Hans Lee

Identification of a leachable compound detrimental to cell growth in single-use bioprocess containers.

Out of the plethora of chemical species extractable at low levels from the materials of construction of single-use bioprocess containers, we have identified one particularly conspicuous compound and shown it to be highly detrimental to cell growth. The compound, bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP), is derived from the breakdown of tris(2,4-di-tert-butylphenyl)phosphite (trade name Irgafos 168®), a common antioxidant additive present in many formulations of polyethylene (one of the polymers commonly used as the material contacting process fluids in bioprocess containers). Cell growth experiments using several mammalian cell lines and growth media spiked with bDtBPP show harmful effects at concentrations well below the parts-per-million range. Cellular response to bDtBPP is rapid, and results in a significant decrease in mitochondrial membrane potential. The migration of bDtBPP from polyethylene-based films is shown to be time- and temperature-dependent. Further, experiments suggest that exposure of oxidized Irgafos 168 to ionizing radiation (such as gamma irradiation) is an important condition for the generation of significant amounts of leachable bDtBPP. LAY ABSTRACT: Biopharmaceuticals are drugs manufactured using cells that are genetically engineered to produce a therapeutic protein. A current trend in biomanufacturing is the replacement of hard-plumbed stainless steel vessels (where these cells are grown) with specialized, pre-sterilized, disposable plastic bags. While this move has significant environmental and cost benefits, the effect of plastics on the biomanufacturing process is not yet completely understood. Here we show that if a chemical compound formed by the breakdown of a common antioxidant additive to plastics leaches into the cell culture liquid, the growth of mammalian cells is strongly inhibited. Some of the factors that promote the generation of this compound, and the conditions that favor migration of the compound into process fluids, are explored here.

A cytotoxic leachable compound from single‐use bioprocess equipment that causes poor cell growth performance

A current trend in the production of biopharmaceuticals is the replacement of fixed stainless steel fluid‐handling units with disposable plastic bags. Such single‐use systems (SUS) offer numerous advantages, but also introduce a new set of materials into the production process and consequently expose biomanufacturers to a new set of risks related to those materials, not to mention reliance on an entirely new supply chain. In the course of developing and conducting a cell‐growth‐based test for suitability of disposable plastic components destined for use in cell culture operations, we discovered that the cytotoxic compound bis(2,4‐di‐tert‐butylphenyl)phosphate (bDtBPP) leaches out of certain bags and into cell culture media in concentrations that are deleterious to cell growth. Specifically, media held in certain bags for several days at 37°C was found to contain bDtBPP, and use of those held‐media samples in cell growth experiments provides data that overlap neatly with cell growth experiments using media spiked directly with bDtBPP, proving that bDtBPP leaching is responsible for the reduced growth attributable to those SUS bags. Overall, this issue represents a risk to the production of biopharmaceuticals in SUS, a risk that must be managed by diligent collaboration among companies along the entire supply chain for SUS components.

Creating a Holistic Extractables and Leachables (E&L) Program for Biotechnology Products

