Abstract
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.
Introduction
An unexpected, black particle (∼300 microns) was visually observed adhering to the interior shoulder of a prefilled syringe containing a biological drug product. The source of the particle was suspected to be from the individual incident syringe rather than from the bulk filling solution because only one syringe from a lot composed of several hundred filled syringes contained the particle.
The source of impurities in finished drug products can be from primary packaging delivery systems (vials, syringes, etc.) and/or from fill finish processing components such as tubing, gaskets, filters, storage containers, and others. Less likely source of impurities in finished drug products is contamination from tools that were used to manufacture process and packaging components. An example is the leaching of tungsten species from glass syringes where the residual tungsten originated from a syringe processing tool used to form a channel for needle insertion (1). To prevent future occurrences, the source, identity, and leachables arising from the black particles were determined.
A polymeric pin used in the syringe manufacturing process was determined to be the source of the black particle. Fourier transform infrared (FTIR) analysis of the pin and black particles were compared. Extractables were generated from the pin and characterized to target potential leachables that may leach upon exposure to drug formulation or normal usage condition (2–8).
Materials and Methods
A prefilled syringe containing solid, visible black particles was an incident sample from Amgen Inc. A polymeric pin was obtained from the syringe manufacturer. Isophthalamide and caprolactam were from Tokyo Chemical Industry “TCI America” (Portland, OR, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively.
Liquid chromatography/mass spectroscopy (LC/MS) was performed on an Agilent 1100 (Santa Clara, CA, USA) high-performance liquid chromatography (HPLC) system coupled to a Thermo Finnegan LTQ (Waltham, MA, USA) ion trap mass spectrometer. The mass spectrometer was tuned using a MRFA (Met-Arg-Phe-Ala) and a caproic acid solution. Analysis was performed in both electrospray ionization and atmospheric pressure chemical ionization modes and UV chromatograms were recorded at 215 nm. Samples (20 μL) were injected onto a C18 column (Phenomenex 00G-4053-B0) heated at 40 °C. The flow rate was 0.2 mL/min and isocratic for 30-min with mobile phase A (0.1% trifluoroacetic acid [TFA] in water) then ramped at 55 min with 10% mobile phase B (0.1% TFA in acetonitrile). The incident sample was injected as is.
A Fourier transform infrared (FTIR) spectroscopy system composed of a Bruker Hyperion 2000 Infrared Microscope with Bruker Vertex 70 as the IR source was used. Data were collected for 128 scans at 4 cm−1 resolution and compared with those in a Hummel library reference included in the BioRad Informatics KnowItAll spectral database. Optical micrographs were taken by the Zeiss-Stemi 2000 stereomicroscope with an attached Zeiss MRC digital camera.
Sample Preparation
The liquid content of the syringe was expelled, and the black material remained adhered to the inner shoulder of the glass surface of the syringe. The syringe plunger was pulled out and the black material was removed using a tungsten microprobe. The material was placed onto a pre-cleaned glass slide for photography. The particle was then transferred to a polished 13 mm × 2 mm NaCl disc with a tungsten probe. The particle was sandwiched between two NaCl discs and compressed in a Spectra-Tech micro-compression cell. Particles that dissolved and leached into the syringe solution were quantitated against a caprolactam or isophthalamide standard curve.
A polymeric pin was extracted with 4 mL of 10 mM sodium acetate, 5% ethanol, pH 4 solution (Solvent A) at 70 °C for 24 h. An extraction control was prepared similarly, but without the polymeric pin. Solvent A was used to mimic the formulation buffer to generate representative extractables.
Results and Discussion
FTIR on Solid Black Particles
Two black particles were observed within the syringe (Figure 1). Based on analysis of IR bands and relative intensities, both black particles are composed of the same material. A database search matched the particle spectrum to poly (meta-xylene adipamide) (Nylon-MXD6) (Figures 2 and 3).
Two black particles observed inside a pre-filled syringe. The larger particle is approximately 300 microns.
Structures of Nylon-MXD6 and Nylon-6.
FTIR spectra of the black material, new polymeric pin, and the Hummel library reference spectrum HP #3 of poly(meta-xylylene adipamide), commonly known as Nylon-MXD6.
The source of Nylon-MXD6 material was traced to the syringe manufacturing process. A polymer pin (approximately 5.5 × 0.5 cm) is inserted into the glass syringe barrel for transportation during normal unit operations. The IR spectra of the particle, polymeric pin, and Nylon-MXD6 (from IR database) matched. However, various nylon subclasses (Nylon-MXD6, 6, 66, 11, 12, etc.) may not be accurately identified by the IR library search algorithms due to visually similar spectroscopic profiles (9, 10). Further analyses of the nylon pin and black particle decomposition products were performed using HPLC and LC/MS to identify the nylon subclasses.
