Feasibility study on the impact of mechanical recycling on the extractables profile and toxicologic safety assessment of small-scale polycarbonate bioreactors

Abstract

The use of recycled plastics in the pharmaceutical industry raises safety questions, especially with regards to leaching substances that could compromise patient safety. This feasibility study evaluates the impact of mechanical recycling on the profile of extractables and the toxicological safety of small-scale polycarbonate bioreactors, using post-industrial polycarbonate as a model. Both new and recycled polycarbonate granules and bioreactor vessels were analyzed for extractable substances using chromatographic methods. Key results include the identification of polycarbonate degradation products, which showed a slight increase in concentration after recycling. The study also found adhesive residues from bioreactor manufacturing and degradation products, with no significant accumulation of potentially harmful substances found during recycling. Model safety assessments, including patient exposure calculations and cellular toxicity assessments, showed that recycled polycarbonate poses minimal toxicological risk under worst-case scenarios. The findings support the possibility of recycling polycarbonate for pharmaceutical applications and highlight the need for strict guidelines to ensure patient safety while promoting sustainable practices.

Graphical abstract

1. Introduction

Plastics are essential in pharmaceutical and biopharmaceutical industries, contributing to the development, manufacturing, and packaging of both existing and new therapeutic products. Packaging ensures the safety and durability of the products and provides essential protection against external influences. (H&K Müller GmbH and Co. KG, 2024) Single-use technologies (SUT) using plastic consumables are favored for their flexibility, and reduced contamination risk. They eliminate the need for cleaning and sterilization, require less space, optimize facility usage and offer quick adaptability to different production scales and types. (Eibl and Eibl, 2019) They also lower environmental impact compared to stainless steel equipment by avoiding water and energy-intensive cleaning processes. (Pietrzykowski et al., 2013)

Today, many of the post-use SUTs are considered hazardous due to contact with biological substances. After use, decontamination is carried out using validated chemical disinfectants or by autoclaving. Once decontaminated, the waste is either incinerated or sent to a landfill. The exact decontamination and waste management procedures may differ among organizations and regions, based on internal waste handling protocols and regulatory guidelines. (Clauss, 2019; Kunststoffe für Medizintechnik: Eigenschaften und Anwendungsbereiche, 2024) Although the quantity of plastic used for biopharmaceutical application is estimated to be less than 0.002% of the overall plastic waste, the industry is willing to include circularity practices. (BPSA Sustainability Committee, n.d.)

Mechanical recycling is typically viewed as the most favorable method to utilize plastic waste due to its superior emission and economic profile. Despite the clear establishment of a waste hierarchy only 16% of global polymer waste is mechanically recycled so far. (Hundertmark et al., 2025) Some countries mechanically recycle up to 35% of polymer waste. (Tsochatzis et al., 2022; Umweltbundesamt, n.d.) A few cases are now emerging in healthcare. For instance, ReMed™ is an innovative recycling program initiated by Novo Nordisk, aimed at collecting and recycling used insulin pens. The plastic from the pens is processed and reused to manufacture furniture, such as chairs. (Novo Nordisk Global, n.d.)

However, integration of mechanically recycled plastic back into the healthcare loop is questioned. (Novo Nordisk Global, n.d.)

Indeed, besides traceability of contaminations, one of the most frequently asked questions about the integration of recycled materials in medical applications concerns the profile of extractables. Some substances can be released by plastic materials, potentially posing a risk in the manufacturing process of biomedicines and to patients if the released materials are harmful to cells and health. (Li et al., 2015) This will be even more the case in cell and gene therapy applications. (Hauk et al., 2023; Aysola et al., 2020) Regulators require proof of product safety and efficacy, with testing for extractables being an essential component. These tests make it possible to detect and minimize potential contamination at an early stage. (Cber Fda, 1999)

A recent study demonstrated the technical feasibility of closed-loop recycling of polycarbonate (PC) bioreactor vessels using cell culture and biocompatibility assays results but did not confirm with extractable data the absence of recycling related compounds that could impose a risk to patient safety. (Barbaroux et al., 2024) (Luu et al., 2022).

In order to map the entire life cycle of plastic and to investigate whether the extractables arise from the material or the various process steps, both new and recycled PC granules were considered. The aim of this feasibility study is to provide a sound worst case basis for performing risk assessments by investigating the extractable profile of PC both in its granular state and in a bioreactor before and after recycling. These assessments are designed to systematically identify, analyze and evaluate potential hazards and risks in order to improve the safety and efficiency of recycling processes. In addition, it will be checked that the use of recycled polycarbonate in bioreactor vessels does not release recycling-related compounds that could impair cell culture and biocompatibility.

2. Materials and methods

2.1. Products and polymer

This study analyzes and compares virgin and recycled Makrolon® grade PC granules as well as virgin and recycled Ambr® 15 Cell Culture sparged microbioreactors (Item no.: 001-7B01) (Fig. 2) made from the same PC as shown in Fig. 1. (Barbaroux et al., 2024)

Fig. 2.

