Lyophilized Peptide and Protein Cold Chain Stability: What the Published Literature Reports

Cold chain dependence is a recurring variable in the published literature on peptide and protein formulation stability, and lyophilization (freeze-drying) is the process most often examined as a way to characterise stability outside refrigerated storage. The studies summarised here span in vitro physicochemical measurements, cell-culture functional assays, and animal immunisation models; none of the findings below should be read as human outcomes. At the level of peptide self-assembly, one in vitro study engineered oligopeptide nanocoacervates from tyrosine-containing sequences [(YR)2R10 + (YR)2D10] reinforced with tannic acid, and reported that the particles retained their count, size below 500 nm, and morphology after exposure to ionic strengths of 10–100 mM, pH 3–10, temperatures of 25–80 °C, biological media, and freeze-drying followed by rehydration [1]. The authors reported that all of the described interactions — π-π stacking, cation-π, hydrogen bonding, and electrostatic — were required for recovery after lyophilization, and described the system as the first peptide-based coacervate reported with lyophilization tolerance [1]. Excipient identity is the most frequently studied variable in freeze-drying of peptide-containing particles. In an in vitro study of polymer microparticles loaded with a peptide self-antigen and a small-molecule immunomodulator, mannitol, trehalose, and sucrose were compared as lyophilization excipients; the authors reported improved caking of the lyophilized product, more complete resuspension, greater product recovery, and smaller changes in particle size and size distribution over time relative to formulations without excipient [2]. In co-cultures of antigen-presenting cells and self-reactive T cells, the excipient-containing lyophilized microparticles retained tolerance-inducing function after storage without refrigeration [2]. An earlier long-term study of recombinant hepatitis B surface antigen particles reported that lyophilized powder retained the original peptide composition across storage at −20 °C, 4 °C, room temperature, and 37 °C for six months, and that lyophilization in the presence of glucose, sucrose, or trehalose — but not mannitol — further improved 4 °C stability of size, shape, and protein content over two years [4]. That same study reported a dissociation between chemical and functional stability: in BALB/c mice, freezing or freeze-drying of the alum-adsorbed vaccine resulted in loss of immunogenicity despite preserved chemical stability [4]. Sugar-stabilised freeze-dried formulations of peptide drugs have been characterised against liquid comparators. For insulin glulisine loaded into amphiphilic cyclodextrin nanoparticles, colloidal parameters of the liquid dispersion were maintained while the insulin itself degraded over 60 days of storage; a lyophilized formulation containing trehalose was reported to extend stability to 30 days at ambient temperature and 90 days at 4 °C, with 95% and greater than 80% insulin recovery respectively, and after intra-intestinal administration in animals insulin glulisine was detected in portal and systemic blood without a lowering of blood glucose [3]. In an in vitro potency study of Escherichia coli-derived (Serine-17) human interferon-beta formulated with human serum albumin, antiviral activity was reported to be stable for two years when lyophilized and refrigerated, with an Arrhenius fit across isothermal temperatures of 25–80 °C and increased thermal stability at higher albumin content [8]. Several animal studies have used lyophilization specifically to interrogate cold chain independence. Influenza vaccine antigens lyophilized in solid bioneedles were reported to remain stable after one month at 60 °C and, on administration to C57BL/6 mice, to induce haemagglutination-inhibition titres comparable to intramuscular whole inactivated vaccine, with lyophilized formulations described as more thermostable than conventional liquid vaccines [5]. An M2e-conjugated gold nanoparticle construct was reported to remain stable in lyophilized form at 4 °C for three months, 37 °C for three months, and 50 °C for two weeks, to reconstitute in water without aggregation, and to induce M2e-specific IgG in mice and ferrets comparable to freshly formulated material [7]. Lipid A-cochleates prepared by emulsification-lyophilization formed a dry product reported as stable at room temperature, re-formed 800 nm cochleates on rehydration, and elicited serum IgG and mucosal IgA responses in orally immunised mice [9]. Lyophilized ovalbumin powders stabilised with trehalose were milled and loaded onto powder-attached microneedles; ex vivo porcine skin studies showed complete insertion and comparable delivery between pre-loaded and on-site loaded arrays, and mouse immunisation produced comparable antigen-specific IgG responses at the same nominal dose [10]. Process engineering has also been studied as an alternative to excipient loading alone. Thin-film freezing followed by lyophilization was reported to convert aluminium-salt-adjuvanted vaccines, including commercial tetanus toxoid, hepatitis B, and human papillomavirus products, into dry powders using as little as 2% w/v trehalose without particle aggregation or loss of immunogenicity on reconstitution, whereas spray freeze-drying approaches described in the same work failed to preserve particle size or immunogenicity [6]. Repeated freeze-thaw of the resulting dry powder was reported not to cause aggregation [6]. In a separate in vitro study, carbohydrate-based ice recrystallization inhibitors were applied to viral vectors during room-temperature storage, freeze-thaw cycling, and lyophilization; N-octyl-gluconamide and the antifreeze glycoprotein analogue OGG-Gal were reported to recover or maintain plaque-forming-unit titres relative to controls, and OGG-Gal reduced temperature-induced aggregation of vesicular stomatitis virus particles [11]. Taken together, the reported pattern across these models is that lyophilization outcomes are formulation-specific: the same drying step preserved peptide composition and function in some systems [1][2][8] while abolishing functional immunogenicity in another [4], and the excipient and freezing method were repeatedly identified as determining variables [2][4][6].