attire geared towards minimizing contamination is required
Components & Containers	Component sterility Container or closure sterility	Monitoring and minimizing the risk of bioburden, microbial/endotoxin contamination on any component or container relating to the final product that may contaminate the sample
Endotoxin Control	Product endotoxin testing Container or closure sterility Equipment mitigation	Product endotoxin contamination must be tested and infrastructure for compliance must be established Containers or closures must be sterile as to not to introduce endotoxins to the product Equipment must be maintained in a specific manner and have the appropriate protocols to minimize the introduction of endotoxins to the environment in which the product is manufactured
Time Limitations	Establishment of time limitations per phase of manufacturing	Time limitations are driven by data pertaining to bioburden or endotoxin exposure Limitations are set to minimize exposure of products to microbial agents or pathogens
Validation	Process simulations Filtration efficacy Equipment	Evaluation of manufacturing process Evaluation of processing controls Evaluation of sterility via media runs Evaluation of filtration derived sterility Equipment qualification, validation, controls, calibration, and maintenance to ensure sterility and appropriate function
Laboratory Controls	Environmental Monitoring Microbial Identification Prefiltration bioburden Alternative microbiological test methods Particle monitoring	Laboratory controls are intended to help monitor, assess compliance, and prove efficacy of protocols Mitigation and alternatives at all phases during the process are necessary to ensure ultimate sterility
Sterility Testing	Microbiological Laboratory Controls Sampling and Incubation Investigation of Sterility Positives	Quality control testing of the products claimed as sterile is required as proof of batch efficacy Protocols for corrective actions are also necessary when contamination is discovered to identify other contaminated samples and the source
Batch Record Review: Process Control Documentation	Written procedures for documentation Process and laboratory data Environmental and personnel Interventions or non-compliant events	Review of documentation is necessary to evaluate the appropriate dictation and tracking of all components contributing to the production of a product This allows for tracking necessary for identifying contamination and any products produced from said contaminated batch

Google Scholar, National Center for Biotechnology Information, and ScienceDirect were searched for both original research and reviews on sterilization techniques. The search terms included degradable polymers, shape memory polymers, polymeric implants, medical device class III regulations, terminal sterilization methods, and novel sterilization methods. Original research on terminal sterilization techniques published after 1994 was prioritized. Additionally, FDA regulatory documents, International Organization for Standardization (ISO) regulations, and selective manufacturing white papers were consulted to corroborate relevant information and understand the contextual framework that guides current clinical translation.

2. FDA ESTABLISHED STERILIZATION METHODS

FDA-approved terminal sterilization methods implemented for most biomedical implants with successful premarket approvals (PMA) and Quality Safety inspections have long been studied for efficacy and safety.²⁰ Class A methods include gamma irradiation, electron-beam (E-beam) irradiation, ethylene oxide (EtO) gas, dry heat, and steam heat sterilization. Consensus standards published by the ISO or other third-party regulatory institutions facilitate industry standardization of Class A sterilization protocols and, if followed appropriately, confer compliance with FDA regulations. Class B methods include hydrogen peroxide (H₂O₂) gas plasma, ozone, and flexible bag methods utilizing EtO.^2,20 In contrast to Class A, no consensus standards exist for Class B methods, however, there are relevant development, validation, and control protocols published that provide guidance. To be considered Class B, adherence to sterilization parameters previously evaluated by the FDA must be maintained to avoid the “novel” classification. PMA applications involving Class B terminal sterilization methods thus require additional documentation of complete validation protocols and thorough descriptions of the sterilization methodology which expand beyond the breadth of Class A application requirements.²⁰

2.1. CLASS A TERMINAL STERILIZATIONS

2.1.1. Gamma Irradiation & E-Beam Irradiation.

Gamma and E-beam irradiations are two low-temperature, high-energy sterilization modalities that are known to have high penetration capability and achieve sterility through both the direct interaction on various cellular components and the induction of free radicals within the contaminating microorganism.^20,34,37,38 The free radicals inflict microbial DNA damage, thereby inhibiting microbial survival.^2,20 Both considered cold sterilizations, gamma and E-beam irradiations share many common features but differ slightly in mechanistic nuances.