Recycled (left) and virgin (right) Ambr® 15 Cell Culture sparged microbioreactors (Barbaroux et al., 2024).

Fig. 1.

Polycarbonate recycling process. Samples used in this study are marked in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The manufacturing process of the microbioreactor consists of several steps, which are shown in Fig. 1. Virgin PC with a melt volume rate of 19 cm³/10 min (300 °C/1.2 kg) was supplied by Covestro Deutschland AG. Virgin microbioreactors (produced from the virgin polycarbonate) were supplied by Sartorius Stedim Lab Ltd. For the recycled samples, microbioreactors (Item no.: 001-5G25) were subjected to autoclaving at 121 °C for 30 min to mimic post-use decontamination. After this process, they were manually disassembled, patches were removed, and the PC vessels were then shredded into flakes using a grinder Dreher S 34/52 with a sieve of 8 mm. Grinding and granulation were conducted using a compounder ZSE 40 L/D 40 from Leistritz Extrusionstechnik GmbH. Extruder temperatures were set between 240 and 300 °C (different heating zones). The throughput was set to around 75 kg/h at 176 rpm. Vessels were injection molded with standard processes parameters and the new, “recycled” bioreactors were built according to standard manufacturing conditions and irradiated with E-beam at 25 kGy. A single lot was analyzed.

2.2. Extraction protocol

Extraction is a process in which an extract is obtained from a liquid or solid material using a suitable extraction agent. This agent can be a pure solvent or a mixture of solvents. The efficiency of the extraction is influenced by various parameters, including diffusivity in the polymer, temperature, contact time, mixing (e.g., by shaking), the surface-to-volume ratio, and the choice of extraction solvent. (Pharmacopeia, 2022)

The extractions were carried out in accordance with Sartorius approach published in Pahl et al. (Pahl et al., 2018). This approach shows best sensitivity and selectivity for single use systems related to extractables. (Dorey et al., 2018) The bioreactors were filled with the extraction solvent ethanol and then sealed with polytetrafluorethylene (PTFE) tape. This extraction solvent is expected to extract the maximum from PC and proved to be a suitable solvent for our specific requirements. Previous studies on PC were performed to identify typical extraction substances of this polymer. (Sartorius Stedim Biotech, 2013) In our study, a single determination extraction was performed to determine the relative effect of the recycling process on the extractable profile. The extraction was carried out at a temperature of 40 °C and a shaking rate of 75 rpm over a period of 21 days.

In this study, a surface-to-volume ratio of 6:1 cm²/mL was established for the granules, (see Table 1). In the case of microbioreactors, the ratio has been adjusted to the specific design characteristics in order to ensure complete coverage of the analytical requirements. Therefore, a ratio of 4:1 cm²/mL was used. Despite the different surface-to-volume ratios, a comparison between the granules and the microbioreactors is possible. This is due to the fact that the extraction took place over a period of 21 days, which is long enough to approach an equilibrium of extractable substances. Another practical reason for choosing the 4:1 ratio for the microbioreactors is that the cell culture tests were carried out with a volume of 15 mL, which refers to the standard filling volume of the Ambr® 15 devices.

Table 1.

Extraction information.

Sample	Surface-to-volume ratios [cm2/mL]	Surface [cm2]	Volume [mL]	Weight [g] of extracted material
PC virgin granules	6:1	240	40	18.99
PC recycled granules	6:1	240	40	17.57
Virgin bioreactor vessel	4:1	63	15	9.37
Recycled bioreactor vessel	4:1	63	15	9.37

2.3. Material characterization and analytics

Due to the large number of plastic additives and the different extractable compounds, different chromatographic tests are used for analytical determination. The methods used are based on various publications. (Pahl et al., 2018) (Menzel et al., 2024) (Menzel et al., 2018) (Pahl et al., 2019) (Menzel et al., 2023).

2.3.1. High-performance liquid chromatography (HPLC) – Ultraviolet (UV) detector

A system from Agilent 1200, featuring a G 1314 A VWD detector and a C18 Nucleosil column (5 μm, 250 mm/4.6 mm) along with a Nucleosil C18 100–5 guard column, was employed. The eluents used were acetonitrile (A) and purified water (B) with a gradient profile: 0 min/A 0%, 20 min/A 60%, 40 min/A 100%, 55 min/A 100%, at a flow rate of 1.0 mL/min. The injection volume was set to 20 μl, and the column temperature was maintained at 40 °C.

