Designing Robust Monoclonal Antibody Drug Products: Pitfalls of Simplistic Approaches for Stability Prediction.

Thermal stability attributes including unfolding onset (Tonset) and mid-point (Tm) are often utilized for efficient development of monoclonal antibody (mAb) products during lead selection and formulation screening workflows. An assumption of direct correlation between thermal and kinetic physical stability underpins this basic approach. While literature reports have substantiated this general approach under specific conditions, clear exceptions have been highlighted alongside. Herein, a set of mAbs formulated under diverse solution conditions to generate a broad array of thermal and kinetic stability profiles were systematically analyzed. Sequence modifications in the Fc region were purposefully engineered to generate a set of low-melting mAbs. A diverse set of excipients were subsequently utilized and shown to modulate the Tm over a wide range. While a general correlation between high Tm and low aggregation rate was observed under accelerated conditions, the predictive utility of Tm under relevant product storage conditions was inadequate at best. Critically, Tm data did not correlate with long-term aggregation rates under refrigerated or room temperature conditions. Even under accelerated conditions, Tm appeared to be a poor predictor of aggregation once it exceeded the solution storage temperature (40°C) by ∼15°C, similar to conditions routinely encountered in the development of canonical mAbs (Tm > 60°C). Pitfalls of simplistic correlative approaches are discussed in the context of practical biologics product development.

Succinate Buffer in Biologics Products: Real-world Formulation Considerations, Processing Risks and Mitigation Strategies.

The succinic acid/succinate system has an excellent buffering capacity at acidic pH values (4.5-6.0), promising to be a buffer of choice for biologics having slightly acidic to basic isoelectric points (pI 6 - 9). However, its prevalence in drug products is limited due to the propensity (risk) of its components to crystallize during freezing and the consequent shift in the pH which might affect the product stability. Most of these previous assessments have been performed under operational conditions that do not simulate typical drug product processing conditions. In this work, we have characterized the physicochemical behavior of succinate formulations under representative pharmaceutical conditions. Our results indicate that the pH increases by ∼ 1.2 units in 25 mM and 250 mM succinate buffers at pharmaceutically relevant freezing conditions. X-ray diffractometry studies revealed selective crystallization of monosodium succinate, which is posed as the causative mechanism. This salt crystallization was not observed in the presence of 2% w/v sucrose, suggesting that this pH shift can be mitigated by including sucrose in the formulation. Additionally, three monoclonal antibodies (mAbs) that represent different IgG subtypes and span a range of pIs (5.9 - 8.8) were formulated with succinate and sucrose and subjected to freeze-thaw, frozen storage and lyophilization. No detrimental impact on quality attributes (QA) such as high molecular weight (HMW) species, turbidity, alteration in protein concentration and sub-visible particles, was observed of any of the mAbs tested. Lastly, drug formulations lyophilized in succinate buffer with sucrose demonstrated acceptable QA profiles upon accelerated kinetic storage stability, supporting the use of succinate buffers in mAb drug products.

Effects of Salts and Surface Charge on the Biophysical Stability of a Low pI Monoclonal Antibody.

The impact of five representative Hofmeister salts (NaCl, KCl, MgCl2, Na2SO4, and NaSCN) on the thermal stability and aggregation kinetics of a slightly acidic monoclonal antibody (mAb) were investigated under different pH conditions. The thermal stability of the mAb was assessed by measuring the lowest unfolding transition temperature, Tm, with differential scanning fluorimetry. MgCl2 and NaSCN significantly decreased Tm at all three charged states of the mAb, but to the greatest extent when the mAb surface charge was net positive. Non-native aggregation kinetics was monitored by measuring Rayleigh light scattering. When the mAb surface charge was net positive or net neutral, the nucleation rate increased non-monotonically with MgCl2 and NaSCN but decreased monotonically with NaCl, KCl, and Na2SO4. By contrast, when the mAb surface was negatively charged, there were only minor changes in the nucleation rate with all salts tested. Furthermore, there was less structural perturbation and slower aggregation rates when the mAb was net negatively charged than when it was net neutrally or positively charged. The observed salt effects on thermal unfolding are consistent with ion-specific mechanisms dominated by short-range amide backbone binding. On the other hand, the salt effects on nucleation rates appear to be influenced by both amide backbone binding and long-range electrostatic binding of ions to charged amino acid side chains.

A stepwise mechanism for aqueous two-phase system formation in concentrated antibody solutions.

