A Risk- and Science-Based Approach to the Acceptance Sampling Plan Inspection of Protein Parenteral Products.

The requirement for visual inspection of pharmaceuticals has been a compendial expectation for over a century, with some advancement of visible particle control strategies in recent years. Current philosophies include a 100% inspection and an Acceptance Sampling Plan inspection. The particles found during these inspections are normally categorized simply by particle size (visible vs. subvisible), particle source (intrinsic vs. extrinsic) and particle type (inherent vs. extraneous). We believe that a more risk- and science-based approach is attainable, which is grounded in forensic data, toxicological/medical opinions and prior knowledge. We have provided an outline for how to determine patient safety impact of visible particles found in parenteral products and potential actions that could be taken within the quality system regarding lot disposition. We believe this approach focuses efforts on patient safety risks, enhances the use of prior knowledge and improves consistency in how particle observations are handled.

Do not drop: mechanical shock in vials causes cavitation, protein aggregation, and particle formation.

Industry experience suggests that g-forces sustained when vials containing protein formulations are accidentally dropped can cause aggregation and particle formation. To study this phenomenon, a shock tower was used to apply controlled g-forces to glass vials containing formulations of two monoclonal antibodies and recombinant human growth hormone (rhGH). High-speed video analysis showed cavitation bubbles forming within 30 µs and subsequently collapsing in the formulations. As a result of echoing shock waves, bubbles collapsed and reappeared periodically over a millisecond time course. Fluid mechanics simulations showed low-pressure regions within the fluid where cavitation would be favored. A hydroxyphenylfluorescein assay determined that cavitation produced hydroxyl radicals. When mechanical shock was applied to vials containing protein formulations, gelatinous particles appeared on the vial walls. Size-exclusion chromatographic analysis of the formulations after shock did not detect changes in monomer or soluble aggregate concentrations. However, subvisible particle counts determined by microflow image analysis increased. The mass of protein attached to the vial walls increased with increasing drop height. Both protein in bulk solution and protein that became attached to the vial walls after shock were analyzed by mass spectrometry. rhGH recovered from the vial walls in some samples revealed oxidation of Met and/or Trp residues.

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.

Aggregation of a monoclonal antibody induced by adsorption to stainless steel.

Stainless steel is a ubiquitous surface in therapeutic protein production equipment and is also present as the needle in pre-filled syringe biopharmaceutical products. Stainless steel microparticles can cause the aggregation of a monoclonal antibody (mAb). The initial rate of mAb aggregation was second order in steel surface area and zero order in mAb concentration, generally consistent with a bimolecular surface aggregation being the rate-limiting step. Polysorbate 20 (PS20) suppressed the aggregation yet was unable to desorb the firmly bound first layer of protein that adsorbs to the stainless steel surface. Also, there was no exchange of mAb from the first adsorbed layer to the bulk phase, suggesting that the aggregation process actually occurs on subsequent adsorption layers. No oxidized Met residues were detected in the mass spectrum of a digest of a highly aggregated mAb, although there was a fourfold increase in carbonyl groups due to protein oxidation.

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.

Monoclonal antibody interactions with micro- and nanoparticles: adsorption, aggregation, and accelerated stress studies.

Therapeutic proteins are exposed to various wetted surfaces that could shed subvisible particles. In this work we measured the adsorption of a monoclonal antibody (mAb) to various microparticles, characterized the adsorbed mAb secondary structure, and determined the reversibility of adsorption. We also developed and used a front-face fluorescence quenching method to determine that the mAb tertiary structure was near-native when adsorbed to glass, cellulose, and silica. Initial adsorption to each of the materials tested was rapid. During incubation studies, exposure to the air-water interface was a significant cause of aggregation but acted independently of the effects of microparticles. Incubations with glass, cellulose, stainless steel, or Fe(2)O(3) microparticles gave very different results. Cellulose preferentially adsorbed aggregates from solution. Glass and Fe(2)O(3) adsorbed the mAb but did not cause aggregation. Adsorption to stainless steel microparticles was irreversible, and caused appearance of soluble aggregates upon incubation. The secondary structure of mAb adsorbed to glass and cellulose was near-native. We suggest that the protocol described in this work could be a useful preformulation stress screening tool to determine the sensitivity of a therapeutic protein to exposure to common surfaces encountered during processing and storage.