Gamma irradiation commonly uses Cobalt 60 radiation, generated by the decay of the radioisotope, to kill microorganisms using the resultant high-energy photons as a sterilant. It is an industry staple used in a wide range of applications for which well-defined operating guidelines exist.² Industry standards are to use a 15–45 kGy (average dose of 25 kGy^2,38) irradiation energy at room temperature (20–22 °C)^37,38 to achieve the regulatory threshold of SAL 10⁻⁶.^{2,20,34,35,39,43} Chemically stable polymers, such as polyethylene (PE), polymethacrylic acid (PMMA), or polysulfides,^2,22 and relatively stable degradable polymer formulations containing polycaprolactone (PCL), polylactic acid (PLA), polyglycolytic acid (PGA), or poly(lactic-co-glycolic acid) (PLGA) have all been subjected to sterilization by gamma irradiation,² although pronounced impacts on some of these polymers, particularly in certain fabricated formats (e.g. fibrous meshes/mats, porous foams), have been reported.^44,45

Many of the polymer composites sterilized by gamma irradiation can also be sterilized with E-beam. E-beam method utilizes ionizing energy from electron beams as opposed to ionizing gamma ray photons to penetrate microbes and induce free radical formation.² Other similarities include their dosimetry practices, for which E-beam is also commonly delivered at an average irradiation dose of 25 kGy and at room temperature.³⁸ However, E-beam may demonstrate decreased penetrance as compared to gamma irradiation, especially for materials with higher densities.^38,46 It should be noted that all radiation-based dosimetry protocols that pertain to dose, time, and number of cycles are target material/product dependent.²

The benefits of these high-energy irradiation sterilizations are the readily available guidelines, minimal regulatory burdens associated with the Class A classification, high penetration, and high sterilization efficacy.^2,20,34 From a compliance perspective, regulations are such that the greater the deviation from standard dosimetry practices, the more complex and extensive the regulatory protocols and oversight may be. From a technical viewpoint, however, drawbacks of these irradiation methods can be extensive depending on the polymer in question. For example, the high energy from gamma rays is capable of inducing polymer chain scission (e.g. via C-C bond cleavage in even chemically stable polymers) and crosslinking (by radical recombination),^29,33,47,48 conferring changes in polymer molecular weight,^49,50 packing crystallinity,⁴⁹ mechanical integrity,^22,29,50,51 degradation rate,^33,50,51 porosity,^34,40,50 and thermosensitive behavior.^33,50 For instance, increased crosslinking of UHMWPE post-gamma irradiation could result in increased strength but decreased wear resistance,²² while the reduced molecular weight of polylactide could expedite degradation.³⁴ Marked reduction in osteoinductive properties has also been documented on allogenic bone grafts post-gamma irradiation sterilization,^8,13,39 likely due to damage to the chemical and structural integrity of osteoinductive/angiogenic protein factors and other naturally occurring biopolymers, resulting in poorer osteointegration of the grafts. Similar effects of E-beam sterilization on polymeric scaffold porosity,^41,50,52 degradation profiles,^34,53,54 mechanical integrity,^50,54 and thermosensitive behaviors have been reported.^50,54 Despite their utility in sterilizing some degradable polymers, the vulnerability of labile linkages and crosslinks such as ester bonds to high energy irradiation should always be considered and evaluated case by case. If these Class A sterilization methods are to be implemented, the physical, mechanical, and biological properties of the material post-sterilization should be fully characterized and reported. This, unfortunately, is not always the case with initial reports of novel biomaterials; substantial differences in functional properties with/without terminal sterilization could present hurdles to their clinical translation. Finally, given the well-known negative impacts of radicals resulting from high energy irradiation on material properties, methods for radical quenching/mitigation that have been actively explored with UHMWPE implants³⁰ (discussed in subsequent “novel methods” section) are also worthy of consideration for more labile polymers that are prone to chain scission.

