Insight

Over the past two decades, biopharmaceuticals have achieved groundbreaking advances in improving the quality of life of patients with various cancers, autoimmune diseases, genetic disorders and other conditions. With the growing demand for biopharmaceuticals, it has become crucial to reduce manufacturing costs without compromising product safety, quality and efficacy. An increasing number of biological products have been continuously developed into lyophilized powder for injection formulations and applied in clinical practice.

Although lyophilization technology offers numerous advantages when applied to biological products, proteins are prone to aggregation or denaturation during the lyophilization process, leading to reduced drug potency. Technically, two main strategies are commonly adopted to mitigate the adverse effects of lyophilization on biological products: adding excipients or optimizing the lyophilization process. This article reviews the latest research progress in lyophilization technologies and emerging novel lyophilization techniques that enhance the stability of biological products and counteract or reduce their denaturation.

Lyophilization is a process in which liquid drug solutions are first frozen into solids at low temperatures, followed by sublimation drying under vacuum to remove ice crystals. Upon completion of sublimation, desorption drying is subsequently carried out in a vacuum environment to eliminate bound water⁽¹⁾. When applied to pharmaceutical products, lyophilization can preserve their original physicochemical properties and biological activity, minimize the loss of active pharmaceutical ingredients. Lyophilized preparations generally feature a porous and loose structure, enabling rapid reconstitution and recovery of biological activity upon rehydration. Their low residual moisture content also facilitates long-term storage, making lyophilization an effective drying method for pharmaceuticals.

The pharmaceutical lyophilization process consists of four core stages: prefreezing, sublimation drying (primary drying), desorption drying (secondary drying), and sealing and storage.

Key technical prerequisites must be met during pharmaceutical lyophilization:

Drugs processed following the above procedures can be stored long-term at room temperature or under refrigeration. When required for use, they can be reconstituted with a suitable solvent to restore their state prior to lyophilization.

Biological products are susceptible to denaturation during lyophilization and subsequent storage. To prevent or minimize denaturation throughout the lyophilization cycle and long-term storage, two mainstream approaches are employed: incorporating lyoprotectants into the formulation and optimizing the lyophilization process.

Lyoprotectants are essential additives in lyophilized formulations that mitigate or prevent protein denaturation induced by freezing and drying. Based on the distinct stresses imposed on proteins during freezing and drying, the protective mechanisms of lyoprotectants are categorized into cryoprotection and desiccation protection. Common types of lyoprotectants include sugars, polyols, amino acids, macromolecular polymers, and surfactants. In practical production, one or multiple protectants are selected and formulated according to the specific denaturation characteristics of different biological products.

Optimization of the lyophilization process is another effective approach to improve the stability of biological products during processing and storage. The key optimization targets cover prefreezing, primary drying (sublimation drying), and secondary drying (desorption drying).

Prefreezing optimization: control of ice crystal size

Controlling ice crystal size during prefreezing is one of the primary methods to enhance the stability of biological products. Geidobler et al. prepared lyophilized formulations of bovine serum albumin and a monoclonal antibody with controlled ice crystal sizes and compared their stability. The results showed that rapid freezing via pre-chilled shelf freezing generated smaller ice crystals, prolonged primary drying time, and yielded lyophilized powders with a larger specific surface area.

Primary drying: regulation of drying temperature

Primary drying is the longest and most complex stage of the entire lyophilization process. The ideal operational state is to raise the temperature to the equilibrium level rapidly under appropriate conditions and minimize the duration of equilibrium temperature maintenance.

Secondary drying: control of heating rate

Secondary drying serves as the final stage of lyophilization. Studies by Tang indicated that a slow heating rate is conducive to ensuring product quality, and this rate is also applicable to the processing of biological products.

Batch lyophilization is the dominant commercial manufacturing method for solid biopharmaceuticals. However, traditional lyophilization is economically unfavorable, characterized by long production cycles, high energy consumption and substantial capital investment, resulting in elevated overall manufacturing costs. This review summarizes emerging innovative drying technologies. Though not yet widely adopted in the biopharmaceutical industry, continuous drying technologies offer new research directions to address the limitations of conventional lyophilization. Covered innovative technologies include rotational lyophilization, continuous lyophilization with suspended vials, active lyophilization, spray freeze drying and dynamic lyophilization, Lynfinity® technology, spray drying, PRINT® technology, Microglassification™ for biopharmaceuticals, as well as key considerations for drying process selection.

Traditional batch lyophilization removes solvents (usually water) from solutions based on the principle of sublimation. A typical batch lyophilizer consists of a drying chamber with multiple shelves, a condenser and a vacuum pump. The process comprises three major phases: freezing, primary drying and secondary drying.

Rotational freezing was first patented by Becker in 1957 (Patent No. DE967120) and later adapted for lyophilization, with subsequent patents granted to Broadwin in 1965 (US3203108A) and Oughton et al. in 1999 (US5964043). By modifying patented technologies and conventional lyophilization workflows, a novel unit-dose continuous lyophilization process was developed. A defining feature of this technology is that vials containing liquid products rotate along their longitudinal axis, hence the name rotational lyophilization.

The process starts with continuous rotational freezing, where vials filled with liquid samples spin rapidly along the longitudinal axis at 2500–3000 rpm for a set period. The axial rotation forms a uniform liquid layer of approximately 1 mm thickness on the inner wall of each vial. Subsequently, the rotating vials are exposed to temperature-controlled sterile cryogenic gas streams such as nitrogen or carbon dioxide. The uniform distribution of frozen product on the vial inner wall creates a large specific surface area, enabling rapid and uniform freezing and heating of the layered sample. Solidification takes about 1–2 minutes, followed by thermal equilibration at −40 ℃ to −60 ℃ for another 10–20 minutes. Cooling parameters in the temperature-controlled chamber are further adjusted to facilitate excipient crystallization and ideal morphological formation.

