Analysis of Silicone Oil in Prefilled Syringes and Biopharmaceutical Drug Products Using High-Performance Liquid Chromatography.

As the packaging of choice for many therapeutic proteins, prefilled syringes have been widely used in biopharmaceutical industry as primary containers, where silicone oil is applied to ensure their proper functionality. Adequate lubrication from sufficient amount of silicone oil and its appropriate distribution across syringe barrels is crucial for successful administration of drug product (DP) from the prefilled syringes; however, silicone oil is also susceptible to leaching from the syringe surface into the formulation with the potential to interact with therapeutic proteins, which could lead to the formation of visible and sub-visible aggregates and/or particles that are potentially immunogenic. Accurate determination and careful control of silicone oil levels in both empty syringes and protein drug products are therefore critical in process development to ensure syringe functionality, drug product quality, and patient safety. On the other hand, analysis of silicone oil can be challenging especially when the analysis is performed on formulated protein drug products, where matrix effects could be significant. It is demonstrated in this study that silicone oil in empty syringes or formulated drug products can be extracted effectively using organic solvents and quantitatively determined using high-performance liquid chromatography (HPLC) coupled with a universal detector. It was also shown that direct extraction of silicone oil from formulated protein drug products can be very challenging, but pretreatment of the protein drug products with pepsin enzymatic digestion facilitated the extraction process, which enabled the analysis of silicone oil in the drug product at low ppm levels.

Chemical and Biophysical Characteristics of Monoclonal Antibody Solutions Containing Aggregates Formed during Metal Catalyzed Oxidation.

PURPOSE: To physicochemically characterize and compare monoclonal antibody (mAb) solutions containing aggregates generated via metal catalyzed oxidation (MCO). METHODS: Two monoclonal IgG2s (mAb1 and mAb2) and one monoclonal IgG1 (rituximab) were exposed to MCO with the copper/ascorbic acid oxidative system, by using several different methods. The products obtained were characterized by complementary techniques for aggregate and particle analysis (from oligomers to micron sized species), and mass spectrometry methods to determine the residual copper content and chemical modifications of the proteins. RESULTS: The particle size distribution and the morphology of the protein aggregates generated were similar for all mAbs, independent of the MCO method used. There were differences in both residual copper content and in chemical modification of specific residues, which appear to be dependent on both the protein sequence and the protocol used. All products showed a significant increase in the levels of oxidized His, Trp, and Met residues, with differences in extent of modification and specific amino acid residues modified. CONCLUSION: The extent of total oxidation and the amino acid residues with the greatest oxidation rate depend on a combination of the MCO method used and the protein sequence.

A Holistic Approach of Extractables and Leachables Assessment of Rubber Stoppered Glass Vial Systems for Biotechnology Products.

Rubber stoppered glass vial systems are widely used as primary containers for storing and delivering therapeutic protein products to patients. Addressing concerns and regulatory expectations related to the risk to biologic drug product quality and patient safety from rubber stoppered glass vial systems requires implementation of an extractable and leachable evaluation program based on material understanding, risk assessment, literature review, and a comprehensive scientifically sound analytical testing methodology. The extractable and leachable study design consisted of twelve drug products filled in twelve different size glass vials capped with laminated and nonlaminated rubber stoppers made from three different rubber formulations. Design of the model solvents was successful as they had little to no analytical interference and mimicked the formulation conditions and generated representative extractables capable of predicting leachables. The extraction conditions of time and temperature were appropriate as not to degrade the test materials or the extractable compounds, and yet generated significant quantities for identification of the extractable compounds with confidence. The extractables testing results were capable of predicting the leachable profiles of the twelve drug products. In each case, the leachable profile was a subset of the extractable profile. The organic and elemental impurities of the leachable profiles of drug products were the end-to-end verification of the quality of the glass vials, rubber stoppers and drug product lifecycles. Overall, the holistic approach was fully successful in the qualification of different vial systems as primary containers and delivery systems for different biotherapeutic products to ensure product quality and patient safety.

A Risk-Based Approach to Evaluate and Control Elemental Impurities in Therapeutic Proteins.

