Acetate- and Citrate-Specific Ion Effects on Unfolding and Temperature-Dependent Aggregation Rates of Anti-Streptavidin IgG1

Abstract

Controlling and predicting unwanted degradation, such as non-native aggregation, is a long-standing challenge for mAbs and other protein-based products. mAb aggregation rates are typically sensitive to temperature, pH, and the addition of excipients. Quantitatively comparing temperature-dependent aggregation rates across multiple possible formulations is a challenge in product development. A parallel temperature initial rate method is used to efficiently and accurately determine initial rates for anti-streptavidin (AS) IgG1 aggregation as a function of pH, [NaCl], and in the presence of acetate versus citrate buffer. Parallel temperature initial rates are shown to agree with results from a traditional, isothermal method and permits direct comparison of the formulations across almost 3 orders of magnitude of aggregation rates. The apparent midpoint unfolding temperatures (through differential scanning calorimetry) and the effective activation energy values (Ea) are generally higher in acetate buffer compared with citrate buffer, which is consistent with preferential accumulation of citrate ions compared with acetate ions that was speculated in previous work (Barnett et al., J Phys Chem B, 2015). Static light scattering and Kirkwood-Buff analysis show that AS-IgG1 has stronger net repulsive protein-protein interactions in acetate compared with citrate buffer, also consistent with increased values of Ea. In an extreme case, aggregation of AS-IgG1 is effectively eliminated across all practical temperatures at pH 4 in 10 mM sodium acetate but proceeds readily in citrate buffer.

Identifying protein aggregation mechanisms and quantifying aggregation rates from combined monomer depletion and continuous scattering

“…After a given time, vials were removed from the PTIR device and quenched on ice to effectively halt the aggregation process. The concentration of remaining monomer ( c m ) and the initial monomer concentration ( c m,0 ) for a given protein solution was determined using size exclusion chromatography as described previously [12,33]. The aggregation initial-rate coefficient ( k obs ) was calculated from Eq.…”

“…From a practical perspective, this required significant user manipulation, user time, sample material, and also did not address the question of whether the mechanisms change as a function of temperature. The present report focuses on an approach to circumvent those limitations by combining two recently developed methods to obtain temperature-dependent measures of aggregation rates: parallel-temperature initial rates (PTIR) with SEC [33], and simultaneous multiple-sample static light scattering (SMSLS) [34]. …”

“…In the PTIR method, one instead determines monomer loss for a single or small number of samples at many temperatures for the same incubation time. It has been shown elsewhere, that in the initial-rate regime the two approaches are quantitatively equivalent, but the PTIR method is more sample sparing and efficient [33]. Because aggregation is determined only for the initial rates regime, no specific aggregation model needs to be assumed, although they can be inferred later (see Discussion).…”

“…$$k_{\mathit{obs}}false(Tfalse)=\frac{1-c_{m}false(Tfalse)/c_{m,0}}{t}$$ and is based on the general result that many mechanisms reduce to zero-order kinetics in the limit of small extents of reaction [33,35]. In the above relations, t is the preselected incubation time, T is the incubation temperature, c m is the concentration of monomer at T for that incubation time, and c m ,0 is the initial concentration prior to heating.…”

“…In the above relations, t is the preselected incubation time, T is the incubation temperature, c m is the concentration of monomer at T for that incubation time, and c m ,0 is the initial concentration prior to heating. In previous work, the ratio of c m to c m,0 was defined as the monomer fraction ( m ) [12,33]. …”