The risk mitigation of extractables and leachables presents significant challenges to regulators and drug manufacturers with respect to the development, as well as the lifecycle management, of drug products. A holistic program is proposed, using a science- and risk-based strategy for testing extractables and leachables from primary containers, drug delivery devices, and single-use systems for the manufacture of biotechnology products. The strategy adopts the principles and concepts from ICH Q9 and ICH Q8(R2). The strategy is phase-appropriate, progressing from extractables testing for material screening/selection/qualification through leachables testing of final products. The strategy is designed primarily to ensure patient safety and product quality of biotechnology products. The holistic program requires robust extraction studies using model solvents, with careful consideration of solvation effect, pH, ionic strength, temperature, and product-contact surface and duration. From a wide variety of process- and product-contact materials, such extraction studies have identified and quantified over 200 organic extractable compounds. The most commonly observed compounds were siloxanes, fatty acid amides, and methacrylates. Toxicology assessments were conducted on these compounds using risk-based decision analysis. Parenteral permitted daily exposure limits were derived, as appropriate, for the majority of these compounds. Analysis of the derived parenteral permitted daily exposure limits helped to establish action thresholds to target high-risk leachables in drug products on stability until expiry. Action thresholds serve to trigger quality investigations to determine potential product impact. The holistic program also evaluates the potential risk for immunogenicity. This approach for primary drug containers and delivery devices is also applicable to single-use systems when justified with a historical knowledge base and understanding of the manufacturing processes of biotechnology products. LAY ABSTRACT: In the development of a drug product, careful consideration is given to impurities that may originate from manufacturing equipment, process components, and packaging materials. The majority of such impurities are common chemical additives used to improve the physicochemical properties of a wide range of plastic materials. Suppliers and drug manufacturers conduct studies to extract chemical additives from the plastic materials in order to screen and predict those that may leach into a drug product. In this context, the term extractables refers to a profile of extracted compounds observed in studies under harsh conditions. In contrast, the term leachables refers to those impurities that leach from the materials under real-use conditions and may be present in final drug products. The purpose of this article is to present a holistic approach that effectively minimizes the risk of leachables to patient safety and product quality.

Extractables Analysis of Single-Use Flexible Plastic Biocontainers

Studies of the extractable profiles of bioprocessing components have become an integral part of drug development efforts to minimize possible compromise in process performance, decrease in drug product quality, and potential safety risk to patients due to the possibility of small molecules leaching out from the components. In this study, an effective extraction solvent system was developed to evaluate the organic extractable profiles of single-use bioprocess equipment, which has been gaining increasing popularity in the biopharmaceutical industry because of the many advantages over the traditional stainless steel-based bioreactors and other fluid mixing and storage vessels. The chosen extraction conditions were intended to represent aggressive conditions relative to the application of single-use bags in biopharmaceutical manufacture, in which aqueous based systems are largely utilized. Those extraction conditions, along with a non-targeted analytical strategy, allowed for the generation and identification of an array of extractable compounds; a total of 53 organic compounds were identified from four types of commercially available single-use bags, the majority of which are degradation products of polymer additives. The success of this overall extractables analysis strategy was reflected partially by the effectiveness in the extraction and identification of a compound that was later found to be highly detrimental to mammalian cell growth. LAY ABSTRACT: The usage of single-use bioreactors has been increasing in biopharmaceutical industry because of the appealing advantages that it promises regarding to the cleaning, sterilization, operational flexibility, and so on, during manufacturing of biologics. However, compared to its conventional counterparts based mainly on stainless steel, single-use bioreactors are more susceptible to potential problems associated with compound leaching into the bioprocessing fluid. As a result, extractable profiling of the single-use system has become essential in the qualification of such systems for its use in drug manufacturing. The aim of this study is to evaluate the effectiveness of an extraction solvent system developed to study the extraction profile of single-use bioreactors in which aqueous-based systems are largely used. The results showed that with a non-targeted analytical approach, the extraction solvent allowed the generation and identification of an array of extractable compounds from four commercially available single-use bioreactors. Most of extractables are degradation products of polymer additives, among which was a compound that was later found to be highly detrimental to mammalian cell growth.

Ambient analysis of leachable compounds from single-use bioreactors with desorption electrospray ionization time-of-flight mass spectrometry

