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.

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. The algorithm used for the discriminant analysis was Mahalanobis distance by principal component analysis with residuals. In addition to product identification, the methodology has the potential to be used for characterizing formulated proteins when exposed to external stresses based on the changes of Mahalanobis distances. LAY ABSTRACT: 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. In this study, we demonstrate a novel methodology that combines Raman spectroscopy with discriminant analysis to rapidly identify formulated proteins non-invasively.

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. In this study, we report the root cause analysis of the failed sterile filtration based on the investigations of both the fouling media and the clogged membranes with multiple microspectroscopic techniques. Cellular particles or fragments were identified in the fouling media and on the surfaces of the clogged membranes, which were presumably introduced to the media from the bacterial contamination. This study demonstrated that microspectroscopic techniques may be used to rapidly identify both microbial particles and inorganic precipitates in the cell culture media.

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. They could be identified by distinct IR bands, with additional confirmation from energy-dispersive X-ray spectroscopy analysis. The particles were readily distinguishable from protein particles by these methods. In addition, particles containing a mixture of protein and fatty acids were also identified, suggesting that the particulation pathways for the two particle types may not be distinct. The techniques and observations described will be useful for the correct identification of proteinaceous versus nonproteinaceous particles in pharmaceutical products.

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

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. In this article, we introduced a classification scheme, and identification procedures and methods, for the glass-related particles. We propose to classify them as glass chip, glass lamella/flake, and silica gel. Eight characteristics for each glass particle type have been identified and described for the visual inspection method. The limitations of the visual method and the need to correlate visual results with forensic analysis are discussed. Using representative examples from each type of glass particle, this study summarized their forensic differentiations based on microscopic methods of optical microscopy, scanning electron microscopy, micro-flow imaging, and spectroscopic methods of dnergy-dispersive spectroscopy and Fourier transform infrared spectroscopy. The mechanisms of glass particle formation are listed as references for drug development scientists to investigate the root causes and improve process control on visible glass particles in parenteral vials. LAY ABSTRACT: 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. In this article, we introduced a classification scheme, and identification procedures and methods, for the glass-related particles. We propose to classify them as glass chip, glass lamella/flake, and silica gel. Using representative examples from each type of glass particle, this study summarized their forensic differentiations based on microscopic and spectroscopic methods. The mechanisms of glass particle formation are listed as references for drug development scientists to investigate the root causes and improve process control on visible glass particles in parenteral vials.

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.

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.

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. The soy hydrolysate samples were measured by near-infrared (NIR) in reflectance mode using an infrared fiber optic probe. The fiber optic probe could easily enable in situ measurement of the soy hydrolysates for convenient raw material screening. The best PLS calibration has a determination coefficient of R (2) = 0.887 utilizing no spectral preprocessing, the two spectral ranges of 10,000-5,376 cm(-1) and 4,980-4,484 cm(-1), and a rank of 6 factors. The cross-validation of the model resulted in a determination coefficient of R (2) = 0.741 between the predicted and actual titer values with an average standard deviation of 72 mg/L. Compared with the resource demanding shake flask model, the combination of NIR and chemometric modeling provides a convenient method for soy hydrolysate screening with the advantage of fast speed, low cost and non-destructive.

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

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. manufacturing facilities, media precipitates were observed in the tests of HTST treatments. The media precipitates may have adverse consequences such as clogging the HTST system, altering operating conditions and compromising the efficacy of viral deactivation, and ultimately affecting the media composition and cell growth. In this study, we report the identification of the composition of media precipitates from multiple media HTST runs using combined microspectroscopic methods including Raman, Fourier transform infrared spectroscopy, and scanning electron microscopy with energy-dispersive X-ray spectroscopy. The major composition in the precipitates was determined to be metal phosphates, including calcium phosphate, magnesium phosphate, and iron (III) phosphate. Based on the composition, stoichiometry, and root-cause study of media precipitations, methods were implemented for the mitigation and prevention of the occurrence of the media precipitation. LAY ABSTRACT: Viral contamination in cell culture media is an important issue in the biopharmaceutical manufacturing industry and may have serious consequences on product quality, efficacy, and safety. High-temperature/short-time (HTST) treatment of cell culture media is one of the proven techniques used in the industry for the prevention and mitigation of media viral contamination. With the HTST method, the formulated media is pasteurized (virus-deactivated) by heating at preset conditions. This paper provides the identification and root-cause study of the media precipitates that adversely affected the HTST process and discusses the possible solutions to mitigate the precipitation problem.