Aqueous two-phase system (ATPS) formation is the macroscopic completion of liquid-liquid phase separation (LLPS), a process by which aqueous solutions demix into 2 distinct phases. We report the temperature-dependent kinetics of ATPS formation for solutions containing a monoclonal antibody and polyethylene glycol. Measurements are made by capturing dark-field images of protein-rich droplet suspensions as a function of time along a linear temperature gradient. The rate constants for ATPS formation fall into 3 kinetically distinct categories that are directly visualized along the temperature gradient. In the metastable region, just below the phase separation temperature, Tph , ATPS formation is slow and has a large negative apparent activation energy. By contrast, ATPS formation proceeds more rapidly in the spinodal region, below the metastable temperature, Tmeta , and a small positive apparent activation energy is observed. These region-specific apparent activation energies suggest that ATPS formation involves 2 steps with opposite temperature dependencies. Droplet growth is the first step, which accelerates with decreasing temperature as the solution becomes increasingly supersaturated. The second step, however, involves droplet coalescence and is proportional to temperature. It becomes the rate-limiting step in the spinodal region. At even colder temperatures, below a gelation temperature, Tgel , the proteins assemble into a kinetically trapped gel state that arrests ATPS formation. The kinetics of ATPS formation near Tgel is associated with a remarkably fragile solid-like gel structure, which can form below either the metastable or the spinodal region of the phase diagram.

Guanidinium can both Cause and Prevent the Hydrophobic Collapse of Biomacromolecules.

A combination of Fourier transform infrared and phase transition measurements as well as molecular computer simulations, and thermodynamic modeling were performed to probe the mechanisms by which guanidinium (Gnd+) salts influence the stability of the collapsed versus uncollapsed state of an elastin-like polypeptide (ELP), an uncharged thermoresponsive polymer. We found that the cation's action was highly dependent upon the counteranion with which it was paired. Specifically, Gnd+ was depleted from the ELP/water interface and was found to stabilize the collapsed state of the macromolecule when paired with well-hydrated anions such as SO42-. Stabilization in this case occurred via an excluded volume (or depletion) effect, whereby SO42- was strongly partitioned away from the ELP/water interface. Intriguingly, at low salt concentrations, Gnd+ was also found to stabilize the collapsed state of the ELP when paired with SCN-, which is a strong binder for the ELP. In this case, the anion and cation were both found to be enriched in the collapsed state of the polymer. The collapsed state was favored because the Gnd+ cross-linked the polymer chains together. Moreover, the anion helped partition Gnd+ to the polymer surface. At higher salt concentrations (>1.5 M), GndSCN switched to stabilizing the uncollapsed state because a sufficient amount of Gnd+ and SCN- partitioned to the polymer surface to prevent cross-linking from occurring. Finally, in a third case, it was found that salts which interacted in an intermediate fashion with the polymer (e.g., GndCl) favored the uncollapsed conformation at all salt concentrations. These results provide a detailed, molecular-level, mechanistic picture of how Gnd+ influences the stability of polypeptides in three distinct physical regimes by varying the anion. It also helps explain the circumstances under which guanidinium salts can act as powerful and versatile protein denaturants.

Beyond the Hofmeister Series: Ion-Specific Effects on Proteins and Their Biological Functions.

Ions differ in their ability to salt out proteins from solution as expressed in the lyotropic or Hofmeister series of cations and anions. Since its first formulation in 1888, this series has been invoked in a plethora of effects, going beyond the original salting out/salting in idea to include enzyme activities and the crystallization of proteins, as well as to processes not involving proteins like ion exchange, the surface tension of electrolytes, or bubble coalescence. Although it has been clear that the Hofmeister series is intimately connected to ion hydration in homogeneous and heterogeneous environments and to ion pairing, its molecular origin has not been fully understood. This situation could have been summarized as follows: Many chemists used the Hofmeister series as a mantra to put a label on ion-specific behavior in various environments, rather than to reach a molecular level understanding and, consequently, an ability to predict a particular effect of a given salt ion on proteins in solutions. In this Feature Article we show that the cationic and anionic Hofmeister series can now be rationalized primarily in terms of specific interactions of salt ions with the backbone and charged side chain groups at the protein surface in solution. At the same time, we demonstrate the limitations of separating Hofmeister effects into independent cationic and anionic contributions due to the electroneutrality condition, as well as specific ion pairing, leading to interactions of ions of opposite polarity. Finally, we outline the route beyond Hofmeister chemistry in the direction of understanding specific roles of ions in various biological functionalities, where generic Hofmeister-type interactions can be complemented or even overruled by particular steric arrangements in various ion binding sites.