Control of elemental impurities in the drug products evolved from the generic visual testing of heavy metals as their sulfides to specific elements of toxicological concern in the final drug products by instrumental analysis. The International Council for Harmonisation (ICH) Q3D (R1) guideline for elemental impurities describes a risk-based approach to identify, assess, and control the potential elemental impurities in drug products within the established permitted daily exposures (PDE). Challenges to this approach include how to assess the risks associated with contributing sources such as utilities, manufacturing equipment, container-closure systems, and excipients. Defining at what stage of development that such assessment should be performed to identify the risk levels can be equally challenging. In this article, we report an approach to control elemental impurities of toxicological concern, compliant to the Q3D (R1) guideline, and a summary of results obtained on multiple protein therapeutic products. This approach follows the elements of Process Validation, i.e., Design, Qualification, and Continuous Verification. The design includes the selection of excipients and their suppliers that meet the Option 1 requirement of Q3D (R1). It also comprises the selection of manufacturing equipment, container-closure systems, and utilities. The qualification includes the testing of the potential sources of elemental impurities, i.e., excipients, utilities, and leachables/extractables from the manufacturing equipment and container-closure systems. The Continuous Verification comes from the testing of representative batches at the initiation of stability studies of clinical or commercial drug product batches and at the end of shelf-life expiry of the drug product, and when changes are made to the manufacturing equipment, sources of excipients and container closure systems, and any formulation changes. Our experience shows that the risk associated with the impurity levels of the ten elements of toxicological concern in the therapeutic protein drug products, parenterally administered, is well below the control threshold (30% PDE) in the drug product recommended by the ICH Guideline. Although our focus is on the injectable therapeutic proteins, this approach can be applied to the products administered via other routes as well.

Holistic Extractables and Leachables Program: Evaluations of Prefilled Syringe Systems for Biotechnology Products.

Prefilled syringes (PFS) are a container and delivery device of choice for storing and administering therapeutic protein products to patients. Addressing concerns and regulatory expectations related to the risk to biologic drug product quality and patient safety from PFS requires implementation of an extractable and leachable program based on understanding of materials, risk assessment, review of existing literature, and testing supported by a sound scientific foundation. Extractables and leachables data generated as part of a thorough and holistic program are presented for five PFS systems, including glass and plastic syringes filled with 12 biologic drug products encompassing the implementation of traditional and single-use biotechnology manufacturing processes. The comprehensive extractables and leachables data presented demonstrate and substantiate a holistic extractable and leachable program designed to ensure product quality and patient safety.

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.

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

UNLABELLED: 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.

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 (R(2) = 0.9951), flame photometry sodium (R(2) = 0.9895), and several elements by inductively coupled plasma mass spectrometry [(sodium (R(2) = 0.9869), boron (R(2) = 0.9796), silicon (R(2) = 0.9426), total (R(2) = 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 (R(2) = 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.

Extractables analysis of single-use flexible plastic biocontainers.

UNLABELLED: 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.

Single burr hole evacuation for traumatic acute subdural hematoma of the posterior fossa in the emergency room.

A 57-year-old man and a 55-year-old man presented with acute subdural hematoma of the posterior cranial fossa due to trauma. Both were comatose preoperatively. Emergent single burr hole evacuation in the posterior cranial fossa was performed in the emergency room immediately after computed tomography. Neurological symptoms improved dramatically just after initiating the burr hole evacuation in both patients. A 57-year-old man became alert and could walk unassisted 1 month after surgery. The other could walk with assistance 4 months after surgery, although psychic disturbance resulting from cerebral contusion remained. Single burr hole evacuation in the emergency room is a useful treatment for acute subdural hematoma of the posterior cranial fossa because the procedure can be performed easily and rapidly, thus achieving reduction of intracranial pressure. Progressing neurological deterioration, reversibility of brainstem function by mannitol administration and the sign of brainstem compression and noncommunicating hydrocephalus are good indicators for this treatment.

A case study of nondelamination glass dissolution resulting in visible particles: implications for neutral pH formulations.