2.1.2. Dry Heat & Steam Sterilization.

Heat-based methods of sterilization induce protein denaturation and coagulation, thereby inhibiting the survival of microbes.^2,20 Although optimal temperature, time, and containment conditions for heat sterilization notably vary based on product properties and microbial targets,² general guidelines have been set. Standard dry heat operating conditions are around 160 °C for 120 min.^2,34 Steam heat, operated in a pressurized environment (typically 103–117 kPa),⁴² can be carried out at a lower temperature of 120 °C to achieve the same sterilization efficacy.^2,34 A wide range of medical implants composed of non-degradable and degradable polymers (e.g., polyurethane, polypropylene (PP), PGA, polysulfone, and silicone) have been subjected to heat sterilization methods.²

Compared to high-energy irradiation methods, heat-sterilization is less likely to generate free radicals.² However, the high temperatures may disrupt physical crosslinks, melt crystalline domains, and cause undesired physical crosslinking upon cooling, thus presenting unique challenges for temperature-sensitive thermoplastic polymers including thermal responsive shape memory polymers.^33,55 The impact of heat sterilization could be catastrophic if critical transition temperatures (T_g, T_m) of the thermosensitive polymers fall within the autoclave temperature range. In addition, steam sterilization may also induce undesired hydrolysis of degradable polymers^2,33,55–57 or porosity changes of porous hydrogels (e.g., hyaluronic acid hydrogels) due to hydration and swelling.⁵⁸ Hydrogel-based coatings⁵⁹ or surface-grafted polymers prone to excessive swelling upon hydration⁶⁰ will also have to be carefully evaluated for their compatibility for steam autoclave sterilization. Finally, due to the ability of heat to denature proteins, heat sterilization is likely incompatible with protein-based synthetic hydrogels or native tissue scaffolds where protein conformation is critical to its function properties.

2.1.3. EtO Gas Sterilization.

EtO is an FDA Class A chemical sterilant that neutralizes microbial contamination by alkylating the nitrogenous backbone of an organism’s DNA.^2,20,33 The boiling point of EtO is 10.4 °C, which allows for a relatively low operating temperature (37–63°C) for EtO gas sterilization.^2,61 Effective EtO gas concentration and percent humidity range from 450–1200 mg/l and 40–80%, respectively. Under these conditions, exposure time varies depending on the product but is reported by the Centers for Disease Control and Prevention (CDC) to range from 1–6 h.⁶¹

The compatibility of EtO sterilization depends on the polymer chemistry and intended use of the product in question. In comparison to irradiation and heat-based methods, the milder operating conditions of EtO sterilization make it generally more compatible with degradable polymers so long as they are not chemically reactive with EtO.² The relatively low operating temperatures also make this method attractive for some heat sensitive polymers and composites.^33,34 Furthermore, gas permeability of EtO sterilization presents the added benefit of enhanced penetration of porous structures, which is particularly pertinent for many polymer-based tissue engineering scaffolds. Literature states that EtO sterilization has indeed been applied to PCL, PLGA, and polyethylene glycol (PEG) hydrogel-based products.²

As a chemical sterilant, however, there are a number of inherent drawbacks of EtO. For example, polymer composites containing nucleophilic functional groups, such as primary amine, carboxyl, thiol, or hydroxyl groups, could covalently react with EtO, resulting in irreversible changes in both chemical composition and possibly structural and mechanical properties of the product.^2,33,62 Like many other sterilization techniques, the impact of EtO on specific polymers is context dependent. For instance. Although statistically significant changes in mechanical properties were not observed in dense, injection-molded PLA and PLA/poly(butylene adipate-co-terephthalate) composites after sterilization with EtO,⁵³ EtO sterilization was shown to significantly alter the fiber orientation and morphology of electrospun PLA membranes.⁶³ Furthermore, residual EtO adsorbed on or trapped within the polymeric material is toxic and carcinogenic due to its ability to react with host proteins and chemically modify host DNA upon implantation.^2,33,53,62 Lastly, EtO sterilization has been shown to compromise the osteoinductivity of demineralized bone graft¹³ and impair cellular adhesion to the treated graft,⁶⁴ limiting its utility in tissue regeneration products containing bioactive protein factors.

