Hydroperoxide generation in irradiation-sterilized SUS and potential risks of protein oxidation

Oxidation in polymeric medical implants sterilized by ionizing radiation is by now a well-studied phenomenon. One byproduct of the multitude of oxidative pathways initiated during irradiation of polymers is the generation of hydroperoxide groups bound to polymer chains or fragments (see Figure 1 and reference below). We have discovered that exposure to irradiation-sterilized SUS containers has the potential to induce measurable amounts of oxidation in some protein therapeutics, especially in the context of stability studies having high surface area to volume conditions and/or low protein concentration formulations. This presentation will discuss efforts in our laboratories to quantify the oxidizing species (e.g. hydroperoxides) in SUS containers and explore the parameters that could lead to variability in the amounts present and available to react with product molecules. Using the information gained, we provide a strategy to assess potential risk of protein oxidation in commercial manufacturing operations. Please click Additional Files below to see the full abstract

The ubiquitous issue of cross-mass transfer: applications to single-use systems

The leaching of chemicals by materials has been integrated into risk management procedures of many sectors where hygiene and safety are important, including food, medical, pharmaceutical, and biotechnological applications. The approaches focus on direct contact and do not usually address the risk of cross-mass transfer of chemicals from one item or object to another and finally to the contacting phase (e.g., culture medium, biological fluids). Overpackaging systems, as well as secondary or ternary containers, are potentially large reservoirs of non-intentionally added substances (NIAS), which can affect the final risk of contamination. This study provides a comprehensive description of the cross-mass transfer phenomena for single-use bags along the chain of value and the methodology to evaluate them numerically on laminated and assembled systems. The methodology is validated on the risk of migration i) of -caprolactam originating from the polyamide 6 internal layer of the overpackaging and ii) of nine surrogate migrants with various volatilities and polarities. The effects of imperfect contacts between items and of an air gap between them are particularly discussed and interpreted as a cutoff distance depending on the considered substance. A probabilistic description is suggested to define conservative safety-margins required to manage cross-contamination and NIAS in routine

AComDim applied to evaluate gamma irradiation impact on multilayer PE based films

The use of single-use systems is becoming increasingly common in the biopharmaceutical and biotechnology industries. These systems are manufactured from polymers such as polyethylene (PE) and ethylene vinyl acetate (EVA). For their future applications, these devices are sterilized by γ-irradiation with a dose between 25 and 45kGy. C. Artandi and W.V. Winkle[i] determined that 25kGy is the dose to be at 40% above the minimum needed to kill the most resistant microorganisms. The purpose of this study is to understand what happens on the surface of polymers after γ-sterilization. Optical spectroscopy are of great interest for chemical analysis and are used to obtain information on the composition of materials, such as polymers. The Fourier Transform Infrared (FTIR) spectroscopy provides information on the fundamental vibrations of the molecules using an excitation in the visible. The surface of films is analyzed after being sterilized with different radiation doses and after a natural ageing of few months to check their composition and stability by FTIR spectroscopy. As the number of data is important, the use of chemometric methods, like Principal Component Analysis (PCA) and AComDim (ANOVA Common Dimensions), has many advantages, such as identification of shift and intensity modification, detection and highlighting of the influential factors and interactions between several elements (γ-doses, aging, and film batches)

Limitation and detection of bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP) from bioprocess container materials

The identification of the bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP) by Hammond et al was a key to understand the detrimental effect on cell growth that may occur in single use systems composed of polyethylene. The growth inhibition depends on the concentration and cell line, while known standard cytotoxicity assays (i.e. USP\u3c87\u3e) are insensitive to the levels shown to have detrimental effects. There is therefore no universal standardized cell growth testing allowing determining an absolute quantitative concentration detrimental to all specific cell lines. The most sensitive cells lines tested showed a dose response that challenges the limit of detection of existing analytical methods. Concentrations of bDtBPP are linked to the secondary degradation via gamma irradiation of a common antioxidant and have been shown to be related both to the starting concentration of the antioxidant and to the thermal processing parameters. Comprehensive understanding of this relationship requires the use of design of experiment. Cell based assays used to support a DoE approach have proven to be cumbersome. This now known and well understood phenomenon presented a challenge for the development of a new film composed of polyethylene. The bDtBPP concentration of concern is below detection thresholds for existing standard cell based assays and analytical techniques such that a physico-chemical analysis had to be designed to support the film development project. Extraction methods and analytical methods were specifically developed by using appropriate polymer swelling solvent and chromatographic tools to extract and detect the bDtBPP in worse case conditions. The detection limit of the bDtBPP in worse case conditions is equivalent to the lowest bDtBPP concentrations to be detrimental to the cell growth for the common cell lines. Data are presented detailing the development of the analytical methods and their usefulness relative to the concentrations shown to be of concern

