Practical advice in the development of a lyophilized protein drug product.

The development of lyophilized protein drug products is a critical and complex task in the pharmaceutical industry, requiring a comprehensive understanding of the myriad of factors affecting product quality, stability, and the efficiency and robustness of the lyophilization process. This review offers practical advice on the critical aspects of lyophilized protein drug product development. Practical considerations across both the early and late stages of development are discussed, underscoring the necessity of a strategic approach from initial development through to commercialization.

Lyophilization as an effective tool to develop AAV8 gene therapy products for refrigerated storage.

Recombinant adeno-associated virus (rAAV) has emerged as the leading gene delivery platform for treatment of monogenic disorders. Currently, for clinical and commercial products, rAAVs are typically formulated and stored below -65 °C as frozen liquid. Their long-term storage is often far from ideal because it may result in shorter drug product (DP) shelf-life compared to recombinant protein-based biologics, and also presents challenges for supply chain and inventory management.

Evaluation of manometric temperature measurement (MTM), a process analytical technology tool in freeze drying, part III: heat and mass transfer measurement.

This article evaluates the procedures for determining the vial heat transfer coefficient and the extent of primary drying through manometric temperature measurement (MTM). The vial heat transfer coefficients (Kv) were calculated from the MTM-determined temperature and resistance and compared with Kv values determined by a gravimetric method. The differences between the MTM vial heat transfer coefficients and the gravimetric values are large at low shelf temperature but smaller when higher shelf temperatures were used.

Evaluation of manometric temperature measurement, a process analytical technology tool for freeze-drying: part II measurement of dry-layer resistance.

The purpose of this work was to study the factors that may cause systematic errors in the manometric temperature measurement (MTM) procedure used to determine product dry-layer resistance to vapor flow. Product temperature and dry-layer resistance were obtained using MTM software installed on a laboratory freeze-dryer. The MTM resistance values were compared with the resistance values obtained using the "vial method.

Measurement of the kinetics of protein unfolding in viscous systems and implications for protein stability in freeze-drying.

Purpose: The aim of the study is to determine the degree of coupling between protein unfolding rate and system viscosity at low temperatures in systems relevant to freeze-drying.

Methods: The cold denaturation of both phosphoglycerate kinase (PGK) and beta-lactoglobulin were chosen as models for the protein unfolding kinetics study. The system viscosity was enhanced by adding stabilizers (such as sucrose), and denaturant (guanidine hydrochloride or urea) was added to balance the stabilizing effect of sucrose to maintain the cold denaturation temperature roughly constant.

The effect of stabilizers and denaturants on the cold denaturation temperatures of proteins and implications for freeze-drying.

Purpose: The aim of the study is to investigate the effects of stabilizers and denaturants on the thermal and cold denaturation temperatures of selected proteins in systems of interest to freeze-drying.

Methods: Beta-lactoglobulin and phosphoglycerate kinase (PGK) were chosen as model proteins. Protein thermal and cold denaturation temperatures were determined by both conventional and modulated differential scanning calorimetry and verified by tryptophan emission spectroscopy in selected systems.

Mechanism of protein stabilization by sugars during freeze-drying and storage: native structure preservation, specific interaction, and/or immobilization in a glassy matrix?

The purpose of this study is to investigate the mechanism of protein stabilization by sugars in the solid state. That is, explore whether the stabilization is controlled by "glass dynamics" or by native structure preservation through "specific interaction" between sugars and protein. The IgG1 antibody (150 kD) and recombinant human serum albumin (rHSA) (65 kD) were formulated with sorbitol, trehalose, and sucrose.

Effect of sorbitol and residual moisture on the stability of lyophilized antibodies: Implications for the mechanism of protein stabilization in the solid state.

Purpose: To investigate the effect of plasticizers on the stability of protein formulations in the solid state and to apply these results to a study of mechanisms of protein stabilization by sugars in the solid sate.

Methods: The IgG1 antibody was formulated with either sucrose or trehalose alone or a mixture of sorbitol with sucrose or trehalose. After lyophilization, the pure protein and sucrose formulations were equilibrated at different relative humidities giving residual moistures from less than 1% to 5% for sucrose systems and up to 17% for pure protein systems.

Freeze-drying process design by manometric temperature measurement: design of a smart freeze-dryer.

Purpose: To develop a procedure based on manometric temperature measurement (MTM) and an expert system for good practices in freeze drying that will allow development of an optimized freeze-drying process during a single laboratory freeze-drying experiment.

Methods: Freeze drying was performed with a FTS Dura-Stop/Dura-Top freeze dryer with the manometric temperature measurement software installed. Five percent solutions of glycine, sucrose, or mannitol with 2 ml to 4 ml fill in 5 ml vials were used, with all vials loaded on one shelf.

Evaluation of glassy-state dynamics from the width of the glass transition: results from theoretical simulation of differential scanning calorimetry and comparisons with experiment.

The purpose of this study is to investigate the quantitative relationship between the width of the glass transition, DeltaTg, and glass fragility or activation energy for structural relaxation. The ultimate objective is the estimation of structural relaxation time as a function of temperature from the width of the glass transition region, allowing characterization of glass dynamics by a single simple measurement. The Moynihan correlation indicates that activation energy for structural relaxation should be inversely proportional to the width of the glass transition, but recent experimental studies suggest this relationship is a poor approximation for glasses of pharmaceutical interest.

The effect of temperature on hydrogen bonding in crystalline and amorphous phases in dihydropyrine calcium channel blockers.

Purpose: To investigate the effect of temperature on hydrogen bonding in a series of structurally related amorphous and crystalline compounds to gain a better molecular-level understanding of structural differences in the various phases.

Methods: FT-infrared spectra of seven 1,4-dihydropyridine calcium channel blockers were obtained at temperatures between 0 and 200 degrees C using a temperature-controlled attenuated total reflection accessory. Thermal behavior was characterized using differential scanning calorimetry.

A spectroscopic investigation of hydrogen bond patterns in crystalline and amorphous phases in dihydropyridine calcium channel blockers.

Purpose: To gain insight into the molecular structure of amorphous compounds by investigating hydrogen bonding patterns and strength in a series of structurally related compounds. Seven 1,4-dihydropyridine calcium channel blockers were evaluated.

Methods: FT-Raman and FT-infrared spectra of the compounds in the crystalline and amorphous states were obtained.