Analytical Challenges Assessing Protein Aggregation and Fragmentation Under Physiologic Conditions.

Therapeutic proteins are administered by injection or infusion. After administration, the physiologic environment in the desired body compartment - fluid or tissue - can impact protein stability and lead to changes in the safety and/or efficacy profile. For example, protein aggregation and fragmentation are critical quality attributes of the drug product and can occur after administration to patients. In this context, the in vivo stability of therapeutic proteins has gained increasing attention. However, in vivo protein aggregation and fragmentation are difficult to assess and have been rarely investigated. This mini-review summarizes analytical approaches to assess the stability of therapeutic proteins using simulated physiologic conditions. Furthermore, we discuss factors potentially causing in vivo protein aggregation, precipitation, and fragmentation in complex biological fluids. Different analytical approaches are evaluated with respect to their applicability and possible shortcomings when it comes to these degradation events in biological fluids. Tracking protein stability in biological fluids typically requires purifying or labeling the protein of interest to circumvent matrix interference of biological fluids. Improved analytical methods are strongly needed to gain knowledge on in vivo protein aggregation and fragmentation. In vitro models can support the selection of lead candidates and accelerate the pre-clinical development of therapeutic proteins.

Stability of monoclonal antibodies after simulated subcutaneous administration.

Changes in the environment from the drug product to the human physiology might lead to physical and/or chemical modifications of the protein drug, such as in vivo aggregation and fragmentation. Although subcutaneous (SC) injection is a common route of administration for therapeutic proteins, knowledge on in vivo stability in the SC tissue is limited. In this study, we developed a physiologic in vitro model simulating the SC environment in patients. We assessed the stability of two monoclonal antibodies (mAbs) in four different protein-free fluids under physiologic conditions. We monitored protein stability over two weeks using a range of analytical methods, in analogy to testing purposes of a drug product. Both mAbs showed an increase of protein aggregates, fragments, and acidic species. mAb1 was consistently more stable in this in vitro model than mAb2, highlighting the importance of comparing the stability of different mAbs under physiologic conditions. Throughout the study, both mAbs were substantially less stable in bicarbonate buffers as compared to phosphate-buffered saline. In summary, our developed model was able to differentiate stability between molecules. Bicarbonate buffers were more suitable compared to phosphate-buffered saline in regards to simulating the in vivo conditions and evaluating protein liabilities.

Assessing Particle Formation of Biotherapeutics in Biological Fluids.

The stability of therapeutic proteins can be impacted in vivo after administration, which may affect patient safety or treatment efficacy, or both. Stability testing of therapeutic proteins using models representing physiologic conditions may guide preclinical development strategy; however, to date only a few studies assessing the physical stability are available in the public domain. In this manuscript, the stability of seven fluorescently labeled monoclonal antibodies (mAbs) was evaluated in human serum and phosphate-buffered saline, two models often discussed to be representative of the situation in humans after intravenous administration. Subvisible particles were analyzed using light obscuration, flow imaging, and imaging flow cytometry. All methods showed that serum itself formed particles under in vitro conditions. Imaging flow cytometry demonstrated that mean particle size and counts of mAbs increased substantially in serum over five days; however, particle formation in phosphate-buffered saline was comparably low. Stability differences were observed across the mAbs evaluated, and imaging flow cytometry data indicated that fluorescently labeled mAbs primarily interacted with serum components. The results indicate that serum may be more suitable as in vitro model to simulate physiologic intravenous conditions in patients closely and evaluate the in vivo stability of therapeutic proteins. Fluorescence labeling and detection methods may be applied to differentiate particles containing therapeutic protein from high amounts of serum particles that form over time.

Role of Formulation Parameters on Intravitreal Dosing Accuracy Using 1 mL Hypodermic Syringes.

PURPOSE: Evaluation of product viscosity, density and aeration on the dose delivery and accuracy for intravitreal injections with commonly used commercially available hypodermic 1 mL syringes. METHODS: Six commercially available hypodermic 1 mL syringes with different specifications were used for the study. Syringes were filled with the test solutions with different densities and viscosities. Syringes were also subjected to shaking stress to introduce aeration in the test solutions in the presence of different surfactant concentrations with and without high antibody concentration. Target intravitreal volumes of 100 µL, 50 µL and 30 µL were tested to assess dosing accuracy in a controlled simulated administration setup using DIN ISO 11040-4 guidelines and Zwick/Roell Z010 TN instrument. RESULTS: With increasing product viscosity, higher volumes and hence doses were delivered especially for very low volumes like 50 µL and 30 µL. No impact of increasing product density was found on the delivered dose. The presence of surfactants or high protein concentration can lead to aeration, which also negatively affects the dose accuracy and precision. CONCLUSION: Formulation parameters like viscosity can have an impact on dose delivery using hypodermic syringes for intravitreal injections and on the resulting glide force.

