Surfactants in Freeze-Dried Biologics: Why They Matter in Lyophilized Formulations

7/17/20269 min read

What Are Surfactants in Lyophilized Biologics?
Why Are Surfactants Added to Freeze-Dried Formulations?
How Proteins Become Damaged During Lyophilization
How Surfactants Protect Biologic Drugs
Common Surfactants Used in Pharmaceutical Lyophilization

How to Select the Right Surfactant
Emerging Trends in Surfactants for Lyophilized Biologics
Practical Considerations During Freeze Drying
Frequently Asked Questions
Conclusion

1. What Are Surfactants in Lyophilized Biologics?

Therapeutic proteins are inherently sensitive molecules. Unlike small-molecule drugs, their biological activity depends on maintaining a highly ordered three-dimensional structure throughout manufacturing, storage, transportation, and administration. Even relatively minor physical stresses can cause proteins to unfold, aggregate, or form visible and subvisible particles, potentially affecting product quality and stability.

One of the most common sources of instability during pharmaceutical lyophilization is exposure to interfaces. During formulation preparation, filling, freezing, primary drying, secondary drying, and reconstitution, proteins repeatedly encounter air-liquid, ice-water, solid-gas, and container surfaces. These interfaces can promote protein adsorption, initiating structural changes that eventually lead to aggregation.

Surfactants are added to many biologic formulations to reduce these interfacial stresses. Rather than stabilizing the protein molecule directly, they preferentially occupy interfaces before proteins can adsorb to them, helping maintain protein integrity throughout manufacturing and storage.

This role differs from that of excipients such as trehalose, sucrose, and certain amino acids, which primarily stabilize proteins in the frozen or dried state. If you are unfamiliar with these formulation strategies, our articles on Cryoprotectants in Lyophilization, Lyoprotectants in Freeze Drying, Role of Sugars (Sucrose & Trehalose), Amino Acids in Lyophilized Formulations, and Stabilization Mechanisms in Freeze-Dried Formulations provide the broader scientific foundation.

2. Why Are Surfactants Added to Freeze-Dried Formulations?

A common misconception is that proteins become unstable simply because they are frozen. In reality, temperature alone rarely explains the physical instability observed during lyophilization.

Throughout the freeze-drying process, proteins experience repeated exposure to newly formed interfaces created by manufacturing operations and phase transitions. Each interface represents a potential site for protein adsorption and unfolding.

Examples include:

Air-liquid interfaces created during mixing, pumping, and filling
Ice-water interfaces generated during freezing
Solid-gas interfaces that develop during primary drying
Glass vial and rubber stopper surfaces
Air-liquid interfaces encountered again during reconstitution

Once proteins adsorb to these surfaces, portions of the molecule may partially unfold. These unfolded molecules can interact with neighboring proteins, initiating aggregation and particle formation. For many biologics, preventing these early events is one of the primary objectives of formulation development.

Understanding how these interfaces develop also requires an understanding of Ice Nucleation in Lyophilization, Ice Crystal Formation and Growth, Freeze Concentration During Lyophilization, and The Three Stages of Lyophilization Explained, since the freezing process largely determines the physical environment encountered during drying.

3. How Proteins Become Damaged During Lyophilization

Protein instability during freeze drying rarely results from a single mechanism. Instead, multiple stresses often occur simultaneously, with their combined effects determining the final stability of the product.

Surface Adsorption

Proteins naturally migrate toward interfaces because these environments can lower the system's free energy. However, adsorption often exposes hydrophobic regions that are normally buried within the folded protein structure. As proteins partially unfold, they become more likely to associate with neighboring molecules, leading to aggregation. Once aggregation begins, it is often irreversible, even after reconstitution. This mechanism explains why controlling interfacial stress is a major focus during biologic formulation development.

Freezing-Induced Stress

During freezing, water crystallizes first while proteins, buffers, salts, and excipients remain concentrated within the unfrozen solution. This phenomenon, known as freeze concentration, increases local protein concentration and simultaneously exposes proteins to expanding ice surfaces. Additional factors such as pH shifts, changes in ionic strength, and altered buffer composition can further contribute to instability.

These events are discussed in greater detail in our articles on Freeze Concentration During Lyophilization, Supercooling in Pharmaceutical Freeze Drying, and Controlled Nucleation: Principles and Technologies.