The proper use of extractables data - aspects beyond extractables-measurment

Appropriate extraction techniques for SUS/SUT and methods for analysis of extractables have been intensively discussed over the last years. Today, several proposals for common methods are available and used to conduct extractables studies in the bio-pharmaceutical industry. Therefore it is expected that the number of available extractables data will significantly rise over the next years and it is worth to (re-)consider their proper use for materials and device qualification and risk assessment. While this exercise is straightforward for container closure systems (CCS), for SUS/SUT the situation is more complex. In CCS applications, a single drug product, in contact with a well-defined container system for long term storage is studied. In contrast to a CCS the number of materials, their dimensions and combinations are highly flexible in SUS/SUT applications. Additionally, SUS/SUT are used under dynamic process-conditions of variable solvents, dwell-times, temperatures, flow-rates etc. In our contribution we will discuss two major questions that persist and cannot be solved by means of analytics alone: How can we obtain extractables data for SUS/SUT devices of different sizes and for complex device combinations (assemblies)? This aspect is critical for the device industry, because a high number of different devices and combinations are requested by our customers. Further, assembled products from Configured to Order (CTO) or Engineered to Order (ETO) processes utilizing various compounds, even such from various suppliers can increase their amount and combinations nearly infinitely. It is easily conceivable that it is technically impossible to conduct individual extractables studies for each possible combination. Another aspect which has to be taken into account in the future, is the proper use of extractables data for extrapolations toward potential leachables required for quality risk-assessment. In this context a publication from Jenke & Rabinow (2017) has to be considered, where they showed that the validity of the “intuitive” approach to scale extractables data just by surface area is questionable. We will show how we can develop methods to overcome these - so far - unsolved problems. The proposed methods will be based on basic physical chemistry principles rather than “intuitive” worst-case assumptions. We will show illustrative examples on how extractables data, obtained by different protocols can be used heuristically in scaling and combination exercises. The limits of the conventional scaling by surface areas are discussed in terms of the influence of equilibrium versus diffusion controlled conditions in long versus short term contact. Furthermore, an example will be shown for a calculation of potential leachables solely built on physical chemistry considerations and avoiding any generic worst-case assumption. Jenke & Rabinow: Proper Accounting for Surface Area to Solution Volume Ratios in Exaggerated Extractions PDA J Pharm Sci and Tech (2017), 71 225-23

Identification of antioxidant by-products based on their specific chemistry and their potential detection during SUS extractable study

Single-use films in biopharmaceutical and biotechnology industries are mainly made from polymers such as PE, EVA and EVOH. Depending upon the environmental aggressiveness during various stages of the polymer lifetime, additives are added to protect them such as substituted hindered phenols acting as antioxidants, melt (processing) stabilizers, and to some extent as photo-antioxidants. Ionizing radiation effects on polymers have been widely investigated. They consist mainly of free radicals production. These free radicals can in turn lead to degradation and or crosslinking phenomena (release of gases, discoloration, changes in mechanical properties and gas permeability, degradation and leaching of polymer additives into solvents, etc.) whose extent depends on many factors. In contrast, there is little information on the effect of ionizing radiation on the additive package properties used in multilayer packaging films. A specific influence on chemical transformations of phenols is induced as well. Strong discoloration of the polymer stabilized with phenolic antioxidants originates for instance mainly from the reaction products of the stabilizers. The color development can be attributed to the formation of conjugated diene compounds, arising as a consequence of trapping of radicals by phenolics. The discoloration depends on the structure and concentration of the phenolic transformation products. As a result of the described complexity, a huge variety of potential extractable compounds can be expected from antioxidants. This work focuses of highlighting the degradation products we may expect from the thermal and radiative degradation of the primary and secondary anti-oxidants and to address complexity of identifying properly the by-products one may detect in extractable study applied to pharmaceutical single use products

On the “Fate of Leachables” in biopharmaceutical up-stream and down- stream processes

Single use systems (SUS), based on polymeric materials, are widely used in the biopharmaceutical manufacturing. Compounds potentially migrating from the polymeric materials into the production process at various stages as leachables can either negatively influence the production process performance (e.g. inhibit cell growth) or can contribute to process related drug impurities, which may potentially have an effect on product quality and/or patient safety. Today, the concept of investigating leachables is mainly focused on the measurement of extractables under worst case- or simulation-conditions plus their assessment including an interpolation towards potential leachables. In particular, in the frame of this assessment of leachables, substance specific properties and the effective process conditions are not taken into account. To better understand the range, the load and the concentration of leachables within a dynamic process and finally in a drug product a paradigm change towards the “Fate of Leachables” concept is required. This “Fate of Leachables” concept is based on underlying physical-chemical principles rather than a sequence of worst-case experiments and conservatively adding up concentrations. Relevant levels of leachables can be quantitatively modeled and predicted based on the knowledge of potential extractables in the materials, their phys.-chem. properties and the effective process conditions (medium composition, volume, flow, surfaces, temperature), i.e. Leachables may reach equilibrium concentration in certain static process steps, whereas in dynamic process steps diffusion controlled leachables levels have to be anticipated and appropriately modeled. Adsorption and desorption processes significantly influence the levels of leachables in any biopharmaceutical manufacturing, e.g. in down-stream filtration, separation and purification steps. Ultrafiltration and diafiltration steps can influence the leachables load by diluting or removing them completely. For all these process steps illustrative examples from SSB research studies and based on existing published data will be discussed together with a quantitative description of the underlying physical-chemical processes to demonstrate the capability of the “Fate of Leachables” concept. To motivate the use of the “Fate of Leachables” concept, for a generic process stream, the level of leachables estimated via a worst case evaluation will be compared with the results based on the “Fate of Leachables” concept

pH evolution in solution after contact with multilayer films after different g- irradiation doses and thus reconciliation of pH and TOC with carboxylic acids detected by ion chromatography

