Quantitation of soluble aggregates by sedimentation velocity analytical ultracentrifugation using an optical alignment system - Aspects of method validation.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) is routinely used for quantitation of soluble aggregates as an orthogonal technique to size-exclusion chromatography (SEC). SV-AUC presents many advantages over the SEC, yet lower precision of aggregate quantitation by SV-AUC often complicates comparison between aggregate values generated by these techniques and subsequent decision making. In an earlier report, we described the development of an optical alignment (OA) system and evaluated the intermediate precision of aggregate quantitation offered by the OA. Here, we determine the limit of detection (LOD) and limit of quantitation (LOQ) which can be achieved with the OA. For a common setup using three cells, the improvement lent by the OA system is almost 2.5-fold compared to the earlier reported limits. In addition, we estimate the contribution of the fitting variability and compare options to further increase the precision of aggregate quantitation by SV-AUC.

An optical alignment system improves precision of soluble aggregate quantitation by sedimentation velocity analytical ultracentrifugation.

Appropriate characterization of soluble aggregates is an important aspect of biologics development and manufacturing, and sedimentation velocity analytical ultracentrifugation (SV-AUC) is often used an orthogonal technique to size-exclusion chromatography (SEC) for this purpose. Precise quantification of low levels of soluble aggregates by SV-AUC can be adversely impacted by improper cell alignment. This report describes the development of an optical system capable of quantifying cell alignment that affords a substantial improvement compared to historical approaches.

Characterization of a cathepsin D protease from CHO cell-free medium and mitigation of its impact on the stability of a recombinant therapeutic protein.

During purification process development of a recombinant therapeutic protein, an endoproteolytic activity endogenous to the Chinese hamster ovary (CHO) cells and leading to degradation at particular hydrophobic amino acid residues (e.g., Phe and Trp) was observed when processing at acidic pH. The presence of residual levels of protease activity in purified protein batches affected the inherent activity of the product when stored as a solution. To develop a robust purification strategy to minimize this undesirable impact, identification and characterization of this protease was essential to ultimately ensure that a solution formulation was stable for many years. A protease was isolated from CHO cell-free medium (CFM) using a combination of immobilized pepstatin-A agarose chromatography and size exclusion chromatography (SEC). The isolated protease has significant proteolytic activity at pH ∼ 3 to neutral pH and was identified as cathepsin D by mass spectrometry. Analytical SEC, chip-based capillary gel electrophoresis, imaged capillary isoelectric focusing (cIEF), and circular dichroism (CD) spectropolarimetry analyses were performed for additional characterization of the protease. The identification and characterization of this protease enabled the development of a robust purification process by implementation of a controlled temperature inactivation unit operation (heat inactivation) that enabled essentially complete inactivation of the protease, resulting in the production of a stable drug product that had not been possible using column chromatography alone. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:120-129, 2018.

Biophysical signatures of noncovalent aggregates formed by a glucagonlike peptide-1 analog: a prototypical example of biopharmaceutical aggregation.

LY307161 is a 31 amino acid analog of glucagonlike peptide-1(7-37)OH susceptible to physical instability associated with pharmaceutical processing. Orthogonal biophysical studies were conducted to explore the origins of this physical instability and to distinguish pharmaceutically desirable states of this aggregating peptide from undesirable ones. Equilibrium sedimentation analysis established that LY307161 exists as a monomer at pH 3, and reversibly self-associates in the pH range 7.5-10.5. Causative factors for physical instability related to lyophilization conditions were investigated. Solution pH, acetonitrile content, and concentration of the peptide prior to lyophilization each impacted physicochemical properties of the resultant powders. A comparative study of two powder samples exhibiting physicochemically disparate properties established that LY307161 forms soluble noncovalent aggregates. FT-IR analyses in the solid and solution states identified a prominent band at 1657-1659 cm(-1) attributed to alpha-helix structure. Noncovalent soluble aggregate exhibited characteristic bands at 1615 and 1698 cm(-1) indicative of intermolecular beta-sheet structure. An agitation-induced, precipitated solid form of LY307161 exhibited a different FT-IR signature indicative of a conformationally distinct species. Circular dichroism and fluorescence spectroscopy, together with dynamic light scattering measurements and dye-aggregate complexation, provided additional insights into the distinctions between aggregated and native LY307161.