Mechanical Stress

Protein instability is not limited to the freeze dryer itself.

Routine manufacturing operations such as:

Mixing
Pumping
Filtration
Filling
Agitation during transport
Reconstitution

continually generate fresh air-liquid interfaces.

Without sufficient interfacial protection, proteins repeatedly adsorb and desorb from these interfaces, increasing the probability of structural changes and aggregate formation before the product even enters the freeze dryer.

4. How Surfactants Protect Biologic Drugs

Surfactants protect proteins primarily by modifying the environment around them rather than by stabilizing the protein's molecular structure directly.

They Occupy Interfaces Before Proteins

Surfactant molecules rapidly migrate to newly formed interfaces because of their amphiphilic structure, which contains both hydrophilic and hydrophobic regions. By coating these interfaces first, surfactants reduce the opportunity for proteins to adsorb and unfold. This simple mechanism explains why relatively low surfactant concentrations can substantially improve formulation robustness.

They Reduce Aggregation

Protein aggregation frequently begins with interfacial adsorption. Because surfactants reduce adsorption, they interrupt one of the earliest stages of aggregate formation.

As a result, appropriately selected surfactants can improve:

Physical stability
Particle control
Long-term storage stability
Reconstitution consistency

However, surfactants cannot reverse aggregation that has already occurred. Their role is preventive rather than corrective.

They Improve Manufacturing Robustness

A formulation that performs well at laboratory scale may behave differently during pilot or commercial manufacturing. Large-scale manufacturing introduces additional transfer lines, pumps, filters, filling equipment, and processing steps, all of which increase the total interfacial area encountered by proteins.

Appropriate surfactant selection therefore improves formulation robustness throughout development, scale-up, and commercial production. This becomes particularly important during Technology Transfer, Process Validation, and Continued Process Verification (CPV).

5. Common Surfactants Used in Pharmaceutical Lyophilization

Surfactants used in pharmaceutical formulations are generally classified according to the electrical charge of their hydrophilic head group. This classification influences how they interact with proteins, excipients, and other formulation components. Although four major classes of surfactants exist, only one class is routinely used in therapeutic protein formulations.

Nonionic Surfactants

Nonionic surfactants carry no net electrical charge and are the preferred choice for most freeze-dried biologics. Because they do not contain charged functional groups, they generally have a lower tendency to interact directly with charged amino acid residues on protein surfaces. Instead, they preferentially adsorb to interfaces, reducing protein adsorption while preserving the protein's native structure.

For this reason, nonionic surfactants have become the industry standard for monoclonal antibodies, recombinant proteins, enzymes, vaccines, and many other biologic drug products.

The most widely used examples include:

Polysorbate 20
Polysorbate 80
Poloxamer 188

These excipients have extensive manufacturing and regulatory experience and remain the benchmark against which many newer surfactants are evaluated.

Anionic Surfactants

Anionic surfactants possess a negative electrical charge.

Common examples include:

Sodium dodecyl sulfate (SDS)
Sodium lauryl sulfate (SLS)

These compounds are highly effective detergents and are widely used in analytical laboratories, protein electrophoresis, and cleaning applications. Their strong interactions with proteins frequently disrupt native protein structure, making them unsuitable for stabilizing most injectable biologics during lyophilization.

Cationic Surfactants

Cationic surfactants carry a positive electrical charge.

Examples include:

Cetyltrimethylammonium bromide (CTAB)
Benzalkonium chloride

These surfactants are valued primarily for their antimicrobial properties and are used in selected pharmaceutical formulations, particularly topical and ophthalmic products.

For therapeutic proteins, however, strong electrostatic interactions with negatively charged protein surfaces can increase the risk of instability. Consequently, cationic surfactants are rarely used in lyophilized biologic formulations.

Amphoteric (Zwitterionic) Surfactants

Amphoteric surfactants contain both positive and negative charges, with their overall charge depending on formulation pH.

Examples include:

CHAPS
Cocamidopropyl betaine

These materials are widely used in biochemical research, particularly for membrane protein studies, because they provide relatively mild detergent properties.

Although scientifically valuable, amphoteric surfactants have only limited application in commercial freeze-dried biologic products.

6. How to Select the Right Surfactant

Selecting a surfactant is rarely as simple as choosing the most commonly used excipient. While Polysorbate 20 and Polysorbate 80 have been successfully incorporated into many commercial biologics, the optimal surfactant always depends on the protein, the formulation, the manufacturing process, and the intended shelf life.