2.3.2. Gas chromatography-mass spectrometry (GC–MS)

GC–MS analyses were conducted using a Clarus 600GC and Clarus 600T MS Turbo, equipped with a USP G27 column and utilizing electron ionization (EI) at 70 eV. For liquid injection, the operating parameters included an injector temperature ramp from 75 °C (held for 0.1 min) to 250 °C at a rate of 200 °C/min, with a 1 μl splitless injection and helium as the carrier gas at a flow rate of 1 mL/min. The oven temperature was programmed from 35 °C (held for 0.1 min) to 300 °C at a rate of 15 °C/min, with a 30 min hold. The transfer line and source temperatures were maintained at 250 °C. The scan range was set from 35 to 700 m/z. TurboMass™ 5.4.2 software was used for data processing. Response factors were set to one which is suitable for direct sample comparison. The GC–MS primarily detected semivolatile compounds. Semi-quantification was performed by using the internal standards (ISTD) 2-fluorobiphenyl (sample concentration 10 μg/mL) for liquid injection. Response factors of the analytes are set to one compared to the ISTD. If a substance is confirmed by HS GC–MS or GC–MS analysis, the authentic reference compound is measured together with the internal standard, and the response factor is determined. Subsequently, the concentration of this confirmed compound in a sample extract is calculated using the peak area ratios of the substance and the internal standard and corrected by the response factor (one-point type calibration). The concentration of all other substances is estimated from peak area ratios of standard substance and the peak in question.

2.3.3. Ultra-performance liquid chromatography–high-resolution mass spectrometry (‘LC-MS’)

A Waters ACQUITY UPLC I-Class system, coupled with a Xevo G2-XS ESI-QTOF mass analyzer and a C18 column (BEH, 1.7 μm, 2.1 mm × 100 mm) with a guard column, was utilized. The gradient was run at a flow rate of 0.5 mL/min and a column temperature of 40 °C, with a total run time of up to 40 min. Acetonitrile (A) and purified water with 10 mmol ammonium acetate (B) served as eluents. The gradient profile was as follows: 0 min/A 5%; 0.5 min/A 5%; 9 min/A 99%; 19.5 min/A 99%; 19.6 min/A 5%; 20 min/A 5%. The injection volume was set to 1 μl, with the column temperature maintained at 40 °C. MS parameters included a mass range of m/z 50–1500, a source temperature of 120 °C, a cone gas flow of 50 l/h, a desolvation gas temperature of 550 °C, and a flow of 950 l/h. The collision mode was ramped from 15 to 40 eV, and the analysis was conducted in both ESI-negative and ESI-positive ionization modes. UNIFI® v1.8.2 was used for data processing. Information obtained from LC-MS, such as structure elucidation, was used for compound identification in HPLC-UV results, considering elution order and signal intensity. This technique was not used for quantification.

2.3.4. Reporting limits

Table 2 shows the reporting limit concentrations for the different analytical techniques. The reporting limit is critical to analytical accuracy as it ensures that only reliable and accurate data is reported. It defines the lowest concentration value at which an analyte can be measured with acceptable precision and is closely related to the sensitivity of the method. In addition, the reporting limit helps to meet regulatory requirements by ensuring that only the prescribed concentrations of substances are included in reports. It contributes to quality control by ensuring data quality within the validated limits of the method. Overall, a reporting limit improves the efficiency of data analysis and clarity of data communication. (König et al., 2010)

Table 2.

Reporting limit concentrations and quantities for the different analytical techniques (Scott, 2007).

Analytical Technique	GC–MS	HPLC-UV	LC-MS
Reporting Limit [μg/mL]	0.10	0.30	0.10
Reporting Limit for PC granules [μg/cm2]	0.020	0.050	0.020
Reporting Limit for Microbioreactor [μg/cm2]	0.030	0.080	0.030

3. Results

3.1. Analytical results

The results of the chromatographic analyses of the virgin granules, virgin bioreactor vessels, recycled granules, and recycled bioreactor vessels, are summarized in the following Table 3. This enables a comparison of the change of the individual extractable substances and extractables profile along the recycling process.

Table 3.

Concentrations of detected extractables compounds and toxicological thresholds for safety assessment.

Compounds	CAS	PDE / TTC	PC virgin granules	virgin bioreactor vessel	PC recycled granules	recycled bioreactor vessel
		[μg/day]	Conc. [μg/cm2]
2-Ethoxytetrahydro-2H-pyran	4819-83-4	120 (European Medicines Agency, 2023a)	n.d.	0.055	n.d.	n.d.
4-Hydroxyacetophenone	99–93-4	120 (European Medicines Agency, 2023a)	<R.L.*	<R.L.*	0.030	<R.L.*
Bisphenol A	80–05-7	4.2[a]	<R.L.*	0.032	0.033	0.041
Branched alkane	n.a.	120 (European Medicines Agency, 2023a)	n.d.	0.075	n.d.	<R.L.*
Chlorobenzene	108–90-7	3600[b]	0.086	0.068	0.12	0.11
Cyclohexanol	108–93-0	120 (European Medicines Agency, 2023a)	0.029	0.23	0.037	0.051
Diethylene glycol	111–46-6	120 (European Medicines Agency, 2023a)	n d	0.083	n.d.	0.076
Erucamide	112–84-5	120 (European Medicines Agency, 2023a)	0.52	n.d.	1.00	0.28
Glue from the Patch	n.a.	120 (European Medicines Agency, 2023a)	n.d.	1.4	n.d.	1.2
Methyl styrene	622–97-9	1963[c]	n.d.	<R.L.*	0.044	<R.L.*
p-tert-Butylphenol	98–54-4	120 (European Medicines Agency, 2023a)	0.024	<R.L.*	0.081	0.089
Phenol	108–95-2	12,500[d]	<R.L.*	0.051	<R.L.*	n.d.
Stearic acid	57–11-4	120 (European Medicines Agency, 2023a)	n.d.	0.077	n.d.	0.097
Triacetin	102–76-1	120 (European Medicines Agency, 2023a)	n.d.	0.17	n.d.	0.063
Unknown 1	n.a.	120 (European Medicines Agency, 2023a)	0.063	<R.L.**	0.15	<R.L.**
Unknown 2	n.a.	120 (European Medicines Agency, 2023a)	<R.L.**	<R.L.**	0.062	0.12