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. The stability of the Fc used is critical for obtaining a successful therapeutic protein. The effects of pH, temperature, and salt on the stabilities of Escherichia coli- and Chinese hamster ovary cell (CHO)-derived IgG1 Fc high-order structure were probed using a variety of biophysical techniques. Fc molecules derived from both E. coli and CHO were compared. The IgG1 Fc molecules from both sources (glycosylated and aglycosylated) are folded at neutral pH and behave similarly upon heat- and low pH-induced unfolding. The unfolding of both IgG1 Fc molecules occurs via a multistep unfolding process, with the tertiary structure and C(H)2 domain unfolding first, followed by changes in the secondary structure and C(H)3 domain. The acid-induced unfolding of IgG1 Fc molecules is only partially reversible, with the formation of high-molecular weight species. The CHO-derived Fc protein (glycosylated) is more compact (smaller hydrodynamic radius) than the E. coli-derived protein (aglycosylated) at neutral pH. Unfolding is dependent on pH and salt concentration. The glycosylated C(H)2 domain melts at a temperature 4-5 °C higher than that of the aglycosylated domain, and the low-pH-induced unfolding of the glycosylated Fc molecule occurs at a pH ~0.5 pH unit lower than that of the aglycosylated protein. The difference observed between E. coli- and CHO-derived Fc molecules primarily involves the C(H)2 domain, where the glycosylation of the Fc resides.

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. Surface characterization of glass container, therefore, is important to evaluate the quality of the glass container. In this work, we demonstrate that differential interference contrast (DIC) microscopy is a powerful, effective, and convenient technique to examine the inner surface morphology of glass vials nondestructively. DIC microscopy does not require the cutting of the glass vial for scanning the inner surface and has sufficient spatial resolution to reveal glass pitting, phase separation, delamination scars, and other defects. Typical surface morphology of pharmaceutical glass vials with different alkalinity are compared and discussed.

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. We conducted surface-enhanced Raman spectroscopy (SERS) experiments to test trace melamine spiked in three raw materials commonly used for protein pharmaceutical formulation and purification, including sucrose, urea, and arginine, with a portable Raman device and gold nanoparticles. The detection limit of 10 ppb in raw material dissolved in 30:70% water/acetonitrile is equivalent to 0.5 ppm in solid raw material. It has excellent linearity in the concentration range measured. The cross-validation regression coefficient R(2) and the standard error of prediction (SEP) are 0.960 and 7.18 ppb, respectively, in sucrose. The R(2) and SEP are 0.958 and 9.15 ppb in urea. It has a relatively lower R(2) = 0.630 and a SEP of 35.0 ppb in arginine, which could be due to the competitive adsorption of arginine molecules to the surfaces of gold nanoparticles. The detection of melamine using the SERS technique is rapid (within 3 minutes), convenient, and requires no extraction procedure, offering an alternative method for screening melamine in raw materials at biopharmaceutical manufacture sites.

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. The protein was found to preferentially adsorb to glass surfaces over siliconized surfaces in PFSs. The presence of adsorbed proteins on glass surfaces was confirmed by in situ Raman spectroscopy. Gold nanoparticle staining patterns revealed that adsorption of proteins to hydrophobic cyclic olefin polymer plastic vials was minimized compared with hydrophilic type I glass vials. Bovine serum albumin (BSA) also preferentially adsorbed to glass surfaces compared with siliconized surfaces as revealed by the gold staining patterns in PFS incubated with BSA, supporting the use of albumin to minimize loss of proteins in glass containers. The method is particularly valuable for high-concentration protein formulations in which adsorption of proteins to containers cannot be easily detected by other methods.