For a number of various uses (storage, mixing, freezing, transportation, formulation, and filling) biopharmaceutical solutions are stored in sterile single-use plastic bags. Material transfers can then occur between containers and contents. These migrations, of different types, depend on the physicochemical characteristics of the material (composition, pH, solubility, viscosity, molecular weight, etc.), the nature of the product (solid, semi-solid and liquid) and the conditions of the material utilization. In the case of single-use polymers, γ-irradiation sterilization of the polymer is often carried out. The interactions could be therefore influenced by the dose and the contact time between the container and the contents. γ-sterilization of single-use systems initiates chemical reactions and complex modifications inside the plastic material, In this study, γ-irradiation doses investigated are up to 270 kGy in order to emphazise the γ-irradiation effect and to better investigate the modifications of commercial PE(Polyethylene)/EVOH(Ethylene Vinyl Alcohol)/PE-film and commercial EVA(Ethylene Vinyl Acetate)/EVOH/EVA film. This study is a part of a global investigation on γ-irradiation on multilayer films Non-specific (TOC, pH, conductivity) or specific (e.g. chromatographic, spectroscopic, gravimetric) analytical methods can be used. several approaches were used to study the impact of γ-irradiation on multilayer films, as ion chromatography to detect and quantify the ionic species, and as pH and conductivity measurements to observe the consequences of the chemical modifications.. There are few references available on the leaching of carboxylic acid species impacting aqueous solutions used in biopharmaceutical applications in contact with plastic single-use systems [[i]]. Stability studies under accelerated or real-time degradation conditions make it possible to define the shelf life and storage conditions in order to guarantee the quality of the product. The aim of the study is to identify and quantify the acid compounds that can be released from the container under normal conditions of use of the materials: the extractables. [[1]] D. Jenke, D and V.J. Barge. Factors affecting the release of extractable acetic acid from multi-layered plastic films containing ethylene vinyl acetate (EVA) and polyethylene (PE) layers. Pharm Outsourcing. 15 (2014) 56-59

Influence of γ-irradiated biopharmaceutical films

Preventing cross-contamination, saving costs and increasing configuration flexibility make the adoption of single-use technologies very attractive for the biopharmaceutical industry. The integrity and the security of bags are due to appropriate flexible and barrier polymeric materials, such as polyethylene (PE) or ethylene vinyl acetate (EVA) and polyethylene-co-vinyl alcohol (EVOH), which are barrier to water vapor and oxygen, respectively. Conventional stainless steel tanks are sterilized by steam sterilization by the end-users, whereas plastic containers are sterilized by gamma-irradiation before delivery. The major advantage of radio-sterilization is the penetration power of the γ-radiation. It is known that γ -sterilization of polyolefin based polymer leads to alterations of the material: changes in the additives or potential damage to the polymer, as reported in the literature. Irradiation of polymeric materials has been proven to initiate radiation chemical reactions inside the polymeric material, leading to either an increase or a decrease in the polymer molecular weight. The effects of γ-irradiation on polymers are well known whereas the effects of γ-irradiation on multilayer films have been little investigated. In the case of multilayer films, the acidity of the stored solution increased after gamma irradiation for instance. In another case oxidation of the solution occurred. Such observations denote the presence of acidic and oxidant compounds, which are issued either from modification of surface of the film or from the migration of by-products from core to surface. A global investigation on γ-irradiation on multilayer films is performed to investigate the γ-irradiation based modifications on PE(Polyethylene)/EVOH(Ethylene Vinyl Alcohol)/PE film and EVA(Ethylene Vinyl Acetate)/EVOH/EVA film to assess the multilayer film robustness. Several approaches could be used to study the impact of γ-irradiation on multilayer films, as ESR (Electron Spin Resonance) to observe the radicals formation, ATR-FTIR (Attenuated Total Reflection-Fourier Transform Infrared) and Raman spectroscopies to observe the structural modifications, the measurement of yellowing, the measurement of O2 transmission rate (O2TR) and water vapor transmission rate (WVTR), the measurement of pH to follow the acidity change of solution contained in the bag and the mechanical test to evaluate the toughness of film. Due to the number of data recorded, chemometric methods, such as Principal Component Analysis (PCA), are applied to enhance the weak variations brought to the γ-irradiation of the multilayer films in the different data sets. Results show that films undergo modifications at microscopic level and that they are not altered from macroscopic and application viewpoints. Results are equivalent from batch to batch assuring then a reproducibility of the films behavior for their integration in single-use systems