Preface
Currently, there is no clear regulatory guideline that defines high-concentration antibody products (HCAPs). In literature, formulations with a protein concentration above 100 mg/mL are generally classified as high-concentration formulations. Formulations where elevated protein concentrations induce significant protein-protein interactions, and the crowded solution environment along with increased viscosity directly compromise formulation stability, can all be categorized as HCAPs.
Summary of FDA-Approved High-Concentration Antibody Products
Since the launch of the first monoclonal antibody drug in 1986, more than one hundred antibody drugs have been approved by the FDA and entered the therapeutic market. After 2015, high-concentration antibody formulations have gradually become the mainstream in antibody drug formulation development.
Advantages of High-Concentration Antibody Formulations
High-concentration formulations have gained widespread popularity, with an increasing number of pharmaceutical companies opting for their development for the following reasons:
Reduced Administration Volume and Improved Convenience
Subcutaneous injection has become a primary administration route for chronic diseases due to its ease of use, improved patient compliance, and reduced medical resource consumption, which generally limits the administration volume to no more than 2 mL. HCAPs perfectly meet the demand for reduced dosing volume. Statistics on 46 approved HCAP products confirm this point, among which 34 are administered via subcutaneous injection.
Meeting Dosing Requirements for Special Anatomical Sites
Three products including Susvimo™, Beovu®, and Vabysmo™ are indicated for the treatment of neovascular (wet) age-related macular degeneration via intravitreal injection. The typical dosing volume limit for intravitreal injection ranges from 20 μL to 50 μL, and only high-concentration antibody formulations can satisfy the requirement for ultra-low administration volume.
Lower Production and Logistics Costs
After bulk drug manufacturing, drug substances require storage and transportation to filling sites for formulation processing. High-concentration solutions feature a smaller volume, substantially lowering management costs in transportation, storage and other links compared with low-concentration counterparts.
Facilitating Chronic Disease Management and Saving Medical Resources
This advantage complements the merits of subcutaneous administration. Patients can self-administer via pre-filled syringes or autoinjectors, enabling greater medication freedom and convenience. It alleviates medical burdens, minimizes disruption to daily life, and conserves public medical resources.
Physical Properties and Developmental Challenges of High-Concentration Formulations
Affected by intense protein-protein interactions in high-concentration systems, the physical and chemical properties of proteins undergo dramatic changes. Solution viscosity and aggregation propensity are two core physical characteristics. Variations in these properties pose substantial challenges to manufacturing processes (elevated filtration pressure in UF/DF processes and increased filling loss), product stability (formation of high-molecular-weight variants and precipitates via aggregation), and drug administration (impaired injectability caused by excessive viscosity).
Solution Viscosity
A prominent observable characteristic with increasing protein concentration is the rise in solution viscosity. Concentration-dependent solution viscosity can be described by the formula:
where η denotes specific viscosity, [η] represents intrinsic viscosity, C is polymer concentration, and Kh stands for the Huggins constant. As indicated by the formula, viscosity increases exponentially with rising protein concentration.
Aggregation Propensity
At elevated protein concentrations, enhanced intermolecular interactions driven by steric effects, van der Waals forces and other factors significantly increase protein aggregation tendency. This leads to the formation of soluble aggregates such as dimers and trimers, as well as insoluble aggregate precipitates.
Key Excipients for Stable Formulation Development
Based on the application of excipients in FDA-registered HCAP formulations, seven major categories of critical excipients are summarized: buffers, sugars and polyols, surfactants, amino acid stabilizers, viscosity reducers, chelating agents, and antioxidants.
Buffers
Solution pH is critical to formulation stability, as proteins only maintain physicochemical stability within a narrow pH range to avoid chemical modification and aggregation. Most instances of protein instability stem from inappropriate buffer pH and ionic strength. For example, excessively low pH causes protein degradation and fragmentation, while excessively high pH induces protein deamidation. Therefore, optimal buffer formulation screening is essential for monoclonal antibody (mAb) formulations, which not only affects long-term storage stability but also the subcutaneous absorption duration after administration.
Histidine, used alone or in combination with other amino acids or inorganic buffers, is the most prevalent buffer system for stabilizing HCAPs (adopted in 32 products), among which 25 rely solely on histidine as the buffer. Enspryng® and Hemlibra® adopt a combined buffer system of histidine and aspartic acid, while Takhzyro® uses phosphate-citrate and histidine.
Notably, Humira® contains no buffer agents, making it the only buffer-free mAb product. Research on buffer-free or self-buffering effects of high-concentration antibody solutions has verified that antibodies themselves can act as buffers at high concentrations, eliminating the need for inorganic or amino acid-based buffers.