Visible particles were unexpectedly observed in a neutral-pH placebo formulation stored in glass vials but were not observed in the same formulation composition that contained protein. The particles were identified as silica gel (SiO2 ) and polysorbate 20, suggesting dissolution of the glass vial. Time course studies were performed to assess the effect of variables such as pH, excipients, storage temperature, and duration on particle formation. Data suggest that glass dissolution occurred during the storage in the liquid state, as shown by increased Si levels in solution. Upon freezing, the samples underwent freeze concentration and likely became supersaturated, which resulted in the appearance of visible silica particles upon thawing. The glass degradation described here is unique and differs from the more commonly reported delamination, defined by the presence of reflective, shard-like glass flakes in solution that are often termed lamellae. This case study underscores the importance of an early assessment (during formulation development) of potential incompatibility of the formulation with the primary container.

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.

Hydroxocobalamin association during cell culture results in pink therapeutic proteins.

Process control of protein therapeutic manufacturing is central to ensuring the product is both safe and efficacious for patients. In this work, we investigate the cause of pink color variability in development lots of monoclonal antibody (mAb) and Fc-fusion proteins. Results show pink-colored product generated during manufacturing is due to association of hydroxocobalamin (OH-Cbl), a form of vitamin B12. OH-Cbl is not part of the product manufacturing process; however we found cyanocobalamin (CN-Cbl) in cell culture media converts to OH-Cbl in the presence of light. OH-Cbl can be released from mAb and Fc-fusion proteins by conversion with potassium cyanide to CN-Cbl, which does not bind. By exploiting the differential binding of CN-Cbl and OH-Cbl, we developed a rapid and specific assay to accurately measure B12 levels in purified protein. Analysis of multiple products and lots using this technique gives insight into color variability during manufacturing.

Novel Mechanism of Glass Delamination in Type 1A Borosilicate Vials Containing Frozen Protein Formulations.

Storing protein formulations in the frozen state typically improves stability during long-term storage as a drug substance or as a drug product. The frozen state minimizes chemical degradation and physical instability. However, the frozen state is not an optimal storage condition for the glass vial itself. A significant issue was observed when small, flake-like pieces of glass particles (lamellae) appeared in vials containing thawed protein product. The occurrence of glass particles during freeze-thaw results in product rejection and potentially, adverse events. In recent years, glass flakes due to chemical delamination have been observed in parenteral liquid formulations after long-term storage, resulting in a number of product recalls. In this study, for the first time, glass delamination is reported in pharmaceutical glass vials containing frozen protein formulation, caused by a novel mechanism involving thermally-induced mechanical stress. In this article, a monoclonal antibody drug product in glass vials and the corresponding placebo vials were studied to identify the contributing factors from the freeze-thaw process, such as freezing temperature, the presence or absence of protein, and other handling conditions. Freezing temperature was found to be the most critical factor. Glass lamellae were only observed when the products were frozen to -70 °C, while freezing only to -30 °C did not cause any lamellae formation even after multiple freeze-thaw cycles. Protein concentration and the handling of the vials were also identified as contributing factors. A concentration gradient which formed after freeze-thaw induced a higher rate of lamellae occurrence in a subsequent freeze-thaw cycle compared to vials without the concentration gradient. Analyses by Fourier transform infrared spectroscopy and scanning electron microscopy/energy dispersive spectroscopy confirmed that the flake-like lamellae were thin, flat glass particles. Defects corresponding to the glass flakes were observed by scanning electron microscopy on the inner surface of the vials that contained lamellae. In addition, inductively coupled plasma mass spectrometry testing did not show elevated levels of silicon in the drug product solution, suggesting that the glass lamellae formed in the frozen vials was a local, event-based phenomenon rather than silica dissolution from the product contact surface or glass degradation caused by corrosive attack. These findings can be explained by the same thermally-induced mechanical stress which caused vial breakage. Frozen protein formulations contracted below -30 °C, causing an inward glass deformation and a subsequent rapid movement of the glass when the frozen plug of drug product solution separated from the vial inner surface at approximately -50 to -60 °C. The mechanical stress released during this separation caused vial breakage. The incidence of vial breakage increased with more concentrated product and higher fill volume-to-vial volume ratios. The same mechanism applies to lamellae formation. As the rapid surface separation occurred, small, thin pieces of glass were pulled from the glass surface by the frozen plug, and, as a result, glass lamellae particles appeared in the drug product solution after thawing. LAY ABSTRACT: In recent years, glass flakes have been observed in parenteral liquid formulations due to chemical delamination during long-term storage, resulting in a number of product recalls. In our study, we discovered a novel mechanism of glass delamination in vials containing frozen protein formulations. This glass delamination mechanism has never been reported before, and we believe this work will benefit the pharmaceutical scientific community, especially the biotechnology and parenteral drug industries. Storing protein formulations in the frozen state typically improves stability during long-term storage as a drug substance or as a drug product. The frozen state minimizes chemical degradation and physical instability. However, the frozen state is not an optimal storage condition for the glass vial itself. In this study, we observed that after thawing, small, flake-like pieces of glass particles (i.e., lamellae) appeared in vials containing frozen protein formulation. To investigate the root cause, we performed a series of freeze-thaw experiments and characterized the lamellae particles, the vial inner surface, and the elemental composition of the solution. The root cause was determined to be mechanical stress caused by thermal contraction of frozen protein formulations below -30 °C. This contraction caused an inward glass deformation on the vial sidewall and, subsequently, the glass vial surface abruptly separated from frozen protein formulation. Under this mechanical stress, small, thin glass pieces were peeled from the vial inner surface by the frozen formulation, causing lamellae formation. The experimental design and results leading to the discovery of the novel glass delamination mechanism are presented in detail in this article.

