Biopharmaceutical products represented by recombinant proteins, antibodies and enzyme drugs are highly susceptible to environmental perturbations such as temperature fluctuation, pH shift, mechanical shear and freeze-thaw cycles. Unfolding of tertiary structure, partial denaturation and molecular aggregation frequently lead to loss of biological activity, shortened shelf‑life and increased adverse reaction risks, which raise substantial barriers for storage, transportation and clinical application. As a short side-chain-free oligopeptide, triglycine can form ordered hydrogen-bond networks around protein molecules, stabilise native spatial conformation and effectively lock biological activity, providing a feasible strategy for stability upgrading of biopharmaceuticals.
Triglycine consists of three consecutive glycine residues, with abundant peptide-bond carbonyl and amide groups distributed along its peptide backbone. These polar groups serve as hydrogen-bond donors and acceptors simultaneously. When incorporated into biopharmaceutical liquid formulations or lyophilised systems, triglycine molecules are distributed in the hydration shell surrounding protein surfaces. They build extensive intermolecular hydrogen-bond networks bridging solvent molecules and exposed polar amino-acid residues of the protein surface. Such hydrogen-bond interactions reinforce the hydration layer of biomacromolecules and restrict excessive movement of flexible loop regions that are prone to unfolding.
Under thermal stress or freeze-thaw stimulation, protein molecules tend to break internal hydrogen bonds and expose hydrophobic core domains, further triggering unfolding and aggregation. The hydrogen-bond network constructed by triglycine competes for hydrogen-bond sites, compensates for partial hydrogen-bond loss caused by external disturbance, and constrains conformational drift of polypeptide chains. It inhibits the exposure of internal hydrophobic regions, suppresses intermolecular hydrophobic aggregation, and maintains the intact native folding state. In lyophilised preparations, triglycine forms hydrogen-bond connections with protein residues during dehydration, partially replacing water-mediated hydrogen-bonds and reducing irreversible structural damage induced by water removal.
Different from traditional polyols and sugars, triglycine possesses moderate molecular flexibility without bulky side-chains. It will not cause excessive steric hindrance when adhering to protein surfaces. It maintains weak-to-moderate non-covalent interactions and avoids undesired rigidification that may impair protein dynamic function. Meanwhile, triglycine exhibits good biocompatibility and low immunogenic potential. It does not interfere with antigen‑binding sites or enzyme active-centre microenvironment at appropriate concentrations, so intrinsic biological potency of biopharmaceuticals can be well preserved.
Nevertheless, its stabilising performance shows obvious concentration-dependent characteristics. Too low triglycine content fails to establish sufficient hydrogen-bond networks, resulting in limited protective effect. Excessively high concentration changes solution osmotic pressure, and dense triglycine clusters may generate abnormal intermolecular interactions, which conversely disturb protein conformation. Triglycine usually acts as an auxiliary stabiliser rather than an independent excipient. It often works synergistically with buffers, sugars and salts to optimise formulation stability. It cannot reverse protein degradation caused by chemical modification such as deamidation or oxidation.
Triglycine enhances biopharmaceutical stability by constructing dense hydrogen‑bond networks around protein molecules. It restrains polypeptide-chain unfolding, alleviates aggregation triggered by heat, freeze-thaw and dehydration, and locks native biological activity of protein-based drugs. As a promising peptide-type formulation additive, it brings new ideas for optimising liquid storage stability and lyophilisation protection of recombinant proteins, antibody drugs and industrial biocatalysts.