2.2. CLASS B TERMINAL STERILIZATIONS

2.2.1. Hydrogen Peroxide Gas Plasma.

Hydrogen peroxide (H₂O₂) is a Class B chemical sterilant.^2,20 Its microbial killing mechanism involves the induction of reactive oxygen species (ROS) which can cause broad damages to the DNA, protein, and membrane components of the contaminating microbes.^2,35 H₂O₂ is typically applied as gas plasma or in a vaporized state at 40–65 °C and 25–50 °C, respectively, for 1–3 h.^2,35 It is more commonly used in surface cleaning than in manufacturing class III medical devices. Examples of its device applications are PMMA bone cements or prostheses composed of PMMA for which H₂O₂ gas plasma may be used as an alternative to EtO sterilization.^2,35,65

Benefits of H₂O₂ sterilization are similar to those of other chemical sterilants, including relatively low operating temperatures and better compatibility with heat sensitive polymers.^2,34 Compared to EtO gas, however, vaporized H₂O₂ has less effective penetration of porous scaffolds^{2,34,35,65,66} Furthermore, although deliberate H₂O₂ incorporation in polymeric scaffold may be leveraged for certain drug delivery⁶⁷ or tissue engineering applications,⁶⁸ the hydroxyl free radical generated during H₂O₂ sterilization has the potential to oxidatively crosslink polymers, resulting in deleterious changes.^35,68,69 Studies characterizing the morphological and biochemical changes induced by H₂O₂ sterilization in UHMWPE revealed changes similar to those induced by gamma irradiation, including embrittlement.⁷⁰ H₂O₂ sterilization has also been observed to induce mechanical and degradative property changes to degradable polymers such as PLA.^34,71,72 Lastly, it is of note that H₂O₂ sterilization was found to be ineffective when paired with cellulose-based products due to the high tendency of cellulose to absorb H₂O₂, thereby reducing its effective concentration.³⁵ Overall, the limitations associated with H₂O₂ sterilization make its applications primarily centered around surface sterilization of non-absorbent polymers.

2.2.2. Ozone.

Ozone (O₃) is another FDA approved Class B sterilant.^2,20 As a reactive oxidizing gas, ozone sterilization is typically applied in cycles for approximately 4.5 h² at 30–35 °C⁶¹. The penetration capacity of ozone gas falls between those of EtO and H₂O₂.^2,73 Appropriate applications of this modality are limited to polymers that are resistant to oxidation and do not react with the sterilant itself. Advantages of this sterilization modality include limited regulatory burden due to the Class B classification,²⁰ relatively low operating temperatures,⁶¹ and good penetration of the gas.² Although a wide range of polymers, including polyvinyl chloride, PE, polyacetals, polyesters, polycarbonate, and PP are often subjected to ozone sterilization,^2,61 studies have demonstrated statistically significant changes in polymer degradation rates, morphologies, and biomechanical properties,⁷⁴ as well as surface chemistry alterations when exposed to ozone,^73,74 making it necessary to evaluate its compatibility case by case, especially for degradable polymers intended for implantations.