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.

Identification of an extraneous black particle in a glass syringe: extractables/leachables case study.

An unexpected, black particle (∼300 microns) was visually observed adhering to the interior shoulder of a prefilled glass syringe containing a biological drug product. The goal of this study was to determine the source, identity, and leachables of the black particle. The particle originated from a polymeric pin used during the syringe manufacturing process. Fourier transform infrared (FTIR) spectra comparison of the black particle and polymeric pin correlated to a database match of Nylon-MXD6 with glass fibers. Liquid chromatography/mass spectroscopy analyses identified Nylon-MXD6 and Nylon-6 photo-oxidized-related compounds in both the pin extract and syringe solution. The black particle originated from the pin and contained glass fibers, Nylon-MXD6, and Nylon-6. All nylon-related compounds were observed at <260 ng/mL (ppb) in the syringe solution. Syringes without black particles contained no detectable levels of nylon-related compounds, suggesting that routine contact between a pin and syringe barrel may not lead to syringe contamination or leachables originating from the pin. Abnormal heat exposure and/or extensive pin usage may have led to pin wear and tear.

Tungsten-induced protein aggregation: solution behavior.

Tungsten has been associated with protein aggregation in prefilled syringes (PFSs). This study probed the relationship between PFSs, tungsten, visible particles, and protein aggregates. Experiments were carried out spiking solutions of two different model proteins with tungsten species obtained from the extraction of tungsten pins typically used in syringe manufacturing processes. These results were compared to those obtained with various soluble tungsten species from commercial sources. Although visible protein particles and aggregates were induced by tungsten from both sources, the extract from tungsten pins was more effective at inducing the formation of the soluble protein aggregates than the tungsten from other sources. Furthermore, our studies showed that the effect of tungsten on protein aggregation is dependent on the pH of the buffer used, the tungsten species, and the tungsten concentration present. The lower pH and increased tungsten concentration induced more protein aggregation. The protein molecules in the tungsten-induced aggregates had mostly nativelike structure, and aggregation was at least partly reversible. The aggregation was dependent on tungsten and protein concentration, and the ratio of these two and appears to arise through electrostatic interaction between protein and tungsten molecules. The level of tungsten required from the various sources was different, but in all cases it was at least an order of magnitude greater than the typical soluble tungsten levels measured in commercial PFS.