<R.L. = below reporting limit, * Reporting limit GC–MS, ** Reporting limit HPLC, n.d. = no detection.

Unpublished data:

^a(P. Parris: Regul. Toxicol. Pharmacol. 2020 (118) 104802).

^b(ICH Q3D (R8) 2021).

^c(Fruth: TE project 23056f01).

^d(Fruth: TESF report 21040f01).

A significant result is the detection of monomer building blocks of the PC, including p- tert- butylphenol. The concentration of p-tert-butylphenol is 0.024 μg/cm² in virgin granules, 0.081 μg/cm² in recycled granules and 0.089 μg/cm² in recycled bioreactor vessel. In addition, chlorobenzene, a solvent used in production of PC, was detected in the extracts. (König et al., 2010) The chlorobenzene concentration is higher in the recycled granules and in the recycled bioreactor vessel compared to the virgin granules and virgin bioreactor vessel. The concentration of bisphenol A (BPA) increased slightly with increasing processing steps from 0.031 μg/cm² in the virgin bioreactor vessel to 0.041 μg/cm² in the recycled bioreactor vessel. In the virgin PC granules, BPA was detected below the reporting limit. Phenol, which can occur as an impurity of the monomer BPA, was detected in the virgin bioreactor vessel with a concentration of 0.051 μg/cm², but not in the extract of the recycled bioreactor vessel. For the granules (both virgin and recycled), the concentration of phenol was below the reporting limit of GC–MS. The GC–MS analysis identified adhesive residues exclusively in the virgin and recycled bioreactor vessel which can result from the built-in pH and DO sensor patches. The optical sensors positioned in the microbioreactors on the floor enable non-invasive measurement of pH and dissolved oxygen (DO). (Fig. 3) (Sartorius Stedim Biotech GmbH, 2013) These measured values play a fundamental role in processes involving living cells. These residues include the adhesive itself, as well as triacetin and diethylene glycol. The study showed that the glue concentration in the microbioreactors is constant, with 1.44 μg/cm² in the virgin bioreactor vessel and 1.21 μg/cm² in the recycled bioreactor vessel. The triacetin concentration in the recycled bioreactor vessel is 0.06 μg/cm², which is only half of the concentration in the virgin bioreactor vessel.

Fig. 3.

Optical sensors in the bioreactor vessel.

The concentrations of 2-ethoxytetrahydro-2H-pyran and diethylene glycol, which could be identified as potential degradation products, were detected exclusively in the virgin bioreactor vessel.

2- ethoxytetrahydro- 2H- pyran with a concentration of 0.055 μg/cm² was detected in the virgin bioreactor vessel, whereas it was not detectable in the recycled bioreactor vessel. Diethylene glycol was detected in the virgin bioreactor vessel at a concentration of 0.083 μg/cm² and in the recycled bioreactor vessel at a concentration of 0.076 μg/cm². The thermal degradation product 4-hydroxyacetophenone was detected in all samples. In the virgin granules, in the virgin and recycled bioreactor vessel, the concentration was below the detection limit of GC–MS. In recycled granules, on the other hand, a concentration of 0.03 μg/cm² was detected. The concentration of erucamide, a slip additive, (Zweifel et al., 2009) varies significantly between samples. It is 0.52 μg/cm² in virgin granules, while it is undetectable in virgin bioreactor vessel. At 1.00 μg/cm², the concentration in recycled granules is high, whereas in recycled bioreactor vessel it is low (0.28 μg/cm²).

Also, stearic acid and branched alkanes were found only in bioreactor vessels, but not in the virgin or recycled granules. Methyl styrene, an impact modifier, was not detected in virgin granules. The highest concentration of 0.044 μg/cm² is found in recycled granules. In the bioreactors vessels are the concentration lower than the reporting limit of GC–MS. Cyclohexanol was detected in all samples, only the concentration in the virgin bioreactor vessel is higher than in the other samples.