RATIONALE Trace levels of bis(2,4-di-tert-butylphenyl)phosphate (BdtbPP) leaching from single-use bioreactors (SUBs) were recently found to be highly detrimental to mammalian cell growth. The traditional approach to detect the leachable requires time-consuming solvent extraction of SUBs. To assist the qualification of SUBs and/or their manufacturing raw materials in biopharmaceutical development and manufacturing, it is essential to develop a rapid and sensitive analytical approach for detecting this leachable and related compounds, which is described in this study. METHODS Native films from several commercially available SUBs were directly examined by desorption electrospray ionization (DESI) time-of-flight mass spectrometry (TOFMS) without sample preparation. As a comparison, the same SUBs were also analyzed by high-performance liquid chromatography (HPLC)/ultraviolet (UV) following the solvent extraction. RESULTS With a suitable spray solvent selected in this study, DESI-TOFMS enabled rapid and sensitive detection of leachable compounds directly from SUBs. Accurate mass measurement from TOFMS allowed ready identification of BdtbPP, its parent analog compound, and other polymer components in the SUBs from their protonated surrogates. The relative abundances of BdtbPP in different SUBs measured by DESI-TOFMS exhibited good correlation with those from the traditional extraction-based approach with HPLC/UV. CONCLUSIONS A rapid and sensitive approach was developed for direct detection of BdtbPP and other leachables from SUBs using DESI-TOFMS. The results are in high accordance with those from conventional approaches, which indicates the usefulness of the proposed method as a qualification tool for SUBs in biopharmaceutical development and also its great potential in the analysis of extractables/leachables in a wide variety of materials, process components, devices and containers used in the pharmaceutical industry.

Characterization of Protein Aggregating Tungstates: Electrospray Mass Spectrometry Analysis of Extracts from Prefilled Syringes and from Tungsten Pins Used in the Manufacture of Syringes

Glass prefilled syringes are increasingly becoming a container of choice for storing and administering therapeutic protein products to patients. Tungsten leaching from a PFS is known to induce protein particle formation, and the source was traced to the tungsten pins used in the manufacturing process of the syringe barrels. Study of the tungstates present in extracts from both tungsten pins used in the syringe manufacturing process and from single syringes from various suppliers was undertaken. Electrospray mass spectrometry was chosen as a technique with the sensitivity to characterize tungstates at levels (∼1 ppm of elemental tungsten) observed in single syringes. Extraction solvents were chosen to simulate the range (pH 4.0–7.0) typically used for therapeutic protein formulation. A commercial product formulation buffer was also used as an extraction solution to characterize tungstate species used for tungsten spiking studies of protein. All pin and syringe extracts from various manufacturers were similar in regards to containing stable Na/K containing lacunary polytungstate ([W11O39]7−) species, which were the main species present in syringe extracts and are different than the metatungstate ([W12O39]6−) species identified in commercially available sodium polytungstate and as the main species in pin extracts. These stable Na/K containing polytungstates species present in pin and syringe extracts are likely formed during the glass manufacturing process at >400 °C and may have the capability to subsequently form larger polytungstate complexes. LAY ABSTRACT: Glass prefilled syringes are a type of container used for storing and administering biotechnology medicines to patients. The manufacturing process for the syringes may lead to very low levels of the metal tungsten being present in the syringes, and thus in the medicine stored in the syringes. The presence of tungsten in certain biotechnology medicines has been shown to cause changes to the medicine. Understanding something that can cause a medicine to change is an important part of producing safe and effective medicines for patients. The study described in this article sought to increase understanding by characterizing the form of tungsten observed in syringes from a number of vendors. Study of the tungsten present in syringes from four vendors indicates the same form of tungsten is observed regardless of the vendor. The study also found that the form of tungsten differed from that expected.

Development of an Inductively Coupled Plasma Mass Spectrometry Method for Quantification of Extracted Tungsten from Glass Prefilled Syringes Used as a Primary Packaging for Pharmaceutical and Therapeutic Protein Products