HPLC and LC/MS Analysis on Syringe Liquid (Leachables) and Pin Extracts
The nylon pin extracts and the syringe solution were evaluated and compared by HPLC and LC/MS techniques. Several HPLC (λ = 215 nm) peaks from the syringe solution correlated with the retention times and masses to those from the extracted pin (Figure 4). This suggests that the impurity peaks in the syringe solution originated from the black particles. The majority of the correlated peaks (compounds 1–12) were identified by LCMS analysis (Tables I and II).
HPLC chromatograms (UV = 215 nm) the pin extract, syringe solution, and blank.
Nylon-MXD6–Related Compounds Leached from Black Particles and Observed in the Syringe Solution
Nylon-6–Related Compounds Leached from Black Particles and Observed in the Syringe Solution
Peaks 1 and 2 ionized in APCI mode with parent masses of 164 and 165 m/z, respectively. The identity of compound 1 was confirmed against a commercially available isophthalamide standard and determined at 175 ng/mL (ppb) in the syringe solution. The isophthalamide standard was heated in water at 95 °C for 30 min to form the partially hydrolyzed 3-carbamoylbenzoic acid (compound 2) compound (Figure 5). Compound 2 in the syringe solution was confirmed as 3-carbamoylbenzoic acid by correlating retention time and mass spectrum profile. Nylon MXD6 polymeric chain is susceptible to cleavage by photo-oxidization (11–13) and the terminal amides may undergo hydrolysis in aqueous solutions, which explain the observation of compounds 1–7. Cyclic monomer (compound 8) and dimer (compound 9) are typical by-products during the polymerization of Nylon-MXD6.
HPLC chromatograms (UV = 215 nm) of partially hydrolyzed isophthalamide and syringe solution.
Caprolactam monomer (compound 10), dimer (compound 11), and tetramer (compound 12) are components commonly associated with Nylon 6 (14) and were also observed. The presence of MXD6 and Nylon 6 decomposition components in the both the syringe solution and pin extract suggests that the polymeric pin material contained Nylon-MXD6 and Nylon 6. The black particle may contain other nylon-related components, but only the peaks present in both the syringe solution and pin extract were determined.
The concentrations of the leached compounds were estimated to provide insight into the extent of leaching from the black material into the formulated drug. The concentration of the nylon decomposition products in the syringe solution was determined by HPLC against standard curves composed of commercially available isophthalamide or caprolactam. Each Nylon-MXD6–related and Nylon-6–related compound was observed at <260 ppb. In normal filled syringes that did not contain a black particle, compounds 1–12 were not detected. Routine contact with a pin and syringe barrel does not lead to syringe contamination or leachables arising from the pin.
Abnormal heat exposure and/or extensive pin usage may have led to pin wear and tear. Pin residues exposed to heated glass syringes may adhere to the inner syringe wall and survived the wash/rinse procedures. The syringe containing the black particle was identified during a visual inspection of each filled syringe. The syringe manufacturer was notified and has implemented measures to prevent future occurrences.
Conclusions
The black particle observed in a prefilled syringe originated from a polymeric pin used during the syringe manufacturing process. FTIR spectra comparison of the black particle and polymeric pin correlated to a Nylon-MXD6 database match. HPLC and LC/MS analyses identified Nylon-MXD6 and Nylon-6 related compounds in both the pin extract and syringe solution. All nylon-related compounds were observed at <260 ng/mL in the syringe solution. The polymer material contains both Nylon-MXD6 and Nylon-6. The solutions from syringes without black particles contained no detectable levels of nylon-related compounds, suggesting that routine contact between a pin and syringe barrel does not lead to syringe contamination or leachables arising from the pin.
Acknowledgements
Authors would like to thank Rob Swift for providing the pin from the supplier and the investigation team Joseph Phillips, David Brems, Linda Narhi, Ron Forster, Janice Davis, Anthony Mire-Sluis, Mitra Cruz, and Martin VanTrieste for their efforts and useful discussions.
- © PDA, Inc. 2010
Jump to section
Related Articles
Cited By...
- Holistic Extractables and Leachables Program: Evaluations of Prefilled Syringe Systems for Biotechnology Products
- Characterization of Protein Aggregating Tungstates: Electrospray Mass Spectrometry Analysis of Extracts from Prefilled Syringes and from Tungsten Pins Used in the Manufacture of Syringes
- The Use of TOC Reconciliation as a Means of Establishing the Degree to Which Chromatographic Screening of Plastic Material Extracts for Organic Extractables Is Complete
- 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