3. NOVEL STERILIZATION TECHNOLOGY

Sterilization methods that do not have sufficient data guiding operational parameters and supporting sterilization efficacy, alter operational parameters beyond what has been previously approved, or combine sterilants/processes in new ways are regulated by the FDA as novel processes. Examples may be new additives, sterilant doses outside of the upper or lower limits of the approved range, or processes for which established guidelines are not sufficiently available.²⁰

Novel classification incurs a significantly greater regulatory burden to the manufacturer of the polymeric implant.²⁰ Undergoing a facilities inspection, submitting comprehensive procedural documentation, and providing extensive validation data are expected. Pursing this pathway may increase the amount of time, infrastructure, and overhead cost required to achieve FDA approval and therefore warrants thorough evaluation and comparison with Class A and B methods.

Novel sterilization methods such as vaporized peracetic acid (PAA), UV light, high intensity or pulsed light, sound waves, and microwave radiation have been subject of recent reviews^2,20 as novel sterilant or alternative light/radiation methods and will not be detailed here. This section will instead highlight recent approaches that are either fundamentally different from typical Class A and Class B methods or aimed at mitigating the negative impact of free radicals or unintended degradations, two key challenges shared by a number of Class A, Class B and emerging novel terminal sterilization methods.

3.1. Supercritical CO₂ as a Novel Sterilization Modality.

Supercritical CO₂ (scCO₂) is an emerging method of sterilization being explored for tissue grafts and degradable polymer scaffolds used for regenerative medicine applications. CO₂ behaves as a supercritical fluid above its critical temperature (304.13 K, 31.0 °C) and critical pressure (7.3773 MPa, 72.8 atm), expanding like a gas but with a density like that of a liquid.⁷⁵ It has been utilized as a green industrial extraction solvent due to its chemical stability, relatively low toxicity and environmental impact.^76,77 The exact mechanisms of action for the microbial destruction by scCO₂ are not fully elucidated, although efficacious penetration^76–78 of scCO₂ into the cellular compartments of microbes and the resultant perturbation to intracellular pH (due to dissolution of CO₂ in water), cell wall integrity, and intracellular enzyme structures have all been postulated as underlying factors (Fig. 2).^76,79 To achieve good bactericidal effects, however, scCO₂ is often combined with one or several additives, such as PAA, H₂O₂ or acetic anhydride; water, if compatible with the polymer of interest, may also be added to enhance scCO₂ diffusion.⁷⁶ These additives, used in lieu of higher pressure or temperature, help the sterilization of endospores not effectively neutralized by scCO₂ alone.^76,80–82

Figure 2.

A schematic diagram of how pressurized CO₂ may exert its lethal action on bacteria. Reproduced with permission from reference 79. Copyright © 2007, Elsevier.

The advantages of scCO₂ sterilization include relatively low operating temperatures, limited oxidizing adjuvants (if used, often in low concentration), good penetration within porous scaffolds, and low toxicity.⁷⁶ These features make it particularly appealing to thermal sensitive polymers as well as tissue grafts. Studies directly comparing the effect of scCO₂, gamma irradiation, and steam sterilizations on collagen and polysaccharide composites show that scCO₂ induced biomechanical changes significantly less than the other methods.⁷⁶ The claim that scCO₂ is a gentler sterilization method was further supported by research that demonstrates decreased mechanical changes in bovine bone samples.⁸³ Limited effects of scCO₂ sterilization on the mechanical integrity of bisphenol A glycidyl methacrylate/triethylene glycol dimethacrylate thermoset composites frequently used in orthopedics and dentistry were also reported and are consistent with this notion.⁷¹