Leachable tungsten is associated with protein aggregation and precipitation in glass prefilled syringes, and this may trigger immunogenicity concerns. Determining the level of leachable tungsten from glass prefilled syringes is critical for assuring quality of certain biopharmaceutical drug products. An inductively coupled plasma mass spectrometry (ICP/MS) quantification method was developed to determine elemental tungsten in syringe extracts. The syringe was extracted using 0.5% ammonium hydroxide (pH 11), heat (75 °C), and sonication. The resulting extraction solution was diluted 10 fold prior to ICP/MS analysis. Syringes from three syringe lots containing known low (average 28.0 ng), medium (average 189.4 ng), and high (average 631.9 ng) levels of tungsten were extracted three times each. All syringes with total tungsten greater than 14 ng had extraction efficiency greater than 90% with the first two extractions combined. The calibration curve range was 0.1–200 μg/L tungsten with iridium as the internal standard, and the correlation coefficient was ≥1.0000. The limit of detection at 0.05 μg/L tungsten and limit of quantification at 0.1 μg/L tungsten were determined as having a signal-to-noise ratio greater than 40 and 80 times compared with the blank, respectively. The ICP/MS method was selective for tungsten and iridium in the presence of other metals. Accuracies of spiked tungsten, at three different levels, in syringe extracts were >99% with precision relative standard deviation (RSD) (n = 5) of ≤1%. The matrix effect of the syringe extract media and carryover of tungsten and internal standard were negligible. Onboard stability of the syringe extracts over three days had a tungsten concentration RSD (n = 3) of ≤1%. Syringe extractions performed with 0.45–0.55% ammonium hydroxide had spike recoveries ≥99% and demonstrated extraction solution robustness. Quantified residual tungsten in syringes extract by ammonium hydroxide and analyzed by ICP/MS was acceptable based on extraction efficiency and method performance. LAY ABSTRACT: Elemental tungsten is a known leachable from glass prefilled syringe used as a ready-to-inject drug device in the pharmaceutical industry. Tungsten is a residual artifact from the manufacturing process of the syringe. The leachable tungsten level is of a concern, as it can affect the quality of the filled drug product. To understand possible leachable quantity of tungsten from the prefilled syringe, a tungsten extraction conditions and quantification method were developed. Double extraction of the syringe with 0.5% ammonium hydroxide (pH 11), heat (75 °C), and sonication was able to efficiently extract 90% of the total tungsten from syringe. An inductively coupled plasma mass spectrometry method was qualified to selectively, accurately, and precisely quantify the extracted tungsten. The developed extraction and quantification method was acceptable in determining possible leachable tungsten from prefilled syringes.

Comparison of Acid Titration, Conductivity, Flame Photometry, ICP-MS, and Accelerated Lamellae Formation Techniques in Determining Glass Vial Quality

Certain types of glass vials used as primary containers for liquid formulations of biopharmaceutical drug products have been observed with delamination that produced small glass like flakes termed lamellae under certain conditions during storage. The cause of this delamination is in part related to the glass surface defects, which renders the vials susceptible to flaking, and lamellae are formed during the high-temperature melting and annealing used for vial fabrication and shaping. The current European Pharmacopoeia method to assess glass vial quality utilizes acid titration of vial extract pools to determine hydrolytic resistance or alkalinity. Four alternative techniques with improved throughput, convenience, and/or comprehension were examined by subjecting seven lots of vials to analysis by all techniques. The first three new techniques of conductivity, flame photometry, and inductively coupled plasma mass spectrometry measured the same sample pools as acid titration. All three showed good correlation with alkalinity: conductivity (R2 = 0.9951), flame photometry sodium (R2 = 0.9895), and several elements by inductively coupled plasma mass spectrometry [(sodium (R2 = 0.9869), boron (R2 = 0.9796), silicon (R2 = 0.9426), total (R2 = 0.9639)]. The fourth technique processed the vials under conditions that promote delamination, termed accelerated lamellae formation, and then inspected those vials visually for lamellae. The visual inspection results without the lot with different processing condition correlated well with alkalinity (R2 = 0.9474). Due to vial processing differences affecting alkalinity measurements and delamination propensity differently, the ratio of silicon and sodium measurements from inductively coupled plasma mass spectrometry was the most informative technique to assess overall vial quality and vial propensity for lamellae formation. The other techniques of conductivity, flame photometry, and accelerated lamellae formation condition may still be suitable for routine screening of vial lots produced under consistent processes. LAY ABSTRACT: Recently, delamination that produced small glass like flakes termed lamellae has been observed in glass vials that are commonly used as primary containers for pharmaceutical drug products under certain conditions during storage. The main cause of these lamellae was the quality of the glass itself related to the manufacturing process. Current European Pharmacopoeia method to assess glass vial quality utilizes acid titration of vial extract pools to determine hydrolytic resistance or alkalinity. As alternative to the European Pharmacopoeia method, four other techniques were assessed. Three new techniques of conductivity, flame photometry, and inductively coupled plasma mass spectrometry measured the vial extract pool as acid titration to quantify quality, and they demonstrated good correlation with original alkalinity. The fourth technique processed the vials under conditions that promote delamination, termed accelerated lamellae formation, and the vials were then inspected visually for lamellae. The accelerated lamellae formation technique also showed good correlation with alkalinity. Of the new four techniques, inductively coupled plasma mass spectrometry was the most informative technique to assess overall vial quality even with differences in processing between vial lots. Other three techniques were still suitable for routine screening of vial lots produced under consistent processes.