An NH moiety is not required for anion binding to amides in aqueous solution.

Herein, we use a combination of thermodynamic and spectroscopic measurements to investigate the interactions of Hofmeister anions with a thermoresponsive polymer, poly(N,N-diethylacrylamide) (PDEA). This amide-based polymer does not contain an NH moiety in its chemical structure and, thus, can serve as a model to test if anions bind to amides in the absence of an NH site. The lower critical solution temperature (LCST) of PDEA was measured as a function of the concentration for 11 sodium salts in aqueous solutions, and followed a direct Hofmeister series for the ability of anions to precipitate the polymer. More strongly hydrated anions (CO3(2-), SO4(2-), S2O3(2-), H2PO4(-), F(-), and Cl(-)) linearly decreased the LCST of the polymer with increasing the salt concentration. Weakly hydrated anions (SCN(-), ClO4(-), I(-), NO3(-), and Br(-)) increased the LCST at lower salt concentrations but salted the polymer out at higher salt concentrations. Proton nuclear magnetic resonance (NMR) was used to probe the mechanism of the salting-in effect and showed apparent binding between weakly hydrated anions (SCN(-) and I(-)) and the α protons of the polymer backbone. Additional experiments performed by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy found little change in the amide I band upon the addition of salt, which is consistent with very limited, if any, interactions between the salt ions and the carbonyl moiety of the amide. These results support a molecular mechanism for ion-specific effects on proteins and model amides that does not specifically require an NH group to interact with the anions for the salting-in effect to occur.

Reversal of the hofmeister series: specific ion effects on peptides.

Ion-specific effects on salting-in and salting-out of proteins, protein denaturation, as well as enzymatic activity are typically rationalized in terms of the Hofmeister series. Here, we demonstrate by means of NMR spectroscopy and molecular dynamics simulations that the traditional explanation of the Hofmeister ordering of ions in terms of their bulk hydration properties is inadequate. Using triglycine as a model system, we show that the Hofmeister series for anions changes from a direct to a reversed series upon uncapping the N-terminus. Weakly hydrated anions, such as iodide and thiocyanate, interact with the peptide bond, while strongly hydrated anions like sulfate are repelled from it. In contrast, reversed order in interactions of anions is observed at the positively charged, uncapped N-terminus, and by analogy, this should also be the case at side chains of positively charged amino acids. These results demonstrate that the specific chemical and physical properties of peptides and proteins play a fundamental role in ion-specific effects. The present study thus provides a molecular rationalization of Hofmeister ordering for the anions. It also provides a route for tuning these interactions by titration or mutation of basic amino acid residues on the protein surface.

Effects of End Group Termination on Salting-Out Constants for Triglycine.

Salting out constants for triglycine were calculated for a series of Hofmeister salts using molecular dynamics simulations. Three variants of the peptide were considered with both termini capped, just the N-terminus capped, and without capping. The simulations were supported by NMR and FTIR measurements. The data provide strong evidence that earlier experimental values of salting out constants assigned to the fully capped peptide (as previously assumed) should have been assigned to the half-capped peptide instead. Therefore, these values cannot be used to directly establish Hofmeister ordering of ions at the peptide backbone, since they are strongly influenced by interactions of the ions with the negatively charged C-terminus.

Molecular mechanisms of ion-specific effects on proteins.

The specific binding sites of Hofmeister ions with an uncharged 600-residue elastin-like polypeptide, (VPGVG)(120), were elucidated using a combination of NMR and thermodynamic measurements along with molecular dynamics simulations. It was found that the large soft anions such as SCN(-) and I(-) interact with the polypeptide backbone via a hybrid binding site that consists of the amide nitrogen and the adjacent α-carbon. The hydrocarbon groups at these sites bear a slight positive charge, which enhances anion binding without disrupting specific hydrogen bonds to water molecules. The hydrophobic side chains do not contribute significantly to anion binding or the corresponding salting-in behavior of the biopolymer. Cl(-) binds far more weakly to the amide nitrogen/α-carbon binding site, while SO(4)(2-) is repelled from both the backbone and hydrophobic side chains of the polypeptide. The Na(+) counterions are also repelled from the polypeptide. The identification of these molecular-level binding sites provides new insights into the mechanism of peptide-anion interactions.