Despite the relatively benign operating conditions, there are still drawbacks to this methodology. For example, varying degrees of dimensional, structural, and mechanical property changes, such as compressive strength⁷⁶ and hyperelasticity,⁸⁴ can occur, particularly in porous, physically crosslinked polymers. Such impacts are primarily due to the ability of the solvent-like scCO₂ to interact with the polar functional groups of polymers, which results in the swelling^75,76 and plasticizing of the polymers.^75,85 For instance, the 3D printed macroporous scaffolds of thermoplastic degradable shape memory polymer containing amphiphilic PLA and PEG blocks developed in our lab⁹ were not compatible with the scCO₂ sterilization due to irreversible dimensional changes and the collapse of the macropores upon scCO₂ processing (unpublished results), likely due to extensive disruption of the crystallinity of the PEG domains or the physical entanglement of PLA. Indeed, the swelling of glassy domains or glassy polymers by scCO₂ is recognized as a common outcome of scCO₂ treatment⁷⁵, which could also result in significant increase in both solute transport and premature release of therapeutic cargos embedded in the polymeric scaffold during sterilization. It should be noted that whereas the processing temperature of the scCO₂ sterilization may not “melt” glassy domains, the plasticizing effect of the solvent-like scCO₂ could still alter the performance of thermal responsive shape memory polymers^75,85,86. Lastly, the addition of H₂O₂, PAA, acetic anhydrides, water, and other processing adjuvants may further incur changes due to chemical incompatibility with the polymer, thus case-by-case evaluations are warranted.

3.2. Addition of Antioxidants as Free Radical Scavengers.

Given the well-understood deleterious impact of free radicals generated by high-energy irradiation sterilization on polymers as well as in vivo tissues (post-implantation), antioxidants have been explored as radical scavengers to mitigate oxidative damages induced by sterilization processes. Vitamin E has been doped within UHMWPE arthroplasty³⁰ to minimize the adverse crosslinking effects induced by ROS byproducts of gamma irradiation or other oxidative sterilization processes. In vitro testing of the vitamin E doped UHMWPE post-high energy irradiation demonstrated decreased wear^22,87–89, improved mechanical properties^22,28,87,90, and enhanced fatigue strength^87,89,91, cumulatively outperforming the non-antioxidant products subjected to the same sterilization treatment. It should be appreciated that many decades of prior research in understanding the impact of oxidative damage of gamma irradiation sterilization on UHMWPE paved the way for the strategy of doping implants with the antioxidant in the early 1990s, and the first vitamin E-bearing UHMWPE commercial product did not come to market until 2007²². Clinical outcomes on vitamin E doped implants have been less consistent, with some clinical studies reporting statistically significant improvement in wear^92,93, while others reporting statistically insignificant differences^94–96 in 2–5 year follow-ups. The variability in reported clinical efficacy^{88,94,95,97–100} calls for continued examination of long-term outcomes in order to ascertain the degree to which in vitro vitamin E doping-related improvements may translate in vivo to benefit patients. This highlights the complexity that any modifications of implant manufacturing and sterilization process could impose, from regulatory and business considerations to scientific and clinical outcomes.

3.3. Control of pH during Steam Sterilization.

Unintended bond cleavage of polymers could not only occur under high-energy irradiation, but also under heat or steam, especially when the chemical nature of the polymer pre-disposes it for cleavages. For instance, hydrolytic degradation of polyesters could occur with steam sterilization, especially when the polymer is hygroscopic in nature or the chemistry is labile to cleavage by design. Santi and colleagues developed a tetra-PEG hydrogel microspheres (MS) system for drug delivery where the therapeutic cargo was covalently conjugated with the MS via a carbamate linker and could be subsequently released along with non-toxic CO₂ via β-eliminative cleavage under physiological or basic conditions (Scheme 1).^96,101 Steam autoclave sterilization of the MS could lead to undesired, premature cleavage as the β-elimination was more likely to occur under the harsh temperature. To overcome this challenge, Santi and colleagues acidified the medium (pH 4) to mitigate base-catalyzed cleavage of carbamate linkers during autoclave with success.¹⁰¹ This study exemplifies how the fundamental understanding of the chemistry at play may be exploited for the development of counter strategies. Such a tailored terminal sterilization method could provide an alternative to manufacturing the MS under aseptic conditions, which are known to be costly. However, it should be noted that although the altered pH is implementable in a Current Good Manufacturing Practices (cGMP) compliant facility as stated by the authors,¹⁰¹ thorough understanding of how this modification may alter the classification of the terminal sterilization by the FDA is necessary.

Scheme 1.