Upon completion of cooling, vials are conveyed to the primary drying chamber and held in thermally conductive sleeves or pockets under preset pressure and temperature conditions. The sleeves wrap around the vial exterior to promote uniform heat transfer via conduction and radiation. The vials are then transferred to the secondary drying chamber for desorption of residual moisture. The entire drying process lasts 30 minutes to 2 hours. Reports indicate that the total processing time can be reduced by 10 to 40 times depending on vial specifications and product formulations.

Overall, continuous rotational lyophilization offers prominent advantages in shortened drying duration, high continuous throughput and enhanced Process Analytical Technology (PAT) compatibility for individual vials. Nevertheless, challenges remain in validating the feasibility of implementing this continuous process under cGMP conditions, as well as scale-up and regulatory qualification.

A novel concept of continuous lyophilization with suspended vials has been recently developed under Patent PCT No. WO2018204484. The lyophilization system consists of modular units for different unit operations connected in series to enable uninterrupted vial flow. Vials are suspended on multi-row tracks and transported sequentially through chambers with distinct temperature and pressure settings, separated by load-lock systems to facilitate seamless transfer between modules.

Filled and partially stoppered vials are continuously loaded into the lyophilizer and delivered to the freezing zone, where freezing is achieved via Vacuum-Induced Surface Freezing (VISF) with spontaneous or controlled nucleation. Heat transfer during freezing is realized through forced air convection or radiative cooling gas. The significantly reduced temperature gradient inside the product promotes the formation of larger, uniform pores via the suspended vial structure, accelerating sublimation and shortening overall drying time compared with conventional lyophilization. In addition, controlled nucleation minimizes heterogeneity in ice crystal formation across vials, resulting in consistent porous structure among batches.

Different from tray-based bulk lyophilization, Hosowaka Micron B.V. developed agitated bulk lyophilization termed “active lyophilization” under Patent No. EP1601919A2. This technology enables the drying of heat-sensitive bulk materials with minimal handling, covering solutions, suspensions, pastes and wet solids. Unlike conventional lyophilized cakes, the final product is obtained as free-flowing powder. Agitation also optimizes the physicochemical properties of certain products, improves heat transfer efficiency and shortens drying cycles. The system configuration includes a jacketed conical vacuum dryer, impeller, collection filter, product collector, condenser and vacuum pump.

Meridion Technologies developed the SprayCon Lab® spray freeze dryer based on two Sanofi Pasteur patents (US10006706B2 and US9347707B2). The liquid feeding vessel and spray freezing chamber are installed above the rotational lyophilizer, connected via cooled pipelines and isolation valves that tightly separate the two process zones. Dried products are conveyed from the drying chamber to collection vessels through delivery pipelines. Other innovative technologies in this category include the continuous sterile spray freeze drying technology from IMA Life and conventional spray drying.

With the advancement of biotechnology, lyophilization has found expanding applications in the biopharmaceutical industry. For different biological products, targeted application of lyoprotectants and process optimization are essential to maintain product stability. In-depth research into lyoprotective mechanisms has driven the continuous improvement and innovation of lyophilization technologies. However, further in-depth exploration is still required for the characterization of biological products such as proteins and their stability mechanisms. Key process parameters during lyophilization, as well as structural and property changes of products after reconstitution, remain underreported and need more systematic investigation. Despite existing challenges, lyophilization remains the most reliable technique for preserving the stability of biological products. The development of high-performance novel lyoprotectants and improvement of biopharmaceutical quality have become core research hotspots in this field.

Batch lyophilization currently dominates the commercial production of most mature biopharmaceutical products. A variety of alternative drying technologies have shown promising prospects for the manufacturing of solid biopharmaceuticals without compromising product safety, quality and efficacy. These emerging technologies are of great significance to the biopharmaceutical industry, as they can not only reduce production time, energy consumption and manufacturing costs for life-saving drugs, but also mitigate drug supply risks during public health emergencies such as the COVID-19 pandemic.

While alternative continuous manufacturing methods feature lower operational costs, the impact of Critical Process Parameters (CPPs) such as temperature and shear force on Critical Quality Attributes (CQAs) serves as the fundamental criterion for drying technology selection. Technologies including rotational lyophilization, spray freeze drying, spray drying, PRINT® and Microglassification™ have demonstrated positive effects on the stability of certain proteins and inhalation biopharmaceuticals, yet their applicability to a broad range of parenteral biopharmaceuticals remains to be verified.

Sufficient stability data from product-specific studies are required to achieve industrial transition from traditional batch lyophilization to continuous production. Similar to CPPs, rational selection of formulation excipients tailored to product characteristics is critical to ensuring stability. Moreover, the molecular interaction mechanisms between biopharmaceuticals and solid-state excipients remain unclear. Advanced characterization techniques combined with Process Analytical Technology (PAT) can facilitate rapid and in-depth analysis of process-product relationships. Although most alternative drying technologies can benefit significantly from PAT integration, their commercial scale feasibility requires further validation.

In terms of scale-up, packaging and regulatory validation, several alternative drying processes offer advantages in reducing the complexity associated with multi-unit operation verification. The commercial application potential of these innovative technologies has been validated in the biopharmaceutical industry, yet multiple scale-up challenges remain unresolved.

Given that lyophilization is a lengthy and rigorous dehydration process, optimizing lyophilization curves, shortening processing cycles and maintaining drug stability pose persistent challenges for pharmaceutical formulation professionals.