Cell Damage in Light Chain Amyloidosis: FIBRIL INTERNALIZATION, TOXICITY AND CELL-MEDIATED SEEDING.

Light chain (AL) amyloidosis is an incurable human disease characterized by the misfolding, aggregation, and systemic deposition of amyloid composed of immunoglobulin light chains (LC). This work describes our studies on potential mechanisms of AL cytotoxicity. We have studied the internalization of AL soluble proteins and amyloid fibrils into human AC16 cardiomyocytes by using real time live cell image analysis. Our results show how external amyloid aggregates rapidly surround the cells and act as a recruitment point for soluble protein, triggering the amyloid fibril elongation. Soluble protein and external aggregates are internalized into AC16 cells via macropinocytosis. AL amyloid fibrils are shown to be highly cytotoxic at low concentrations. Additionally, caspase assays revealed soluble protein induces apoptosis, demonstrating different cytotoxic mechanisms between soluble protein and amyloid aggregates. This study emphasizes the complex immunoglobulin light chain-cell interactions that result in fibril internalization, protein recruitment, and cytotoxicity that may occur in AL amyloidosis.

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.

Concomitant coiling reduces metalloproteinase levels in flow diverter-treated aneurysms but anti-inflammatory treatment has no effect.

BACKGROUND AND PURPOSE: Flow diverters (FD) can cause rare but devastating delayed aneurysm ruptures in which matrix metalloproteinases (MMPs) have been potentially implicated. Concomitant coiling or anti-inflammatory medications have been proposed to prevent the risk of delayed ruptures. The aim of this study was to evaluate concomitant coiling and ciclosporin in regulating the expression of MMPs in FD-treated aneurysms. MATERIALS AND METHODS: Elastase-induced aneurysms were created in 20 rabbits. Aneurysms were treated with (1) FD alone; (2) FD with concomitant coiling; (3) FD+ ciclosporin; or (4) left untreated as controls. At sacrifice, MMP levels were analyzed by zymography. Kruskal-Wallis one-way non-parametric ANOVA was performed for each enzyme. If significant results were observed for the Kruskal-Wallis test, pairwise group comparisons were performed using Dunn's test with Bonferroni multiple-testing correction. RESULTS: Significant differences were observed among groups for pro-MMP9 (p=0.0337). Pairwise comparison demonstrated higher levels of pro-MMP9 with concomitant coiling compared with untreated aneurysms (p=0.012), with higher though not significantly different levels of pro-MMP9 in FD with concomitant coiling versus FD alone. While not statistically significant, trends were noted regarding differences in active-MMP9 across groups, with a lower level of active-MMP9 with concomitant coiling compared with the other FD groups. No significant differences were observed for pro- or active-MMP2 across groups, or for FD + ciclosporin compared with FD alone. CONCLUSIONS: FD implantation increases the level of pro-MMP9 expression in aneurysms. Provocative trends regarding modulation of active-MMP9 expression with concomitant coiling suggest the need for larger confirmatory preclinical studies. Anti-inflammatory treatment with ciclosporin appears to have a minimal biological effect. TRIAL REGISTRATION NUMBER: R01NS076491.

Use of MMV as a Single Worst-Case Model Virus in Viral Filter Validation Studies.

Typical platform processes for biopharmaceutical products derived from animal cell lines include a parvovirus filtration unit operation to provide viral safety assurance of the drug product. The industry has adopted this platform unit operation and gained a wider understanding of its performance attributes, leading to the possibility of streamlined approaches to virus clearance validation. Here, the concept of virus validation on a parvovirus-grade filter with a single worst-case model virus is presented. Several lines of evidence, including published literature and Amgen's own data, support the use of a parvovirus, such as mouse minute virus (MMV), as a worst-case model virus to assess virus removal by parvovirus filters. The evidence presented includes a discussion of the design and manufacture of virus filters with a size exclusion mechanism for removal. Next, the characteristics of different model viruses are compared and a risk assessment on the selection of the relevant model viruses for clearance studies is presented. Finally, a comprehensive summary of literature and Amgen data is provided, comparing the clearance of larger viruses against MMV. Together, this analysis provides a strong scientific rationale for the use of a single, worst-case model virus for assessing virus removal by parvovirus filters, which will ultimately allow for more efficient and streamlined viral clearance study designs. LAY ABSTRACT: Demonstrating the virus clearance capability of a purification process is an important aspect of biopharmaceutical process development. A key component of the viral safety of the process is the inclusion of a parvovirus-grade filter as an effective and robust virus removal step. Traditional methodologies for viral clearance studies have been based on a conservative, data-intensive approach, but recent trends in the field of virus clearance and process development show evolution towards streamlined and more efficient study designs that are based on understanding the mechanism of viral clearance by downstream unit operations. The publication of scientific datasets and awareness of the underlying mechanisms involved with these unit operations have fueled this trend. Here, the concept of virus validation on a parvovirus-grade filter using a parvovirus as single, worst-case model virus is presented. Multiple lines of evidence are provided to support this proposal, including a review of published literature and Amgen historical data. The adoption of this approach provides benefits in terms of cost savings for executing viral clearance studies, but it also simplifies the necessary dataset and focuses on only supplying value-added information to demonstrate the viral safety of the process.

Multipronged approach to managing beta-glucan contaminants in the downstream process: control of raw materials and filtration with charge-modified nylon 6,6 membrane filters.

(1→3)-ß-D-Glucans (beta-glucans) have been found in raw materials used in the manufacture of recombinant therapeutics. Because of their biological activity, beta-glucans are considered process contaminants and consequently their level in the product needs to be controlled. Although beta-glucans introduced into the cell culture process can readily be removed by bind-and-elute chromatography process steps, beta-glucans can also be introduced into the purification process through raw materials containing beta-glucans as well as leachables from filters made from cellulose. This article reports a multipronged approach to managing the beta-glucan contamination in the downstream process. Raw material screening and selection can be used to effectively limit the level of beta-glucan introduced into the downstream process. Placement of a cellulosic filter upstream of the last bind-and-elute column step or effective preuse flushing can also limit the level of contaminant introduced. More importantly, this article reports the active removal of beta-glucan from the downstream process when necessary. It was discovered that the Posidyne(®) filter, a charge-modified nylon 6,6 membrane filter, was able to effectively remove beta-glucans from buffers at relatively low pH and salt concentrations. An approach of using low beta-glucan buffer components combined with filtration of the buffer with a Posidyne membrane has been successfully demonstrated at preparative scale. Additionally, the feasibility of active removal of beta-glucan from in-process product pools by Posidyne membrane filtration has also been demonstrated. Based on the data presented, a mechanism for binding is proposed, as well as a systematic approach for sizing of the Posidyne filter.