Dipicolinic acid as a novel spore-inspired excipient for antibody formulation

Abstract

Ionic excipients are commonly used in aqueous therapeutic monoclonal antibody (mAb) formulations. Novel excipients are of industrial interest, with a recent focus on Arg salt forms and their application as viscosity reducing and stabilizing additives. Here, we report that the calcium salt of dipicolinic acid (DPA, pyridine-2,6-dicarboxylic acid), uniquely present in nature in the core of certain bacterial spores, reduces the viscosity of a mAb formulated at 150mg/mL, below that achieved by Arg hydrochloride at the same concentration (10mM). DPA also reduced the reversible phase separation of the same formulation, which characteristically occurs for this mAb upon cooling to 4°C. Differential scanning calorimetry and differential scanning fluorimetry did not reveal a conformation destabilisation of the mAb in the presence of 10mM DPA, or by the related quinolinic acid (QA, pyridine-2,3-dicarboxylic acid). However, fluorescence spectrophotometry did reveal localised (aromatic) conformational changes to the mAb attributed to DPA, dependent on the salt form. While precise mechanisms of action remain to be identified, our preliminary data suggest that these DPA salts are worthy of further investigation as novel ionic excipient for biologics formulation.

Bacterial Spores: Mechanisms of Stability and Targets for Modern Biotechnologies

“…As indicated above, the content of this biomarker is up to 15% of the dry weight of spores and its content varies depending on the species, strain of spore-forming bacteria and sporulation conditions [38,39,42,48]…”

“…The presence of several additional membranes compared to a vegetative cell is a barrier against the penetration of water and substances dissolved in it. Dehydration protects the inside proteins from denaturation and irreversible aggregation at extremely high temperatures, which mediates the ability of spores to survive for a long time (tens, hundreds or more years) without a supply of nutrients under conditions when vegetative cells die [19,[37][38][39].…”

“…High heat resistance of endospores is provided by dipicolinic (pyridine-2,6-dicarboxylic) acid (DPA), which forms a chelate complex with calcium and other divalent cations and reduces the water content in spores to a very low level [21,39,42,43]. DPA accounts for up to 15% of the total endospore mass and is not found in vegetative cells [7,19,21,38,44,45]. Despite the fact that DPA was found in bacterial spores quite a long time ago [43], the function of this interesting chemical structure has been not finally determined [19,44,45].…”

“…The leading role of DPA in ensuring dehydration [37], creating and maintaining metabolic rest [7], as well as stabilization of basic nuclear proteins undisputed [38,44] and minimizing the likelihood of their denaturation, aggregation, and lysis [7,37,38,44] is indisputable. During germination, DPA is released from spores, providing the reverse process of hydration of the nucleus and the formation of vegetative cells [19,37].…”

“…During germination, DPA is released from spores, providing the reverse process of hydration of the nucleus and the formation of vegetative cells [19,37]. The unique properties of DPA of endospores have allowed modern biotechnologists to consider it as a promising excipient (excipient) to increase the stability and highly effectiveness of biopharmaceutical liquid protein preparations and prevent their non-specific aggregation, for example, antibodies [38].…”