In Table 4, the PC oligomers are presented separately because their quantification is not possible due to the lack of suitable standard substances. Instead, the areas of oligomers detected during the LC-MS analysis are reported. Table 4 illustrates that the area of oligomers is subject to fluctuations during the recycling process, with the recycled microbioreactor having a lower amount of oligomers compared to the virgin granules. Since there is no trend to observe, this will not be discussed further.

Table 4.

Peak Area of PC oligomers (LC-MS response) and toxicological thresholds for safety assessment.

Compounds	CAS	PDE / TTC	PC virgin granules	virgin bioreactor vessel	PC recycled granules	recycled bioreactor vessel
		[μg/day]	Area [counts]
Bisphenol A carbonate (1:2) - diphenyl	20,325–64-8	120 (European Medicines Agency, 2023a)	5934	65,634	2957	414
Bisphenol A carbonate (2:3) - diphenyl	n.a.	120 (European Medicines Agency, 2023a)	1635	32,653	n.d.	n.d.
Bisphenol A carbonate (3:4) - diphenyl	n.a.	120 (European Medicines Agency, 2023a)	4527	n.d.	4181	2673
Bisphenol A carbonate cyclic trimer (3:3)	811–43-8	120 (European Medicines Agency, 2023a)	29,326	15,609	68,390	21,169

n.d. = no detection.

With HPLC-UV two unknowns were detected. Unknown 1 was detected in the virgin and recycled granules up to 0.015 μg/cm² but not in the recycled bioreactor vessel. Unknown 2 was found in the recycled granules and bioreactor vessel up to 0.12 μg/cm².

4. Discussion

The detailed description of a successful recycling process as well as the impact of recycling on the performance of the bioreactors can be found in Barbaroux et al. (Li et al., 2015) This study uses samples from the same recycling batch. Fig. 2 shows a picture of specimen of PC Ambr® 15 before and after recycling. Indicating a significant yellowing after recycling. However, the yellowing does not correspond to recycling related degradation of the material but rather to the irradiation. (James and Chung, 2025)

The use of a standardized surface-to-volume ratio contributed to the reproducibility and comparability of the results. The chosen extraction conditions ensured that the polymers remained largely intact.

The degradation products p-tert-butylphenol and 4-hydroxyacetophenone, were produced by degradation due to irradiation or thermal degradation during the recycling process.

p-tert-Butylphenol is used as a chain breaker to control the molecular weight of PC and was detected in the recycled bioreactor vessel, indicating a cleavage of the chain ends and thus a shortening of the polymer chains. (Fig. 4) (Menzel et al., 2018).

Fig. 4.

Polymerization, chain termination of Polycarbonate and formation of 4-tert-Butylphenol (Menzel et al., 2018).

4-Hydroxyacetophenone is formed by the methyl cleavage of the isopropylidene bond of the PC, followed by the formation of a peroxide bond. This process is continued by the cleavage of alkoxy radicals by means of irradiation and subsequent hydrolysis. (Fig. 5) (Menzel et al., 2018) Both degradation products were detected in the virgin granules and in the virgin bioreactor vessel in low concentrations or below the detection limit. The increased concentration of 4-hydroxyacetophenone and p- tert-butylphenol in recycled granules and in recycled bioreactor vessel suggests that additional degradation took place due to the thermal processing steps during the recycling process. These conclusions are further supported by the change in melt flow rate after recycling (reported by Barbaroux et. Al. (Li et al., 2015)) which increases from 20 g/10 min to 29 g/10 min (300 °C/1.2 kg). This increase is a clear indication of the shortened polymer chains allowing the melted material to flow more easily.

Fig. 5.

Degradation of polycarbonate (Menzel et al., 2018).

Besides the degradation products of PC, other substances are identified such as the additives and adhesive residues in the virgin and recycled microbioreactors but not in the resin. No additional accumulation of these substances was detected in the recycled bioreactor vessel. This indicates that these substances are depleted during the recycling process.

Analysis of the extracts has revealed the presence of impurities such as erucamide in the granules but not in the bioreactor vessels. It is assumed that these substances originate from the packaging of the granulates, since these granules stored in polypropylene bags were used for extractable analysis. (Zweifel et al., 2009)

Branched alkanes were found only in the microbioreactors, but not in the granules. These substances originate from the sparger in the bioreactor vessel that is made of polypropylene.

The degradation of PC can be tracked by analyzing the bisphenol A (BPA) concentration.

It is noticeable that the area of the oligomers in the recycled material is smaller than in the original virgin material of the bioreactor vessel. This indicates that there has been a reduction in oligomers during the recycling process which could indicate possible decomposition or transformation of the oligomers. This observation is particularly relevant because it allows conclusions to be drawn about the effectiveness of the recycling process and the quality of the recycled material. A detailed study of these fluctuations can provide valuable insights into optimizing the recycling process and improving material quality.