Determining Maximum Allowable Rubber Stopper Displacement for Container Closure Integrity (CCI).

Sterile pharmaceuticals require they be developed and manufactured using suitable container closure systems to maintain sterility until product opening. Characterizing container closure integrity (CCI) in relation to rubber stopper displacement was controversially discussed during the Annex 1 revision process. An automated inspection system can reject units with displaced rubber stoppers, and the related acceptance criteria for such in-process testing can be established by adequate studies. In this manuscript, we describe a novel helium leak CCI testing method to study the relation of rubber stopper displacement and CCI. Ten different commonly used vial-rubber stopper combinations were characterized, which led to robust test results. Pronounced differences between the different vial-rubber stopper combinations were observed, clearly showing that the combination of different stoppers, vials, and caps led to significant differences in allowable stopper displacement for routine manufacture.

Characterization of Polymeric Syringes Used for Intravitreal Injection.

Intravitreal (IVT) injection is currently the state of the art for drug delivery to the back of the eye. Drug Products (DP) intended for IVT injections usually pose challenges such as a very low injection volume (e.g. 50 µL) and high injection forces. DPs in vials are typically transferred and injected using disposable polymer syringes, which can feature a silicone oil (SO) coating. In our syringe in-use study, we compared dead volume, total SO content and SO layer distributions of three IVT transfer injection syringes. We assessed multiple potential impact factors such as protein concentration, needle gauge, injection speed, surfactant type and the impact of the in-use hold time on sub-visible particle (SvP) formation and injection forces. Pronounced differences were observed between the syringes regarding SvP generation. Siliconized syringes showed higher SvP counts as compared to non-siliconized syringes. In some cases injection forces exceeded 20 N, which caused needles to burst off during injection. The syringes also showed relevant differences in total SO content and dead volume. In conclusion, specific consideration in the selection of an adequate transfer injection syringe are required. This includes extensive testing and characterization under intended and potential in-use conditions and the development of in-use handling procedures.

Tracking the physical stability of fluorescent-labeled mAbs under physiologic in vitro conditions in human serum and PBS.

In recent years, the stability of biotherapeutics in vivo has received increasing attention. Assessing the stability of biotherapeutics in serum may support the selection of adequate molecule candidates. In our study, we compared the physical stability of 8 different monoclonal antibodies (mAbs) in phosphate-buffered saline (PBS) and human serum. mAbs were Alexa Fluor 488-labeled and characterized with respect to fragmentation, aggregation, and proteinaceous particle formation. Samples were analyzed using size-exclusion chromatography, light obscuration, and flow imaging. In addition, novel methods such as flow cytometry and fluorescence microscopy were applied. mAbs were selected based on their hydrophobicity and isoelectric point. All mAbs studied were inherently less stable in human serum as compared to PBS. Particle size and particle counts increased in serum over time. Interestingly, certain mAbs showed significant levels of fragmentation in serum but not in PBS. We conclude that PBS cannot replicate the physical stability measured in serum. The stability of labeled mAbs in human serum did not correlate with their hydrophobicity and isoelectric point . Serum stability significantly differed amongst the tested mAbs.

Evaluation of Different Quality-Relevant Aspects of Closed System Transfer Devices (CSTDs).

PURPOSE: Health care professionals can be exposed to hazardous drugs such as cytostatics during preparation of drugs for administration. Closed sytem transfer devices (CSTDs) were introduced to provide protection for healthcare professional against unintended exposure to hazardous drugs. The interest in CSTDs has significantly increased after USP <800> monograph was issued. The majority of the studies published so far on CSTDs have focused on their "containment" function. However, other important attributes for CSTDs with potential importance for product quality impact are not yet fully evaluated. METHODS: In the current study, we evaluated four sytems from different suppliers, in combination with different container closure systems (CCS), using solutions of different viscosity and surface tension. The different CSTD / CCS combinations were tested for (a) containment (integrity) using a highly sensitive helium leak test, (b) the force required for mounting the vial adaptor, (c) contribution to visible and subvisible particles as well as (d) the hold-up volume. RESULTS: Results show that the majority of CSTDs may have leaks varying in size, and that some of them generated visible particles due to stopper coring and subvisible particles, both due to silicon oil and particulate contaminations of the Devices. Finally, the holdup volume was up to 1 mL depending on the CSTD type, vial size and solution viscosity. CONCLUSION: These results show that there is a need to evaluate the compatibility of CSTD systems to select the best system for the intended use and that CSTDs may adversely impact product quality and delivered dose.