Technical decision-making with higher order structure data: specific binding of a nonionic detergent perturbs higher order structure of a therapeutic monoclonal antibody.

Robust higher order structure (HOS) characterization capability and strategy are critical throughout biopharmaceutical development from initial candidate selection and formulation screening to process optimization and manufacturing. This case study describes the utility of several orthogonal HOS methods as investigational tools during purification process development. An atypically high level of residual detergent in a development drug substance batch of a therapeutic monoclonal antibody triggered a root cause investigation. Several orthogonal biophysical techniques were used to uncover and characterize a specific interaction between the detergent and the antibody. Isothermal titration calorimetry (ITC) was used to quantify the molar ratio and affinity of the binding event, and circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC) were used to evaluate corresponding impacts on secondary/tertiary structure and thermal stability, respectively. As detergents are used routinely in biopharmaceutical processing, this case study highlights the value and power of HOS data in informing technical investigations and underlines the importance of HOS characterization as a component of overall biopharmaceutical analytical control strategy.

Zinc binding drives the folding and association of the homo-trimeric gamma-carbonic anhydrase from Methanosarcina thermophila.

Carbonic anhydrase from the archeon Methanosarcina thermophila (Cam) is a homo-trimeric enzyme, the left-handed beta-helical subunits of which bind three catalytic Zn(2+) ions at symmetry-related subunit interfaces. The observation of activity for holo-Cam at nanomolar concentrations provides a minimal estimated free energy of folding and assembly of the trimeric holo-complex of approximately 70 kcal (mol trimer)(-1) at standard state. Although the direct measurement of stability by chemical denaturation was precluded by the irreversible unfolding of the holo-enzyme, the reversible unfolding of metal-free apo-Cam is well described by a three-state model involving the folded apo-trimer, the folded monomer and the unfolded monomer. The monomer is estimated to have a stability of 4.0 +/- 0.3 kcal (mol monomer)(-1). The association to form apo-trimer contributes 13.2 +/- 0.4 kcal (mol trimer)(-1), a value confirmed by analytical ultracentrifugation measurements. Far- and near-UV circular dichroism data show a progressive increase in secondary and tertiary structure as the apo-monomer is converted to holo-trimer. The literature value for the free energy of binding of one Zn(2+) ion to a canonical active site, 16.4 kcal mol(-1), is consistent with the presumption that the >45 kcal (mol trimer)(-1) generated by the binding of three ions represents the major contribution to the stability of the holo-trimeric Cam.

Opalescent appearance of an IgG1 antibody at high concentrations and its relationship to noncovalent association.

PURPOSE: Therapeutic antibodies are often formulated at a high concentration where they may have an opalescent appearance. The aim of this study is to understand the origin of this opalescence, especially its relationship to noncovalent association and physical stability. METHODS: The turbidity and the association state of an IgG1 antibody were investigated as a function of concentration and temperature using static and dynamic light scattering, nephelometric turbidity, and analytical ultracentrifugation. RESULTS. The antibody had increasingly opalescent appearance in the concentration range 5-50 mg/ml. The opalescence was greater at refrigerated temperature but was readily reversible upon warming to room temperature. Turbidity measured at 25 degrees C was linear with concentration, as expected for Rayleigh scatter in the absence of association. In the concentration range 1-50 mg/ml, the weight average molecular weights were close to that expected for a monomer. Zimm plot analysis of the data yielded a negative second virial coefficient, indicative of attractive solute-solute interactions. The hydrodynamic diameter was independent of concentration and remained unchanged as a function of aging at room temperature. CONCLUSIONS: The results indicate that opalescent appearance is not due to self-association but is a simple consequence of Rayleigh scatter. Opalescent appearance did not result in physical instability.