5. Safety assessment

Safety assessment is a key process in the pharmaceutical industry that aims to systematically identify, analyze, and evaluate potential hazards and risks to patient safety. This process is critical to ensure the safety and efficacy of pharmaceutical products and to protect the health of patients. As part of the risk assessment, the chemical composition, the manufacturing processes, and possible sources of contamination are examined. A special focus is laid on the evaluation of potentially toxic impurities. The risk assessment includes the steps of hazard identification, exposure assessment, dose-response assessment, and risk characterization. The process of a risk assessment is described by M. Aysola et al. in “BPSA- Extractables/ Leachables considerations for Cell and Gene Therapy Drug Product Development.”. (Aysola et al., 2020)

Please note that this is a theoretical worst case model assessment solely based on ethanol extractables data and static exposure calculations. The authors are aware that therapeutic cells are commonly cultivated in aqueous media and under dynamic conditions, e.g. in a perfusion bioreactor, resulting in significantly lower cell exposure than for static conditions. (Hauk et al., 2023; Hauk et al., 2024)

5.1. Direct patient exposure

The process of stem cell therapy starts with the collection of stem cells through apheresis, after the donor has been given medication to mobilize the cells. The harvested stem cells are then cultured in a bioreactor vessel system and may be genetically modified if needed. Following quality control and enrichment, the necessary number of cells is calculated and prepared in a solution for infusion. A worst-case approach is being used to calculate the maximum amount of extractables that could be given to a patient from the bioreactor, excluding any downstream purification steps. Exemplarily, the daily dose given to a patient is determined using Eq. (1) related to CD34+ cells, which are to be considered as a model cell type for illustration of a stem cell therapy. (Klump, 2025; Chen et al., 2013)

The following hypothetic dose scheme is considered: The calculation of dosed cells is based on the target cell count, which ranges from 2 to 5 × 10⁶ cells/kg, the patient's body weight (e.g., 75 kg), and the viable cell density in the bioreactor vessel (>20 × 10⁶ cells/mL). This cell solution is administered to the patient via infusion, with subsequent monitoring to assess the therapy's effectiveness and any potential side effects. (Müller-Tidow and Dreger, 2025; Ashihara et al., 2002) There are about 2 to 5 million human cells per kilogram of body weight. (Ashihara et al., 2002) With an average body weight of 75 kg for a man, this results in a total of about 262.5 million cells. The infusion volume is calculated by dividing the total number of cells in a 75 kg man by the viable cell density of CD34+ cells (20–50 million cells/mL). (Chen et al., 2013) (Rees-Manley, 2026) This gives an arbitrary infusion volume of 7.5 mL, which is considered as one worst case product batch for one Ambr® bioreactor. (Eq. (1).

In this study, a risk analysis of the medium that will be administered to the patient will be demonstrated without taking the PERL distribution between cells and liquid phase into account. (Hauk et al., 2023) In order to assess the potential hazard to the patient the patient exposure is calculated using the data of Table 3.

Exposure is calculated based on the daily dose administered and the surface area and working volume of the microbioreactor module. (Hauk et al., 2021) (Hauk et al., 2021) This is a worst-case assumption, as it does not take into account any possible reduction in extracted substances through downstream purification processes. The aim of the risk assessment is to check whether the amount of substance administered is toxicologically relevant. Safety limits such as the permissible daily dose (PDE) and the toxicological threshold (TTC) are used for this purpose. (International Council for Harmonization (ICH): Q3C (R6) - Guideline for Residual Solvents, 2019; European Medicines Agency, 2023b) Comparing these limits with patient exposure gives the safety margin, which must be ≥1 for a successful assessment. (I. 60812, 2006)

In Table 5 the safety margin of the entire extractables profile is summarized. The calculation of the patient exposure and safety margin are performed below using BPA exemplarily. The concentration of BPA in the recycled bioreactor vessel is 0.041 μg per cm². The calculation of the patient exposure value is carried out according to Eq. (2). This results in a patient exposure value for BPA of 1.29 μg per day $(\mathit{Patient\; Exposure\; Value}=\frac{0.041\mathit{\mu g}/\mathrm{cm}2\times 63\mathrm{cm}2}{15\mathit{ml}}\times \frac{7.5\mathit{ml}}{\mathit{day}}=1.29\mathit{\mu g}/\mathit{day}$), which means that under “worst-case” assumptions, the patient would be exposed with 1.29 μg of BPA per day.

Table 5.

Safety margin of all compounds.

Parris et al. determined a PDE value for BPA of 4.2 μg per day. (see Table 3) (Parris et al., 2020) and therefore a safety margin of 3 ($\mathit{Safety\; margin}=\frac{4.2\mathit{\mu g}/\mathit{day}}{1.29\mathit{\mu g}/\mathit{day}}=3$) can be calculated. Although this calculated safety margin indicates an acceptable risk, it is noteworthy that the hazardous potential of BPA is still not conclusively discussed and assessed. (Tsai, 2006)

This method of risk assessment is applicable to all extractable substances and is intended to determine the “safety profile” of extracts from recycled materials. The color coded heat maps in Table 5 illustrates the safety threshold of each extractable substance: green indicates that the limit has not been exceeded (safety threshold ≥1), while red indicates that the limit has been exceeded (safety threshold <1) in our model application scenario. Substances that are not detected or are below the reporting limit are not taken into account and are also given a green color code, as they are considered acceptable.

