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.

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.

Framework Mutations of the 10-1074 bnAb Increase Conformational Stability, Manufacturability, and Stability While Preserving Full Neutralization Activity.

The broadly neutralizing anti-HIV antibody, 10-1074, is a highly somatically hypermutated IgG1 being developed for prophylaxis in sub-Saharan Africa. A series of algorithms were applied to identify potentially destabilizing residues in the framework of the Fv region. Of 17 residues defined, a variant was identified encompassing 1 light and 3 heavy chain residues, with significantly increased conformational stability while maintaining full neutralization activity. Central to the stabilization was the replacement of the heavy chain residue T108 with R108 at the base of the CDR3 loop which allowed for the formation of a nascent salt bridge with heavy chain residue D137. Three additional mutations were necessary to confer increased conformational stability as evidenced by differential scanning fluorimetry and isothermal chemical unfolding. In addition, we observed increased stability during low pH incubation in which 40% of the parental monomer aggregated while the combinatorial variant showed no increase in aggregation. Incubation of the variant at 100 mg/mL for 6 weeks at 40°C showed a 9-fold decrease in subvisible particles ≥2 µm relative to the parental molecule. Stability-based designs have also translated to improved pharmacokinetics. Together, these data show that increasing conformational stability of the Fab can have profound effects on the manufacturability and long-term stability of a monoclonal antibody.

CHO cell production and sequence improvement in the 13C6FR1 anti-Ebola antibody.

From March 2014 through February 2015, the Ebola virus spread rapidly in West Africa, resulting in almost 30,000 infections and approximately 10,000 deaths. With no approved therapeutic options available, an experimental antibody cocktail known as ZMapp™ was administered to patients on a limited compassionate-use basis. The supply of ZMapp™ was highly constrained at the time because it was in preclinical development and a novel production system (tobacco plants) was being used for manufacturing. To increase the production of ZMapp™ for an uncertain future demand, a consortium was formed in the fall of 2014 to quickly manufacture these anti-Ebola antibodies in Chinese hamster ovary (CHO) cells using bioreactors for production at a scale appropriate for thousands of doses. As a result of the efforts of this consortium, valuable lessons were learned about the processing of the antibodies in a CHO-based system. One of the ZMapp™ cocktail antibodies, known as c13C6FR1, had been sequence-optimized in the framework region for production in tobacco and engineered as a chimeric antibody. When transfected into CHO cells with the unaltered sequence, 13C6FR1 was difficult to process. This report describes efforts to produce 13C6FR1 and the parental murine hybridoma sequence, 13C6mu, in CHO cells, and provides evidence for the insertion of a highly conserved framework amino acid that improved the physical properties necessary for high-level expression and purification. Furthermore, it describes the technical and logistical lessons learned that may be beneficial in the event of a future Ebola virus or other pandemic viral outbreaks where mAbs are considered potential therapeutics.

Differential binding of heavy chain variable domain 3 antigen binding fragments to protein A chromatography resins.

This work examines the binding of 15 different VH3 IgGs and their corresponding F(ab')2 fragments to two different protein A chromatography resins: MabSelect(®), which utilizes a recombinant protein A ligand, and MabSelect SuRe(®) (SuRe), which utilizes a tetrameric Z domain ligand. The results show that VH3 F(ab')2 fragments can exhibit a variety of binding behaviours for the two resins. Contrary to previously published data, a subset of these molecules show strong interaction with the Z domain of SuRe(®). Furthermore, the results show that sequence variability of residue 57 in the VH3 heavy chain CDR2 domain correlates with binding behaviour on MabSelect(®) and SuRe(®). Site-directed mutagenesis of this residue confers gain or loss of VH3 F(ab')2 binding to these resins in 3 mAbs, demonstrating that it plays a key role in both recombinant protein A and Z domain interaction. A fourth mAb with a longer CDR2 loop was not affected by mutation of residue 57, indicating that CDR2 domain length may alter the binding interface and lead to the involvement of other residues in protein A binding.

Characterization of cysteine related variants in an IgG2 antibody by LC-MS with an automated data analysis approach.

In this communication, a high-throughput method for automated data analysis of cysteine-related product quality attributes (PQAs) in IgG2 antibodies is reported. This method leverages recent advances in the relative quantification of PQAs to facilitate the characterization of disulfide variants and free sulfhydryls (SHs) in IgG2 antibodies. The method uses samples labeled with a mass tag (N-ethyl maleimide [NEM]) followed by enzymatic digestion under non-reducing conditions to maintain the cysteine connectivity. The digested IgG2 samples are separated and detected by mass spectrometry (MS) and the resulting peptide map is analyzed in an automated fashion using Pinpoint software (Thermo Scientific). Previous knowledge of IgG2 disulfide structures can be fed into the Pinpoint software to create workbooks for various disulfide linkages and hinge disulfide variants. In addition, the NEM mass tag can be added to the workbooks for targeted analysis of labeled cysteine-containing peptides. The established Pinpoint workbooks are a high-throughput approach to quantify relative abundances of unpaired cysteines and disulfide linkages, including complicated hinge disulfide variants. This approach is especially efficient for comparing large sets of similar samples such as those created in comparability and stability studies or chromatographic fractions. Here, the high throughput method is applied to quantify the relative abundance of hinge disulfide variants and unpaired cysteines in the IgG2 fractions from non-reduced reversed-phase high-performance liquid chromatography (nrRP-HPLC). The LC-MS data analyzed by the Pinpoint workbook suggests that the nrRP-HPLC separated peaks contain hinge disulfide isoforms and free cysteine pairs for each major disulfide isoform structure.

Postmortem findings from Dugong (Dugong dugon) submissions to the University of Queensland: 1997-2010.

To better record and characterize mortality in the declining population of dugong (Dugong dugon) in southeast Queensland, Australia, animals were collected and brought to the University of Queensland for postmortem examination. Fifty-five animals were examined over a 14-yr period. Human activities commonly caused the animal death. Several deaths were attributed to primary or secondary infections and idiopathic and degenerative diseases. A significant proportion of animals were found to have nonspecific signs of chronic debility, but the causes of disease and mortality in these cases remains to be identified.