Particle Analysis of Biotherapeutics in Human Serum Using Machine Learning.

In recent years, an increasing number of studies assessed the stability of biotherapeutics in biological fluids. Such studies aim to simulate the conditions encountered in the human body and investigate the in vivo stability under in vitro conditions. However, on account of complexity of biological fluids, standard pharmaceutical methods are poorly suited to assess the stability of biotherapeutics. In this study, a fluorescent-labeled therapeutic immunoglobulin G (IgG) was analyzed for proteinaceous particles after mixing with human serum and after incubation at 37°C for 5 days. Samples were analyzed using standard pharmaceutical methods (light obscuration and dynamic imaging). Moreover, we developed a fluorescence microscopy method allowing to semiquantitatively detect IgG particles in serum. Several hundred IgG particles were detected after exposure to serum. Moreover, particle counts and particle size increased in serum over time. The results showed that an IgG may form particles on mixing with serum and novel methods such as fluorescence microscopy are required to gain insight on the stability of biotherapeutics in biological fluids. Furthermore, we showed distinct advantages of machine learning over traditional threshold-based methods by analyzing microscopy images. Machine learning allowed simplifying particles in regards to count, size, and shape.

Method to Predict Glass Vial Fogging in Lyophilized Drug Products.

Glass fogging is a phenomenon occurring in lyophilized drug products and can be described as a thin product layer deposited on the inner surface of the glass container, in the area not covered by the lyo cake itself. It is often considered a cosmetic defect; however, the loss of container closure integrity is a potential consequence of the fogging's expansion to the vial neck region, making this a potential critical defect. Thus, a method for predicting the extent of vial fogging before the actual freeze-drying is of particular interest for the pharmaceutical industry. For that reason, we evaluated a simple method ("simulated fogging") applicable to drug product formulations in a specific container closure system. Two different vial types with different surface hydrophilicity were tested using 3 model protein formulations, comparing the simulated fogging test and the degree of fogging after actual lyophilization. The simulated fogging method could predict fogging and showed a correlation to fogging in lyophilized drug product glass vials. We observed that all formulations showed fogging in the hydrophilic vials. By contrast, hydrophobic vials prevented fogging, however, interestingly with remaining defects of so-called droplet formation. Other than extent of fogging, no additional differences of lyophilized cake properties or other product quality attributes were observed between products using the different glass vial types tested.

Subcutaneous Injection Volume of Biopharmaceuticals-Pushing the Boundaries.

Administration into the subcutaneous (SC) tissue is a typical route of delivery for therapeutic proteins, especially for frequent treatments, long-term regimens, or self-administration. It is currently believed that the maximum volume for SC injections is approximately 1.5 mL. Larger SC injection volumes are considered to be associated with injection pain and adverse events at the injection site. However, no controlled clinical studies and actual evidence exist to support this assumption. In this review, we discuss current and publically available data related to SC administration volumes. We conclude that injection volumes higher than 3.5 mL are worth exploring if required for the development of efficacious drug treatments. Studying tissue back pressure, injection site leakage, local tolerability, and injection-related adverse events, such as injection pain, should be considered for the development of higher SC injection volumes.

Understanding the freezing of biopharmaceuticals: first-principle modeling of the process and evaluation of its effect on product quality.

Freezing and thawing are important process steps in the manufacture of numerous biopharmaceuticals. It is well established that these process steps can significantly influence product quality attributes (PQA). Herein, we describe a physico-mathematical model to predict product temperature profiles based on the freezing program as input parameter in a commercial freeze-thaw module. Applying this model, the time from first nucleation until the last point to freeze (LPF) reaching -5°C and the time from -5°C at LPF to -30°C at LPF was varied to study the effect on PQA in a full factorial design. Effects of process parameter settings on a typical fully formulated, highly concentrated monoclonal antibody (mAb) solution as well as highly concentrated mAb solution formulated with buffer only were investigated. We found that both process phases affected PQA, such as aggregates by size-exclusion chromatography, polydispersity index by dynamic light scattering, and number of subvisible particles and turbidity in a complex way. In general, intermediate cooling and freezing times resulted in overall optimized PQA. Fully formulated mAb solution containing cryoprotectant and nonionic surfactant was significantly less affected by freezing-thawing than mAb solution formulated in buffer only.