Response of a concentrated monoclonal antibody formulation to high shear.

There is concern that shear could cause protein unfolding or aggregation during commercial biopharmaceutical production. In this work we exposed two concentrated immunoglobulin-G1 (IgG1) monoclonal antibody (mAb, at >100 mg/mL) formulations to shear rates between 20,000 and 250,000 s(-1) for between 5 min and 30 ms using a parallel-plate and capillary rheometer, respectively. The maximum shear and force exposures were far in excess of those expected during normal processing operations (20,000 s(-1) and 0.06 pN, respectively). We used multiple characterization techniques to determine if there was any detectable aggregation. We found that shear alone did not cause aggregation, but that prolonged exposure to shear in the stainless steel parallel-plate rheometer caused a very minor reversible aggregation (<0.3%). Additionally, shear did not alter aggregate populations in formulations containing 17% preformed heat-induced aggregates of a mAb. We calculate that the forces applied to a protein by production shear exposures (<0.06 pN) are small when compared with the 140 pN force expected at the air-water interface or the 20-150 pN forces required to mechanically unfold proteins described in the atomic force microscope (AFM) literature. Therefore, we suggest that in many cases, air-bubble entrainment, adsorption to solid surfaces (with possible shear synergy), contamination by particulates, or pump cavitation stresses could be much more important causes of aggregation than shear exposure during production.

Precipitation of a monoclonal antibody by soluble tungsten.

Tungsten microparticles may be introduced into some pre-filled syringes during the creation of the needle hole. In turn, these microcontaminants may interact with protein therapeutics to produce visible particles. We found that soluble tungsten polyanions formed in acidic buffer below pH 6.0 can precipitate a monoclonal antibody within seconds. Soluble tungsten in pH 5.0 buffer at about 3 ppm was enough to cause precipitation of a mAb formulated at 0.02 mg/mL. The secondary structure of the protein was near-native in the collected precipitate. Our observations are consistent with the coagulation of a monoclonal antibody by tungsten polyanions. Tungsten-induced precipitation should only be a concern for proteins formulated below about pH 6.0 since tungsten polyanions are not formed at higher pHs. We speculate that the heterogenous nature of particle contamination within the poorly mixed syringe tip volume could mean that a specification for tungsten contamination based on the entire syringe volume is not appropriate. The potential potency of tungsten metal contamination is highlighted by the small number of particles that would be required to generate soluble tungsten levels needed to coagulate this antibody at pH 5.0.

Mechanistic studies of glass vial breakage for frozen formulations. I. Vial breakage caused by crystallizable excipient mannitol.

The process of freeze-thaw not only subjects bioproducts to potentially destabilizing stress, but also imposes challenges to retain container integrity. Shipment and storage of frozen products in glass vials and thawing of the vials prior to use at clinics is a common situation. Vial integrity failure during freeze-thaw results in product loss and safety issues. Formulations of biomolecules often include crystallizable excipients, which can cause glass vial breakage during freeze-thaw operations. In this study, mannitol formulations served as models for mechanistic investigation of root causes for vial breakage. Several parameters and their impacts on vial breakage were investigated, including mannitol concentration (5% and 15%), different freeze-thaw conditions (fast, slow, and staging), fill configurations (varying fill volume/vial size ratio), and vial tray materials (plastic, stainless steel, corrugated cardboard, aluminum, and polyurethane foam). The results in this study were subjected to a statistical proportion test. The data showed that large fill volumes strongly correlated with higher percentage of vial cracks. Furthermore, the 15% mannitol was found to cause more breakage than 5% mannitol, especially with fast temperature gradient. Significantly more thawing vial breakage occurred in the fast compared to slow freeze-thaw with all types of vial trays. The freezing breakage was substantially lower than the thawing breakage using the fast temperature gradient, and the trend was reversed with the slow temperature gradient. An intermediate hold at -30 degrees C prior to further decrease in temperature proved to be a practical approach to minimize mannitol-induced vial breakage. Thermal mechanical analysis (TMA) and strain gage techniques were employed to gain mechanistic insights, and it was found that the primary causes for mannitol-induced vial breakage were partial crystallization during freezing and "secondary" crystallization of non-crystallized fraction during thawing. The strain on the vial's axial direction was significantly higher than the hoop direction, typically resulting in bottom lens of the vial coming off. Without a -30 degrees C hold, rapid volume expansions due to initial crystallization and secondary crystallization of mannitol were observed in TMA profiles, and these expansions were more apparent in 15% mannitol compared to 5% mannitol. With the introduction of a -30 degrees C hold step, abrupt expansions diminished in TMA profiles, suggesting that most of the mannitol crystallization occurred concurrently with ice solidification during the -30 degrees C holding step and, thus, secondary crystallization during thawing was minimal and the sudden expansion event was eliminated. Therefore, vial breakage during both freezing and thawing was reduced.