The recycling process described poses negligible toxicological risk compared to using virgin material according to the conditions outlined in the selected patient exposure scenario, as shown by the heat maps in Table 5. All margins of safety are greater than 1, indicating that the dose is less than PDE, even under the most adverse conditions.

$${\mathrm{V}}_{\mathrm{T}}=\frac{\text{Viable Cell Density}\ast {\mathrm{m}}_{\text{patient}}}{{\mathrm{c}}_{\text{cell}}}$$ (1)

V_T = Infusion volume [mL/day]; $c_{\mathit{cell}}$ = Concentration [cell number/kg]; $m_{\mathit{patient}}$= body weight [kg];

Viable Cell Density = Total number of CD34+ cells [cell number/mL]

$$\text{Patient Exposure Value}=\frac{\mathrm{c}\times \mathrm{A}}{{\mathrm{V}}_{\mathrm{L}}}\times {\mathrm{V}}_{\mathrm{T}}$$ (2)

Patient exposure value [μg/day]; c = concentration of extractables[μg/cm²]; A = surface area [cm²].

V_L = work volume [mL]; V_T = volume per day [mL/day]

$$\text{Safety margin}=\frac{\mathrm{PDE}}{\text{Patient Exposure Value}}\text{or}$$

$$\text{Safety margin}=\frac{\mathrm{TTC}}{\text{Patient Exposure Value}}$$ (3)

safety margin; PDE = Permitted Daily Exposure [μg/day]; Patient exposure value [μg/day]; TTC = Threshold of Toxicological Concern [μg/day].

5.2. Exposure to cells (product quality)

At the cellular level, it is crucial to analyze the toxicological effects of the extractables to identify potential hazards to cell health and function during cultivation in the bioreactor. A comprehensive risk assessment includes the identification and quantification of extractables as well as the assessment of their biological relevance and potential effects on cellular systems. This analysis is essential to ensure the quality of biopharmaceutical “cell-based-product” and to ensure patient safety. (Aysola et al., 2020)

In the cellular context, we consider the highest concentration of a substance at which no negative effects on the cells are detected as relevant for toxicological assessment. Since no data are available for the CD34+ cell line, we used published biological data of a surrogate cell line (U2OS human cell line). (Pahl et al., 2024) The authors are aware that U2OS cell line is not a highly sensitive, but a robust cell line for which a huge toxicological data base exists.1 This publication reports as highest concentration without observable biological effects for BPA 11.4 μg/mL and for Phenol 4.7 μg/mL.2 Therefore, the risk assessment is carried out using these two substances as examples. (Thangaraju and Varthya, 2022)

In this case “exposure” is just the maximum concentration a substance can reach in the bioreactor. To determine this value, the extractables concentration [μg/cm²] given in Table 3 is multiplied by the area of the microbioreactor [63 cm²] and then divided by the in-use volume. The safety margin is calculated with Eq. (4). (Hauk et al., 2021). For phenol, which was not detected in the recycled bioreactor vessel we used the reporting limit for exposure calculation.

The values calculated in Table 6 and Table 7 have a safety margin above 1.

$${\mathit{safety\; margin}}_{\mathit{cell}}=\frac{\mathit{highest\; concentration\; without\; biologic\; effect}}{\mathit{exposure\; value}}$$ (4)

highest concentration without biologic effect [μg/mL]; Exposure value [μg/mL].

Table 6.

Overview of the safety margin_cell for bisphenol A.^⁎, ^⁎⁎

${\mathit{safety\; margin}}_{\mathit{cell}}=\frac{11.4\mathit{\mu g}/\mathit{ml}}{\mathit{exposure\; value}}$
PC virgin granules	virgin bioreactor vessel	PC recycled granules	recycled bioreactor vessel
${\mathit{safety\; margin}}_{\mathit{cell}}=\frac{11.4\mathit{\mu g}/\mathit{ml}}{\le 0.30\mathit{\mu g}/{\mathit{ml}}^{\ast \ast }}$	${\mathit{safety\; margin}}_{\mathit{cell}}=\frac{11.4\mathit{\mu g}/\mathit{ml}}{0.13\mathit{\mu g}/\mathit{ml}}$	${\mathit{safety\; margin}}_{\mathit{cell}}=\frac{11.4\mathit{\mu g}/\mathit{ml}}{0.14\mathit{\mu g}/\mathit{ml}}$	${\mathit{safety\; margin}}_{\mathit{cell}}=\frac{11.4\mathit{\mu g}/\mathit{ml}}{0.17\mathit{\mu g}/\mathit{ml}}$
${\mathit{safety\; margin}}_{\mathit{cell}}$≥38	${\mathit{safety\; margin}}_{\mathit{cell}}$≈ 88	${\mathit{safety\; margin}}_{\mathit{cell}}$≈ 82	${\mathit{safety\; margin}}_{\mathit{cell}}$≈ 67

^⁎Reporting limit GC–MS.

