The Development of a Novel Aflibercept Formulation for Ocular Delivery.

Aflibercept is a recombinant fusion protein that is commercially available for several ocular diseases impacting millions of people worldwide. Here, we use a case study approach to examine alternative liquid formulations for aflibercept for ocular delivery, utilizing different stabilizers, buffering agents, and surfactants with the goal of improving the thermostability to allow for limited storage outside the cold chain. The formulations were developed by studying the effects of pH changes, substituting amino acids for sucrose and salt, and using polysorbate 80 or poloxamer 188 instead of polysorbate 20. A formulation containing acetate, proline, and poloxamer 188 had lower rates of aggregate formation at 4, 30, and 40°C when compared to the marketed commercial formulation containing phosphate, sucrose, sodium chloride, and polysorbate 20. Further studies examining subvisible particles after exposure to a transport stress and long-term stability at 4°C, post-translational modifications by multi-attribute method, purity by reduced and non-reduced capillary electrophoresis, and potency by cell proliferation also demonstrated a comparable or improved stability for the enhanced formulation of acetate, proline, and poloxamer 188. This enhanced stability could enable limited storage outside of the cold chain, allowing for easier distribution in low to middle income countries.

Automated multi-attribute method sample preparation using high-throughput buffer exchange tips.

RATIONALE: The multi-attribute method (MAM) has become a valuable mass spectrometry (MS)-based tool that can identify and quantify the site-specific product attributes and purity information for biotherapeutics such as monoclonal antibodies (mAbs) and fusion molecules in recent years. As we expand the use of the MAM at various stages of drug development, it is critical to enhance the sample preparation throughput without additional chemical modifications and variability. METHODS: In this study, a fully automated MAM sample preparation protocol is presented, where rapid desalting in less than 15 minutes is achieved using miniaturized size-exclusion chromatography columns in pipette tips on an automated liquid handler. The peptide samples were analyzed using an electrospray ionization (ESI) orbitrap mass spectrometer coupled to an ultra-high-performance liquid chromatography (UHPLC) system with a dual column switching system. RESULTS: No significant change was observed in product attributes and their quantities compared with manual, low-artifact sample preparation. The sample recovery using the buffer exchange tips was greatly enhanced over the manual spin cartridges while still demonstrating excellent reproducibility for a wide variety of starting sample concentrations. Unlike a plate desalting system, the individual columns provide flexibility in the number of samples prepared at a time and sample locations within plates. CONCLUSIONS: This automated protocol enables the preparation of up to 96 samples with less "at-bench" time than the manual preparation of a smaller batch of samples, thereby greatly facilitating support of process development and the use of the MAM in quality control.

Evaluation of Crystal Zenith Microtiter Plates for High-Throughput Formulation Screening.

Formulation screening for biotherapeutics can cover a vast array of excipients and stress conditions. These studies consume quantities of limited material and, with higher concentrated therapeutics, more material is needed. Here, we evaluate the use of crystal zenith (CZ) microtiter plates in conjunction with FluoroTec-coated butyl rubber mats as a small-volume, high-throughput system for formulation stability studies. The system was studied for evaporation, edge effects, and stability with comparisons to type 1 glass and CZ vials for multiple antibodies and formulations. Evaporation was minimal at 4°C and could be reduced at elevated temperatures using sealed, mylar bags. Edge effects were not observed until 12 weeks at 40°C. The overall stability ranking as measured by the rate of change in high molecular weight and percent main peak species was comparable across both vials and plates at 4°C and 40°C out to 12 weeks. Product quality attributes as measured by the multi-attribute method were also comparable across all containers for each molecule formulation. A potential difference was measured for subvisible particle analysis, with the plates measuring lower particle counts than the vials. Overall, CZ plates are a viable alternative to traditional vials for small-volume, high-throughput formulation stability screening studies.

Coformulation of Broadly Neutralizing Antibodies 3BNC117 and PGT121: Analytical Challenges During Preformulation Characterization and Storage Stability Studies.

In this study, we investigated analytical challenges associated with the formulation of 2 anti-HIV broadly neutralizing antibodies (bnAbs), 3BNC117 and PGT121, both separately at 100 mg/mL and together at 50 mg/mL each. The bnAb formulations were characterized for relative solubility and conformational stability followed by accelerated and real-time stability studies. Although the bnAbs were stable during 4°C storage, incubation at 40°C differentiated their stability profiles. Specific concentration-dependent aggregation rates at 30°C and 40°C were measured by size exclusion chromatography for the individual bnAbs with the mixture showing intermediate behavior. Interestingly, although the relative ratio of the 2 bnAbs remained constant at 4°C, the ratio of 3BNC117 to PGT121 increased in the dimer that formed during storage at 40°C. A mass spectrometry-based multiattribute method, identified and quantified differences in modifications of the Fab regions for each bnAb within the mixture including clipping, oxidation, deamidation, and isomerization sites. Each bnAb showed slight differences in the levels and sites of lysine residue glycations. Together, these data demonstrate the ability to differentiate degradation products from individual antibodies within the bnAb mixture, and that degradation rates are influenced not only by the individual bnAb concentrations but also by the mixture concentration.

Development of a quantitative mass spectrometry multi-attribute method for characterization, quality control testing and disposition of biologics.

Regulatory agencies have recently recommended a Quality by Design (QbD) approach for the manufacturing of therapeutic molecules. A QbD strategy requires deep understanding at the molecular level of the attributes that are crucial for safety and efficacy and for insuring that the desired quality of the purified protein drug product is met at the end of the manufacturing process. A mass spectrometry (MS)-based approach to simultaneously monitor the extensive array of product quality attributes (PQAs) present on therapeutic molecules has been developed. This multi-attribute method (MAM) uses a combination of high mass accuracy / high resolution MS data generated by Orbitrap technology and automated identification and relative quantification of PQAs with dedicated software (Pinpoint). The MAM has the potential to replace several conventional electrophoretic and chromatographic methods currently used in Quality Control to release therapeutic molecules. The MAM represents an optimized analytical solution to focus on the attributes of the therapeutic molecule essential for function and implement QbD principles across process development, manufacturing and drug disposition.