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. Foreign microparticles consisting of mixtures pose unique challenges for identification as their spectra are difficult to interpret and general database searches usually return unsatisfactory results. Moreover, sample separation through purification and other manipulations is time consuming and often difficult for these microparticles due to their small sizes and the limited quantities of the components. Here we demonstrate an applicable methodology that combines multiple microscopic and microspectroscopic techniques to identify a heterogeneous microparticle without the need for sample purification or chemical separation. This methodology primarily combines Raman, infrared, and energy dispersive X-ray microspectroscopic techniques to obtain complementary spectral information for the identification of heterogeneous particles. With this methodology, the mixed microparticle investigated in this study was determined to consist of polyisobutylene, hydrated magnesium silicate, titanium dioxide, and silica, likely originating from the vial stopper material.

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. The air gap formed in the syringe funnel area (rich in residual tungsten) becomes accessible to solutions when the vacuum is pulled. Leachable tungsten deposits that were not removed by the supplier's wash process are concentrated in this small area. Extraction procedures used to measure residual tungsten in empty syringes would under-report the tungsten quantity unless the funnel area is wetted during the extraction. Improved syringe barrel forming and washing processes at the supplier have lowered the residual tungsten content and significantly reduced the risk of protein aggregate formation. This experience demonstrates that packaging component manufacturing processes, which are outside the direct control of drug manufacturers, can have an impact on the drug product quality. Thus close technical communication with suppliers of product contact components plays an important role in making a successful biotherapeutic.

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.

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. However, there have been few sophisticated analytical techniques that can qualitatively and quantitatively characterize in-situ the morphology, thickness, and distribution of silicone oil in PFSs. In this paper, we demonstrate the application of three optical techniques including confocal Raman microscopy, Schlieren optics, and thin film interference reflectometry to visualize and characterize silicone oil distribution in PFS. The results showed that a container coating process could produce unevenly distributed silicone oil on the glass barrel of PFSs. An insufficiency of the amount of silicone oil on the glass barrel of a PFS can cause stalling when the device is preloaded into an auto-injector. These analytical techniques can be applied to monitor the silicone oil distribution in PFSs.

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. In this paper we demonstrate that a visible laser Raman microscope was particularly useful to identify micro-particles that were inside glass containers such as glass syringes, vials, and test tubes, which are commonly used as containers for aqueous formulated drugs. The examples include the identifications of a droplet-like particle inside a pre-filled glass syringe, a fibrous particle inside a glass test tube, and a white particle inside a glass vial; all of these examples usually demand challenging or time-consuming sample manipulation for other techniques. The Raman microscopic technique was shown to be able to solve these challenging micro-particle identifications due to its ability to carry out detection in situ. Particularly in the example of micro-droplet identification, the Raman microscopic technique was the only choice for a fast and successful particle detection. For all three identifications, Raman in situ detection has significantly accelerated particle analysis and avoided potential sample secondary contamination or losses owing to none or minimal sample manipulation.

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. Three cysteines are not hydrogen bonded and exposed. One cysteine is moderately hydrogen bonded and buried. This explains the earlier observation that only three cysteines were detectable using DTNB titration. No characteristic Raman band of disulfide bond was observed in the Raman spectra of rh-IL-1ra in both solution and in crystals. It rules out the supposition that there is one disulfide bond in rh-IL-1ra crystals based on X-ray diffraction. Raman and UVRR spectra of rh-IL-1ra exhibit canonical marker bands of tryptophan. They do not support the proposal that there is cation-pi interaction involving tryptophans in solutions and crystals. These results demonstrate that Raman spectroscopy offers certain advantages over X-ray diffraction for studies of detailed local environment and intermolecular interactions of side chains of proteins in solution and in crystals.