Among the 46 HCAPs:
- 6 products have a pH range of 4.5–5.0
- 12 products have a pH range of 5.1–5.5
- 21 products have a pH range of 5.6–6.0
- 4 products have a pH range of 6.1–6.5
- 1 product has a pH range of 6.6–7.0
- 2 products have a pH range of 7.1–7.5
Controlling asparagine deamidation and aspartic acid isomerization is one of the core strategies to maintain antibody stability in liquid formulations, especially for residues located in the complementarity-determining region (CDR). Under physiological pH, the rate-limiting step of deamidation and isomerization is the formation of aspartic acid intermediates. Neutral pH promotes asparagine deamidation, while slightly acidic conditions (pH ≤ 5.0) accelerate aspartic acid isomerization. Hence, most antibody formulations are optimized to a pH range of 5–6 to mitigate chemical modification and achieve optimal product stability.
Sugar and Polyol Stabilizers
Of the 46 HCAPs, 18 adopt sucrose, 5 use trehalose, 4 use sorbitol, 2 use mannitol, and 17 contain no sugars or polyols. Disaccharides and polyols stabilize protein conformation via the preferential exclusion mechanism, serving dual functions as osmotic pressure regulators and protein stabilizers.
Sucrose is the most widely used stabilizer in both lyophilized (LYO) and liquid (SOL) formulations due to its non-crystallizable property and ability to maintain an amorphous state during freezing.
Surfactants
Surfactants minimize protein adsorption and adhesion to packaging and container surfaces, reducing aggregation and particulate formation. They mitigate interfacial stress imposed on antibodies during manufacturing processes such as filtration, mixing and filling, as well as stress generated during transportation across production facilities, warehouses and hospitals. Additionally, surfactants alleviate mechanical stress and suppress aggregation induced by protein-protein interactions.
Only three types of surfactants are currently used in commercial antibody formulations: Polysorbate 80 (28 cases), Polysorbate 20 (10 cases), and Poloxamer 188 (3 cases). It should be noted that although Polysorbate 20 and 80 are widely applied in HCAPs, they are complex chemical mixtures prone to degradation via oxidation and hydrolysis pathways, which adversely affect protein quality, safety and stability. Moreover, visible protein-silicone oil particles have been observed in some protein formulations containing Poloxamer 188 under long-term storage at 2–8 °C. Therefore, comprehensive formulation development and characterization are required during surfactant screening.
Amino Acid Stabilizers
Amino acids exert versatile functions in antibody formulations: histidine acts as a buffer, glycine and proline serve as tonicity modifiers, arginine functions as a stabilizer and viscosity reducer, and methionine acts as an antioxidant. Statistical data demonstrate that rational combination of multiple amino acids facilitates the development of stable HCAP formulations.
Viscosity Reducers
Viscosity is a key consideration in HCAP development. When antibodies are formulated at concentrations above 100 mg/mL, protein-protein interactions trigger an exponential increase in solution viscosity, often exceeding the pharmacologically acceptable limit for subcutaneous injection. Incorporating viscosity reducers into formulations can weaken intermolecular interactions, thereby enhancing the stability of high-concentration mAb solutions and reducing viscosity.
Studies have shown that uncharged amino acids including glycine, alanine, phenylalanine and tryptophan reduce viscosity by modulating hydrophobic interactions. Amino acid salts and common inorganic salts such as arginine hydrochloride, histidine hydrochloride, lysine hydrochloride, sodium chloride, sodium sulfate and sodium acetate can also be used as viscosity-lowering excipients for high-concentration mAb formulations.
Antioxidants and Chelating Agents
Oxidative degradation is a critical stability concern for antibody formulations. The most susceptible oxidizable amino acid residues in antibodies include Met, Tyr, Trp, His and Cys. The addition of chelating agents (e.g., EDTA, DTPA) and antioxidant amino acids (e.g., methionine) can inhibit oxidation and stabilize protein solutions.
6. Conclusion
A retrospective analysis of 46 FDA-approved high-concentration antibody drugs reveals that histidine is the most commonly used buffer, sucrose dominates as an osmotic pressure regulator and carbohydrate stabilizer, and Polysorbate 80 is the primary surfactant. Most HCAPs employ two or more amino acids as excipients. Rational combination of these excipients at optimized concentrations synergistically inhibits protein aggregation, reduces solution viscosity, and improves overall product stability.
7. Prospect
Emerging research on immunological disease therapy demands higher dosing doses (>30–50 mg/kg), driving pharmaceutical enterprises to focus on developing formulations with protein concentrations exceeding 200 mg/mL. Relevant literature has reported formulation systems with concentrations over 400 mg/mL that maintain favorable stability at room temperature.
Building on conventional subcutaneous injection technology, Halozyme’s ENHANZE® drug delivery technology enables larger administration volumes with significant advantages in drug absorption efficiency and bioavailability.
With the continuous development of novel excipients and the discovery of new excipient combinations, the conventional definition of high-concentration formulations will be redefined, and current developmental challenges associated with HCAPs will be progressively resolved.