For this reason, surfactant selection is typically performed alongside broader formulation development rather than as an isolated exercise.

Protein Characteristics

Every therapeutic protein behaves differently. Some proteins readily adsorb to interfaces, while others are more susceptible to aggregation caused by freezing, oxidation, or changes in pH. Properties such as molecular size, surface hydrophobicity, isoelectric point, conformational stability, and aggregation propensity all influence how much interfacial protection is required.

Consequently, a surfactant that performs well for one monoclonal antibody may provide little benefit for another protein with different physicochemical characteristics.

Manufacturing Process

The manufacturing process itself determines how many interfaces a protein encounters before and after lyophilization. Processes involving extensive pumping, mixing, recirculation, sterile filtration, or prolonged holding times expose proteins to repeated air-liquid and solid-liquid interfaces. These stresses can significantly increase the likelihood of adsorption and aggregation.

As manufacturing scales from laboratory development to commercial production, the cumulative interfacial area generally increases, making surfactant performance even more important during Technology Transfer, Process Validation, and Continued Process Verification (CPV).

Compatibility with Other Excipients

Surfactants rarely function independently.

Their performance depends on interactions with other formulation components, including:

Sugars such as sucrose and trehalose
Buffers
Amino acids
Salts
Bulking agents
Stabilizing polymers

Changing one excipient can alter the behavior of the entire formulation. For this reason, surfactants are typically optimized as part of the complete formulation rather than evaluated in isolation.

Readers interested in the broader formulation strategy may also find our articles on Cryoprotectants in Lyophilization, Lyoprotectants in Freeze Drying, Buffer Selection in Lyophilization, Role of Sugars (Sucrose & Trehalose), and Formulation Development for Lyophilized Products useful.

Container Closure System

Protein adsorption is not limited to the freeze-drying process. Glass vial surfaces, rubber stoppers, silicone oil used in prefilled syringes, tubing, and stainless-steel equipment can all introduce additional interfaces during manufacturing and storage.

These interactions are routinely considered during formulation development because they can influence both protein stability and visible or subvisible particle formation.

Long-Term Stability

A surfactant should remain chemically stable throughout the intended shelf life of the product. Formulation scientists therefore evaluate not only protein stability but also the stability of the surfactant itself under long-term and accelerated storage conditions.

Common evaluation parameters include:

Oxidative degradation
Hydrolytic degradation
Particle formation
Protein compatibility
Changes during stability studies

The goal is to ensure that both the biologic and its excipients remain stable throughout the product lifecycle.

7. Emerging Trends in Surfactants for Lyophilized Biologics

For many years, Polysorbate 20 and Polysorbate 80 have been regarded as the industry standard for biologic formulations. Their widespread use reflects decades of manufacturing experience and extensive regulatory acceptance.

However, experience has also shown that polysorbates are not chemically inert. Under certain formulation and storage conditions, they may undergo oxidation or hydrolysis. These degradation pathways can generate free fatty acids and peroxide species, potentially contributing to particle formation, protein instability, or changes in product quality over extended storage periods.

Although these issues do not affect every formulation, they have encouraged increased research into alternative surfactants with improved chemical stability.

One area of active investigation involves alkyl glycosides, such as n-dodecyl-β-D-maltoside (DDM), which have demonstrated promising interfacial protection in some protein formulations. Other next-generation nonionic surfactants are also being explored to improve oxidative stability while maintaining compatibility with therapeutic proteins.

Despite this growing interest, newer surfactants must demonstrate long-term safety, manufacturability, analytical compatibility, and regulatory acceptability before they can achieve widespread commercial adoption. Consequently, polysorbates remain the benchmark against which emerging surfactants continue to be evaluated.

8. Practical Considerations During Freeze Drying

Surfactants are an important component of biologic formulations, but they should never be viewed as a solution to every stability challenge. Successful formulation development requires balancing formulation composition, process parameters, analytical characterization, and manufacturing considerations.

More Surfactant Is Not Necessarily Better

Increasing surfactant concentration beyond the optimal level does not always improve protein stability. Excessive concentrations may influence formulation properties, analytical measurements, or interactions with other excipients without providing additional protection against interfacial stress.

