Erwin Freund

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. Biotechnol. Bioeng. 2009;103: 936–943.

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.

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.

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. Biotechnol. Bioeng. 2010;105: 121–129.

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.

Production of particles of therapeutic proteins at the air–water interface during compression/dilation cycles

Particles in protein therapeutics are undesirable because they may have the potential for causing adverse immunogenicity in patients. Agitation-induced exposure to the air–water interface during manufacturing, shipping, and administration can cause particle formation in therapeutic protein products. We systematically studied how application of surface pressure during periodic interfacial compressions caused a model monoclonal antibody to form particles. Above a critical interfacial compression ratio of 5 we observed a dramatic increase in the rate of protein particle formation. During continuous interfacial compression/dilation cycles, particle numbers increased but the particle size distribution remained unchanged. When cyclic compressions were halted, particles did not nucleate additional particles or grow further in bulk solution suggesting that they are formed only at the air–water interface. In fact, we found that particles in the bulk slowly decreased in number upon standing. The rate of particle formation was only weakly dependent on both the bulk protein concentration and the period of cyclical interfacial compressions. These observations are consistent with the interfacial aggregation of proteins during periods of high surface pressure, followed by collapse of the adsorbed layer and detachment of protein particles from the interface into the bulk.

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.

Automation and High-Throughput Technologies in Biopharmaceutical Drug Product Development with QbD Approaches

Quality by Design (QbD) of biopharmaceuticals assumes that desired properties of the final drug product can be predicted with the help of various analytical and characterization methods during the development process. This should be translated into a sound, scientific understanding of drug behavior to design a stability space with clearly defined parameters that produce acceptable product quality attributes. In the QbD framework, the combination and interaction of multiple input variables that have been demonstrated to provide assurance of quality is called the design space. Approaches for identifying the design space in the biopharmaceutical industry generally rely on statistical design of experiments (DOEs) which can be resource intensive (Horvath et al., Mol Biotechnol 45(3):203–206, 2010; Ng and Rajagopalan, Quality by design for biopharmaceuticals: perspectives and case studies, Wiley, Hoboken, 2009). In cases where the number of variables is large, performing a full factorial DOE may be resource intensive. The automated high-throughput methods have been found to be useful in exploration of multiple conditions, defined by so many variables. In this chapter, we describe the use of QbD for applications of automation and high-throughput technology in biopharmaceutical processes from early molecular assessment to the late stages of commercial drug development. High-throughput techniques reinforce the QbD approach not only by providing a large pool of data points using minimal resources but also by obtaining comparable and structural results, which allow thorough statistical analysis.

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.

Visible and Subvisible Protein Particle Inspection Within a QbD-Based Strategy

In spite of significant progress in many analytical technologies, nondestructive particle characterization remains a challenge. Guidance on foreign particle matter in conventional therapeutics dates back many years in contrast to protein aggregate particles present in the relatively recent biotechnology protein therapeutic products. In this chapter the focus will be on protein aggregates that manifest themselves as both visible and subvisible particles. Protein aggregation is undesired and when unavoidable must be controlled. This requires the ability to characterize this attribute, ideally by size and number and its distribution amongst a manufactured drug product lot as a function of time. Once quantitation is available, in-process controls can be established to ensure that those aggregation levels observed in the manufacture of clinical materials are not exceeded in the commercial product during its shelf life.