^⁎⁎Reporting limit HPLC.

Table 7.

Overview of the safety margin_cell for phenol.^⁎, ^⁎⁎

${\mathit{safety\; margin}}_{\mathit{cell}}=\frac{4.7\mathit{\mu g}/\mathit{ml}}{\mathit{exposure\; value}}$
PC virgin granules	virgin bioreactor vessel	PC recycled granules	recycled bioreactor vessel
${\mathit{safety\; margin}}_{\mathit{cell}}=\frac{4.7\mathit{\mu g}/\mathit{ml}}{\le 0.10\mathit{\mu g}/{\mathit{ml}}^{\ast }}$	${\mathit{safety\; margin}}_{\mathit{cell}}=\frac{4.7\mathit{\mu g}/\mathit{ml}}{0.21\mathit{\mu g}/\mathit{ml}}$	${\mathit{safety\; margin}}_{\mathit{cell}}=\frac{4.7\mathit{\mu g}/\mathit{ml}}{\le 0.10\mathit{\mu g}/{\mathit{ml}}^{\ast }}$	${\mathit{safety\; margin}}_{\mathit{cell}}=\frac{4.7\mathit{\mu g}/\mathit{ml}}{\le 0.10\mathit{\mu g}/{\mathit{ml}}^{\ast }}$
${\mathit{safety\; margin}}_{\mathit{cell}}$≥ 47	${\mathit{safety\; margin}}_{\mathit{cell}}$≈ 22	${\mathit{safety\; margin}}_{\mathit{cell}}$≥ 47	${\mathit{safety\; margin}}_{\mathit{cell}}$≥ 47

^⁎Reporting limit GC–MS.

^⁎⁎Reporting limit HPLC.

6. Conclusions

Mechanical recycling is highlighted as a potentially crucial step towards sustainable practices, as it could provide a solution to reduce greenhouse gas emissions and conserve resources without compromising product quality. Yet, high quality and regulatory requirements, especially with regards to extractables, hinder the use of recycled material in biopharmaceutical applications. This feasibility study evaluates the extractables profile of PC in microbioreactors over the entire life cycle of a closed loop recycling process. Ethanol was used as a worst-case extraction solution that effectively extracts both polar and nonpolar substances from the plastic samples.

A toxicological safety assessment of patient risk and of a hypothetic therapeutic cell line was conducted for comparison of recycled versus non-recycled polycarbonate.

The findings demonstrate that the recycling process introduces certain degradation products, such as p- tert- butylphenol and 4-hydroxyacetophenone, which are indicative of chain length reduction and polymer degradation. In addition, the recycling process may effectively remove substances, such as adhesive residues, and preserve the chemical integrity of the recycled material. The recycling process does not compromise the quality and safety of pharmaceutical products.

Of particular note is that BPA, a compound with a known endocrine disruption potential, does slightly accumulate during recycling up to 0.041 g/cm² in recycled microbioreactors. Yet the model toxicological safety assessment showed that the exposure administered to a patient under worst case conditions is below the PDE. Additionally, these concentrations do not pose significant risks to cells in the provided exposure scenario. (Aysola et al., 2020) Yet, it must be mentioned, that further toxicological evaluations of BPA as an endocrine disruptor for individual applications is beyond the scope of this feasibility study.

The results support the cautious adoption of recycling practices in pharmaceutical applications, emphasizing their safety and efficiency. (Thangaraju and Varthya, 2022)

This feasibility study confirms that mechanically recycled materials could be used continuously from an extraction perspective. It underscores the need for strict guidelines tailored to specific plastics in order to ensure patient safety and product quality in the pharmaceutical and biopharmaceutical sectors. Additionally, the study advocates further research to enhance the recycling process and improve material quality, particularly addressing variations in oligomer concentrations during recycling. Overall, the study endorses the use of recycled PC in pharmaceutical applications, underscoring its potential for sustainable practices while maintaining safety and effectiveness.

CRediT authorship contribution statement

Janice Zitoun: Writing – original draft, Visualization, Methodology, Investigation, Formal analysis. Jannik Dippel: Writing – review & editing, Supervision. Ina Pahl: Writing – review & editing. Magali Barbaroux: Writing – review & editing, Resources. Armin Hauk: Writing – review & editing.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Janice Zitoun, Jannik Dippel, Ina Pahl, Magali Barbaroux, Armin Hauk, reports a relationship with Sartorius Stedim Biotech GmbH that includes: employment.

Data availability

No data was used for the research described in the article.