Mechanistic studies of glass vial breakage for frozen formulations. II. Vial breakage caused by amorphous protein formulations.

In an accompanying article we have described parameters that influence vial breakage in freeze-thaw operations when using crystalizable mannitol formulations, and further provided a practical approach to minimize the breakage in manufacturing settings. Using two diagnostic tools-thermal mechanical analysis (TMA) and strain gage, we investigated the mechanism of mannitol vial breakage and concluded that the breakage is related to sudden volume expansions in the frozen plug due to crystallization events. Glass vial breakage has also been observed with a number of frozen protein formulations consisting of only amorphous ingredients. Therefore, in this study, we applied the methodologies and learnings from the prior investigation to further explore the mechanism of vial breakage during freeze-thaw of amorphous protein products. It was found that temperature is a critical factor, as breakage typically occurred when the products were frozen to -70 degrees C, while freezing only to -30 degrees C resulted in negligible breakage. When freezing to -70 degrees C, increased protein concentration and higher fill volume induced more vial breakage, and the breakage occurred mostly during freezing. In contrast to the previous findings for crystallizable formulations, an intermediate staging step at -30 degrees C did not reduce breakage for amorphous protein formulations, and even slightly increased the breakage rate. The TMA profiles revealed substantially higher thermal contraction of frozen protein formulations when freezing below -30 degrees C, as compared to glass. Such thermal contraction of frozen protein formulations caused inward deformation of glass and subsequent rapid movement of glass when the frozen plug separates from the vial. Increasing protein concentration caused more significant inward glass deformation, and therefore a higher level of potential energy was released during the separation between the glass and frozen formulation, causing higher breakage rates. The thermal expansion during thawing generated moderate positive strain on glass and explained the thaw breakage occasionally observed. The mechanism of vial breakage during freeze-thaw of amorphous protein formulations is different compared to crystallizable formulations, and accordingly requires different approaches to reduce vial breakage in manufacturing. Storing and shipping at no lower than -30 degrees C effectively prevents breakage of amorphous protein solutions. If lower temperature such as -70 degrees C is unavoidable, the risk of breakage can be reduced by lowering fill volume.

Protein formulation and fill-finish operations.

One of the challenges for the successful commercialization of therapeutic proteins is to maintain the safety and efficacy of the protein during the manufacturing process, storage, and administration. To achieve this, the purified form of the protein drug is usually "formulated" with carefully selected excipients. The operations that occur subsequent to protein purification, such as freezing of the purified protein bulk, thawing of the bulk, formulation (excipient addition), sterile filtration, filling, freeze-drying, and inspection are commonly referred as "formulation and fill-finish operations". This review is focused on the protein formulation and fill-finish operations, critical process parameters at each operation, and the process considerations required for maintaining safety and efficacy of the drug during manufacturing and storage. Since proteins have complex molecular structures that can influence the protein stability, the reader is first introduced to salient concepts related to protein structure. This is followed by a review of the possible protein-degradation mechanisms and how a variety of external factors can contribute to protein degradation during the in vitro processing of the protein drug. The reader is then introduced to each of the formulation and fill-finish operations mentioned above, the possible degradations during each unit-operation, and process considerations necessary to avoid those degradations.