Detection of adulteration in acetonitrile.

To address the increasing concern that acetonitrile may be intentionally adulterated to meet the shortfall in global supplies resulting from a downturn in its manufacturing, three analytical techniques were examined in this study. Gas Chromatography with Thermal Conductivity Detection (GC-TCD), Near Infrared (NIR) spectroscopy and Fourier Transform Infrared (FT-IR) spectroscopy were assessed for their ability to detect and quantify potential adulterants including water, alternative organic solvents, and by-products associated with the production of acetonitrile. The results of the assessment of the three techniques for acetonitrile adulteration testing are discussed.

Development of Conductivity Method as an Alternative to Titration for Hydrolytic Resistance Testing Used for Evaluation of Glass Vials Used in Pharmaceutical Industry

The European Pharmacopeia surface test to analyze the hydrolytic resistance is a common industrial method to understand and ensure the quality of produced glass vials. Hydrolytic resistance is evaluated by calculating the alkalinity of water extract from autoclaved vials by titration. As an alternative to this titration technique, a conductivity technique was assessed, which directly measures the ions in the water extract. A conductivity meter with a 12 mm diameter electrode was calibrated with a 100 μS/cm conductivity standard and carryover minimized by rinsing the probe in a water beaker per analysis. The limit of quantification at 1 μS/cm was determined as having a signal-to-noise ratio of 3 compared with the water blank. The conductivity method was selective for glass-composing elements (boron, sodium, aluminum, silicon, potassium, and calcium) within the vial extract. Accuracies of spiked conductivity standard within the range of 1 to 100 μS/cm were ±7% and had linearity with coefficient of determination (R2) of ≥0.9999. Intraday precision had a relative standard deviation (RSD) (n = 5) of ≤6% for spiked conductivity standard within the range of 1 to 100 μS/cm. Interday precision had a RSD (n = 4) of ≤6% for 10 vials from three glass vial lots. Conductivity of water extracts from nine sets of seven lots of glass vials had a precise linear relationship [R2 = 0.9876, RSD = 1% (n = 9)] with titration volumes of the same lots. Conductivity results in μS/cm could be converted to titration volumes in milliliters by a conversion factor of 0.0275. The simplicity, sample stability, and individual vial analysis of the conductivity technique were more advantageous than the current titration technique. LAY ABSTRACT: The quality of glass vials used as primary containers in the pharmaceutical industry is of concern due to recent observations of glass flake-like delamination, or lamellae, under specific storage conditions. The current European Pharmacopoeia method to assess glass vial quality utilizes acid titration of vial extract pools to determine hydrolytic resistance or alkalinity. As an alternative to the European Pharmacopoeia method, the vial extracts were analyzed for conductivity, which directly determines the level of ions that were readily extracted from the vial surfaces. Lower quality glass would have greater surface defects that lead to higher ions extracted and higher conductivity value. The conductivity method was found to be suitable to measure the ions in water extracts and showed strong correlation with alkalinity. The advantage of the conductivity method over the alkalinity method was greater ease, lower volume requirements, stability, and flexibility in analysis.