Surfactant concentration should therefore be established experimentally during formulation optimization.

Surfactants Cannot Compensate for Poor Process Design

A well-chosen surfactant cannot overcome an inadequately developed freeze-drying cycle. If freezing conditions generate unsuitable ice crystal morphology, if primary drying exceeds the product's collapse temperature, or if excessive residual moisture remains after secondary drying, formulation instability may still occur regardless of surfactant selection.

For this reason, surfactant optimization should always be considered alongside Cycle Development in Pharmaceutical Lyophilization, Shelf Temperature in Lyophilization, Chamber Pressure in Freeze Drying, Product Temperature in Lyophilization, and Residual Moisture in Lyophilized Products.

Monitor Surfactant Stability Throughout Product Development

Because surfactants themselves may degrade over time, modern development programs routinely monitor surfactant integrity in parallel with protein stability. Changes in surfactant quality can sometimes be mistaken for protein instability if appropriate analytical methods are not employed.

Consequently, understanding both protein degradation pathways and excipient degradation pathways is essential for successful root cause investigations.

Formulation Optimization Is Always Multifactorial

Rarely does a single excipient determine the success of a lyophilized formulation.

Protein stability typically reflects the combined effects of:

Protein properties
Sugar selection
Buffer composition
Amino acid selection
Surfactant concentration
Freezing conditions
Drying cycle design
Storage conditions

The objective is therefore to optimize the formulation as an integrated system rather than maximizing the performance of any individual excipient.

9. Frequently Asked Questions

Do surfactants stabilize proteins directly?

Not usually. Their primary function is to reduce adsorption at interfaces such as air-liquid, ice-water, and container surfaces. This helps preserve the native protein structure by preventing one of the earliest steps in the aggregation pathway.

Why are Polysorbate 20 and Polysorbate 80 used so frequently?

These nonionic surfactants provide effective interfacial protection, have extensive manufacturing and regulatory experience, and are compatible with many therapeutic proteins. Their long history of successful use has made them the standard choice for numerous commercial biologic products.

Can surfactants prevent every type of protein aggregation?

No. Surfactants primarily reduce aggregation initiated by interfacial stress. Aggregation caused by oxidation, deamidation, inappropriate pH, excessive heat, or other degradation mechanisms generally requires additional formulation or process strategies.

Are surfactants required in every lyophilized biologic?

No. Some proteins remain sufficiently stable without surfactants, whereas others depend heavily on them. Their inclusion should always be supported by formulation studies and experimental evidence rather than routine practice.

Why are ionic surfactants rarely used in biologic formulations?

Anionic and cationic surfactants can interact strongly with charged regions of protein molecules, increasing the likelihood of structural changes or aggregation. Nonionic surfactants generally provide effective interfacial protection while minimizing these interactions, making them the preferred choice for most therapeutic proteins.

10. Conclusion

Surfactants play a specialized but essential role in the development of freeze-dried biologics. Rather than stabilizing proteins directly, they protect proteins from one of the most significant sources of instability encountered during manufacturing and lyophilization—interfacial stress.

Their successful application depends on much more than selecting a familiar excipient. Protein characteristics, formulation composition, manufacturing processes, freeze-drying conditions, container closure systems, and long-term storage requirements all influence surfactant performance. Understanding these relationships enables formulation scientists to make informed decisions that improve product robustness throughout development and commercial manufacturing.

As biologic therapies continue to evolve, surfactants will remain an integral part of formulation science. At the same time, continued research into next-generation surfactants and improved formulation strategies is expected to further enhance the stability of future lyophilized biologic products.

Disclaimer
The information presented in this article is intended exclusively for educational and informational purposes as part of the Lyophilization Core scientific knowledge base. It is designed to support the understanding of pharmaceutical lyophilization science, engineering principles, formulation development, process development, and manufacturing concepts.

This content should not be interpreted as regulatory guidance, GMP instructions, manufacturing procedures, process validation protocols, engineering specifications, or professional consulting advice. The suitability of any lyophilization process, formulation, equipment, or operating condition must be evaluated based on product-specific scientific data, validated procedures, applicable regulatory requirements, and qualified scientific and engineering judgment.

Pharmaceutical development and commercial manufacturing should always be conducted in accordance with applicable Good Manufacturing Practices (GMP), relevant regulatory guidance, approved quality systems, and site-specific standard operating procedures.