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

Unlabelled: 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.

Rapid Identification and Characterization of Formulated Protein Products by Raman Spectroscopy Coupled with Discriminant Analysis.

Unlabelled: The rapid identification of protein drug products for packaging and receiving can significantly reduce disposition cycle time, and thereby improve the efficiency and productivity of the supply chain to better meet the needs of patients. In this feasibility study, we demonstrate a novel methodology that combines Raman spectroscopy with discriminant analysis that can be used for rapid identification or verification of finished products. With this methodology, Raman spectra of formulated therapeutic proteins were collected non-invasively with the samples either in a quartz cuvette or in the original glass vials, and analyzed without subtraction of buffer or placebo solutions.

Microspectroscopic investigation of the membrane clogging during the sterile filtration of the growth media for mammalian cell culture.

Growth media for mammalian cell culture are very complex mixtures of several dozens of ingredients, and thus the preparation of qualified media is critical to viable cell density and final product titers. For liquid media prepared from powdered ingredients, sterile filtration is required prior to use to safeguard the cell culture process. Recently one batch of our prepared media failed to pass through the sterile filtration due to the membrane clogging.

Free fatty acid particles in protein formulations, part 1: microspectroscopic identification.

We report, for the first time, the identification of fatty acid particles in formulations containing the surfactant polysorbate 20. These fatty acid particles were observed in multiple mAb formulations during their expected shelf life under recommended storage conditions. The fatty acid particles were granular or sand-like in morphology and were several microns in size.

Classification of glass particles in parenteral product vials by visual, microscopic, and spectroscopic methods.

Unlabelled: Glass vials have been used as primary containers for parenteral drugs including biopharmaceuticals. Different types of glass-related particles, although in low occurrence rate, may be adventitiously introduced in these parenterals. Proper classification and investigations of these glass-related particles may help to understand their formation, improve process control, reduce glass-related particles, and deliver safe parenteral drugs to patients.

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.

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

Unlabelled: 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.

Screening soy hydrolysates for the production of a recombinant therapeutic protein in commercial cell line by combined approach of near-infrared spectroscopy and chemometrics.

Soy hydrolysates are widely used as the major nutrient sources for cell culture processes for industrial manufacturing of therapeutic recombinant proteins. The primary goal of this study was to develop a spectroscopy based chemometric method, a partial least squares (PLS), to screen soy hydrolysates for better yield of protein production (titers) in cell culture medium. Harvest titer values of 29 soy hydrolysate lots with production yield between 490 and 1,350 mg/L were obtained from shake flask models or from manufacture engineering runs.

Identification and root cause analysis of cell culture media precipitates in the viral deactivation treatment with high-temperature/short-time method.

Unlabelled: High-temperature/short-time (HTST) treatment of cell culture media is one of the proven techniques used in the biopharmaceutical manufacturing industry for the prevention and mitigation of media viral contamination. With the HTST method, the formulated media is pasteurized (virus-deactivated) by heating and pumping the media continuously through the preset high-temperature holding tubes to achieve a specified period of time at a specific temperature. Recently, during the evaluation and implementation of HTST method in multiple Amgen, Inc.

Effect of pH, temperature, and salt on the stability of Escherichia coli- and Chinese hamster ovary cell-derived IgG1 Fc.

The circulation half-life of a potential therapeutic can be increased by fusing the molecule of interest (an active peptide, the extracellular domain of a receptor, an enzyme, etc.) to the Fc fragment of a monoclonal antibody. For the fusion protein to be a successful therapeutic, it must be stable to process and long-term storage conditions, as well as to physiological conditions.

Nondestructive detection of glass vial inner surface morphology with differential interference contrast microscopy.

Glass particles generated by glass dissolution and delamination of the glass container for pharmaceutical products have become a major issue in the pharmaceutical industry. The observation of glass particles in certain injectable drugs, including several protein therapeutics, has recently resulted in a number of product recalls. Glass vial surface properties have been suggested to play a critical role in glass dissolution and delamination.

