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.

Modeling and robust pooling design of a preparative cation-exchange chromatography step for purification of monoclonal antibody monomer from aggregates.

This study has implemented and calibrated a model that describes the separation of the monomer of monoclonal antibodies from the dimer and larger oligomers on preparative-scale using cation-exchange chromatography. A general rate model with temperature dependent diffusion was coupled to a pH- and temperature-dependent steric mass action model. The model was shown to predict the retention of the monomer, dimer, and oligomer at low loadings for different pH levels and temperatures. Additionally, the model was shown to adequately predict the elution behavior of the monomer and soluble aggregates at high loadings within the same ranges with some limitations. The model was not able to accurately describe the shape of the product break-through curves or the slight levels of co-elution of the dimer and oligomer with the monomer at higher pH. The model was used to predict how 12 process variations impact the separation. The model is used to establish an elution end collection criterion such that the step can robustly provide the target purity of monomers.

Caprylic acid precipitation method for impurity reduction: an alternative to conventional chromatography for monoclonal antibody purification.

We report the use of caprylic acid based impurity precipitation as (1) an alternative method to polishing chromatography techniques commonly used for monoclonal antibody purification and (2) an impurity reduction step prior to harvesting the bioreactor. This impurity reduction method was tested with protein A purified antibodies and with cell culture fluid. First, the operational parameters influencing precipitation of host cell proteins and high molecular weight aggregate in protein A pools were investigated. When used as a polishing step, the primary factor affecting purification and yield was determined to be pH. Caprylic acid precipitation was comparable to polishing IEX chromatography in reducing host cell protein and aggregate levels. A virus reduction study showed complete clearance of a model retrovirus during caprylic acid precipitation of protein A purified antibody. Caprylic acid mediated impurity precipitation in cell culture showed that the impurity clearance was generally insensitive to pH and caprylic acid concentration whereas yield was a function of caprylic acid concentration. Protein A purification of caprylic acid precipitated cell culture fluid generated less turbid product pool with reduced levels of host cell proteins and high molecular weight aggregate. The results of this study show caprylic acid precipitation to be an effective purification method that can be incorporated into a production facility with minimal cost as it utilizes existing tanks and process flow. Eliminating flow through chromatography polishing step can provide process intensification by avoiding the process tank volume constraints for high titer processes.

The use of native cation-exchange chromatography to study aggregation and phase separation of monoclonal antibodies.

This study introduces a novel analytical approach for studying aggregation and phase separation of monoclonal antibodies (mAbs). The approach is based on using analytical scale cation-exchange chromatography (CEX) for measuring the loss of soluble monomer in the case of individual and mixed protein solutions. Native CEX outperforms traditional size-exclusion chromatography in separating complex protein mixtures, offering an easy way to assess mAb aggregation propensity. Different IgG1 and IgG2 molecules were tested individually and in mixtures consisting of up to four protein molecules. Antibody aggregation was induced by four different stress factors: high temperature, low pH, addition of fatty acids, and rigorous agitation. The extent of aggregation was determined from the amount of monomeric protein remaining in solution after stress. Consequently, it was possible to address the role of specific mAb regions in antibody aggregation by co-incubating Fab and Fc fragments with their respective full-length molecules. Our results revealed that the relative contribution of Fab and Fc regions in mAb aggregation is strongly dependent on pH and the stress factor applied. In addition, the CEX-based approach was used to study reversible protein precipitation due to phase separation, which demonstrated its use for a broader range of protein-protein association phenomena. In all cases, the role of Fab and Fc was clearly dissected, providing important information for engineering more stable mAb-based therapeutics.

Hsp31, the Escherichia coli yedU gene product, is a molecular chaperone whose activity is inhibited by ATP at high temperatures.

The Escherichia coli chromosome contains several uncharacterized heat-inducible loci that may encode novel molecular chaperones or proteases. Here we show that the 31-kDa product of the yedU gene is an efficient homodimeric molecular chaperone that is conserved in a number of pathogenic eubacteria and fungi. Heat shock protein (Hsp) 31 relies on temperature-driven conformational changes to expose structured hydrophobic domains that are likely responsible for substrate binding. Complementing the function of refolding, remodeling, and holding chaperones, Hsp 31 preferentially interacts with early unfolding intermediates and rapidly releases them in an active form after transfer to low temperatures. Although Hsp 31 does not appear to exhibit intrinsic ATPase activity, binding of ATP at high temperatures restricts the size or availability of the substrate binding site, thereby modulating chaperone activity. The possible role of ATP in coordinating the function of the cellular complement of molecular chaperones is discussed.

Medium-dependence of the secondary structure of exendin-4 and glucagon-like-peptide-1.

Exendin-4 is a natural, 39-residue peptide first isolated from the salivary secretions of a Gila Monster (Heloderma suspectum) that has some pharmacological properties similar to glucagon-like-peptide-1 (GLP-1). This paper reports differences in the structural preferences of these two peptides. For GLP-1 in aqueous buffer (pH 3.5 or 5.9), the concentration dependence of circular dichroism spectra suggests that substantial helicity results only as a consequence of helix bundle formation. In contrast, exendin-4 is significantly helical in aqueous buffer even at the lowest concentration examined (2.3 microM). The pH dependence of the helical signal for exendin-4 indicates that helicity is enhanced by a more favorable sequence alignment of oppositely charged sidechains. Both peptides become more helical upon addition of either lipid micelles or fluoroalcohols. The stabilities of the helices were assessed from the thermal gradient of ellipticity (partial differential theta(221)/partial differential T values); on this basis, the exendin helix does not melt appreciably until temperatures significantly above ambient. The extent of helix formation for exendin-4 in aqueous buffer (and the thermal stability of the resulting helix) suggests the presence of a stable helix-capping interaction which was localized to the C-terminal segment by NMR studies of NH exchange protection. Solvent effects on the thermal stability of the helix indicate that the C-terminal capping interaction is hydrophobic in nature. The absence of this C-capping interaction and the presence of a flexible, helix-destabilizing glycine at residue 16 in GLP-1 are the likely causes of the greater fragility of the monomeric helical state of GLP-1. The intramolecular hydrophobic clustering in exendin-4 also appears to decrease the extent of helical aggregate formation.