β-eliminative linker cleavage. Reproduced with permission from reference 101. Copyright © 2020, John Wiley & Sons, Ltd.

4. ASEPTIC MANUFACTURING

Aseptic manufacturing and processing is an alternative when available terminal sterilizations are incompatible with a product in question.²¹ ISO, cGMP, and other regulatory agencies provide guidelines for companies to achieve compliance and approval of their aseptic procedures. The decision to pursue the aseptic alternative should be an informed one because the regulatory burden and capital necessary to build the aseptic manufacturing infrastructure is substantial. Without terminal sterilization, rigorous measures must be put in place to minimize the risk of pathogenic contamination.^21,36

The extensive regulations pertain, but are not limited to, microbial and particulate concentrations associated with equipment, operational environment, and components relating to manufacturing or product composition.²¹ Table 2 outlines some key aspects of aseptic regulations in the FDA guidance document for sterile drug products produced by aseptic processing, highlighting the extensive infrastructure and quality management necessary to achieve successful aseptic manufacturing. Although the specific fabrication flow of polymeric implants may vary significantly case by case, such high levels of regulations throughout the production are expected. As such, implementing aseptic processing as the sole means of sterility assurance for polymeric regenerative medicine products should only be considered after a thorough evaluation of terminal sterilization compatibility and consideration of its incurred regulatory burden.

5. CONCLUSION

Sterilization technology and regulations are both complex and extensive. Despite the diversity in methodology, conventional terminal sterilization methods originally developed for metallic, ceramic, and chemically stable polymeric implants present barriers to sterilizing labile degradable functional polymers and tissue grafts for regenerative medicine applications. To preserve the chemical, structural and mechanical integrity of the polymer-based implant and its surgical handling and in vivo degradation/drug release profiles, a salient choice of terminal sterilization modality should be made based on an understanding of both the unique nature of the polymer and the pros/cons of each sterilization method and associated regulatory classification (Scheme 2).

Scheme 2.

Pros and cons of different classifications of terminal sterilization methods versus the aseptic manufacturing alternative.

General polymeric formulation/scaffold considerations include chemical compositions (e.g., presence of labile linkages sensitive to cleavage during sterilization or functional groups that may react with a chemical sterilant), unique thermomechanical behavior (e.g., transition temperature compatibility with the sterilization temperature implemented), and dimensional stability/porosity (e.g., whether irreversible swelling may occur by steam autoclave or scCO₂). Once incompatible terminal sterilization modalities are excluded (e.g., autoclave for thermal sensitive materials; scCO₂ for porous polymers that swell/plasticize extensively; EtO for polymers with nucleophilic functional groups), priority should be given to methods with Class A or Class B designations. In the case of macroporous, thermal-sensitive, degradable shape memory polymer scaffolds, multiple established modalities may all present challenges. In such a case, different methods may need to be evaluated to identify the one resulting in minimal or acceptable levels of alterations to key materials properties and implemented consistently for all preclinical in vitro and animal studies.

As highlighted in this review, infrastructure, capital, time, compliance protocols, and burden of proof are but a few areas affected by regulatory classifications. Furthermore, these implications may not be confined solely to the approval processes but may extend throughout the product’s lifetime. Managing this process is undoubtedly complex and calls for interdisciplinary collaboration. Identifying a terminal sterilization modality/condition that sufficiently satisfies regulatory requirements while preserving product functionality could be a substantial challenge. Indeed, many novel biomaterials have a hard time transitioning from research laboratories to the market due to either belated findings of incompatibility with conventional Class A/B sterilization methods or the tremendous regulatory burdens associated with the novel sterilization modalities implemented during laboratory research or the aseptic processing alternative. Deliberate integration of the regulatory considerations discussed in this review early in the material design/innovation may prove critical to streamlining ultimate clinical translations. Interdisciplinary collaboration between research and development, regulatory, and leadership teams is likely required to thoroughly evaluate options and tackle the challenge.