Detection of trace melamine in raw materials used for protein pharmaceutical manufacturing using surface-enhanced Raman spectroscopy (SERS) with gold nanoparticles.

Melamine, a nitrogen-rich molecule, was found as an adulterant in pet foods in 2007 in North America and in milk products in 2008 in China. These scandalous abuses of melamine have alarmed the biopharmaceutical industry and the FDA and alerted them to potential adulteration and contamination of melamine in raw materials used to make protein therapeutics. Highly sensitive analytical methods are needed to screen melamine adulteration and contamination in raw materials.

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.

Direct visualization of protein adsorption to primary containers by gold nanoparticles.

Adsorption of proteins to primary containers can result in protein loss, protein denaturation, or aggregation. We report a simple and effective method to directly detect and visualize adsorption of proteins to container surfaces by staining adsorbed proteins with gold nanoparticles, which bind proteins nonspecifically. The gold nanoparticle staining method was applied to study adsorption to siliconized glass prefilled syringes (PFSs) of a therapeutic protein in a liquid formulation.

Identification of a mixed microparticle by combined microspectroscopic techniques: a real forensic case study in the biopharmaceutical industry.

Identification of foreign microparticles in drug products is one of the first steps in evaluating the nature of particle contamination and its consequences for product quality. To characterize various foreign particles, we use spectral database search methods as well as a number of microscopic and microspectroscopic techniques. Here, we report a case study involving the identification and root-cause investigation of a microparticle consisting of four compounds.

Root Cause Analysis of Tungsten-Induced Protein Aggregation in Pre-filled Syringes.

Particles isolated from a pre-filled syringe containing a protein-based solution were identified as aggregated protein and tungsten. The origin of the tungsten was traced to the tungsten pins used in the supplier's syringe barrel forming process. A tungsten recovery study showed that the vacuum stopper placement process has a significant impact on the total amount of tungsten in solutions.

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.

Distribution of silicone oil in prefilled glass syringes probed with optical and spectroscopic methods.

Prefilled glass syringes (PFSs) have become the most commonly used device for the delivery of recombinant protein therapeutics in parenteral formulations. In particular, auto-injectors preloaded with PFSs greatly facilitate the convenient and efficient self-administration of protein therapeutics by patients. Silicone oil is used as a lubricant in PFSs to facilitate the smooth motion of the plunger during injection.

Raman microscopic applications in the biopharmaceutical industry: in situ identification of foreign particulates inside glass containers with aqueous formulated solutions.

Particle identification is an important analytical procedure for quality control and assurance in the biopharmaceutical industry. Rapid and reliable identification of micro-particles helps in evaluating the nature of particle contamination and its consequences on the product quality regulated by internal and external standards. Raman microscopy is one of the microspectroscopic techniques that can be used to identify micro-particles with the advantage of in situ detection.

Conformation and side chains environments of recombinant human interleukin-1 receptor antagonist (rh-IL-1ra) probed by raman, raman optical activity, and UV-resonance Raman spectroscopy.

The conformation and local environments of the side chains cysteines and aromatics of recombinant human interleukin-1 receptor antagonist (rh-IL-1ra) have been studied by visible Raman, Raman optical activity (ROA) and UVRR spectroscopy. The results reveal that the secondary structure of rh-IL-1ra is predominantly beta-sheet, which is consistent with conclusions from multinuclear NMR in solutions and X-ray diffraction analysis of crystals. It confirms that all four cysteines are in reduced state.

Raman spectroscopy of protein pharmaceuticals.

Recent advances in optical and spectroscopic technologies have enabled a plethora of Raman spectrometers that are suitable for studies of protein pharmaceuticals. Highly sensitive Raman spectrometers have overcome the handicap of the fundamentally weak Raman effect that hampered their applications to protein pharmaceuticals in the past. These Raman spectrometers can now routinely measure protein therapeutics at the low concentration of 1 mg/mL, which is on par with other spectroscopic methods such as CD, fluorescence and FTIR spectroscopies.