Why Replacement Air Matters in Therapeutic Protein Infusions

When medication is withdrawn or infused from a rigid glass bottle, something must replace the volume of liquid leaving the container. That replacement is usually air.

Conventional vented administration systems allow air to enter the bottle, but depending on the configuration, that air may be introduced below the medication’s surface. It must then travel through the liquid as bubbles before reaching the bottle’s headspace.

I2F takes a different approach.

I2F was developed to route filtered replacement air directly to the headspace of a rigid medication bottle rather than introducing it below the medication’s surface. The device does not eliminate the natural air–liquid interface already present above the medication. It changes the path the incoming air takes to reach that space.

Why might that distinction matter?

Published research has established that bubbles are not simply harmless pockets of air moving through a liquid. Each bubble creates a mobile air–liquid interface. For structurally sensitive therapeutic proteins, those moving interfaces can become a source of physical stress.

The effect of routine replacement-air bubbling during actual clinical infusion has not yet been fully measured. However, the underlying science raises an important question:

If dynamic air–liquid interfaces can affect therapeutic proteins, should replacement air be repeatedly introduced through the medication when it can instead be directed to the headspace?

What Research Has Established About Air–Liquid Interfaces

Therapeutic proteins are structurally complex medications. Unlike many small-molecule drugs, proteins depend on a carefully folded three-dimensional structure to maintain their stability and biological function. That structure can be influenced by temperature, light, mechanical agitation, formulation conditions, contact with surfaces, and exposure to interfaces.

An air–liquid interface is the boundary where air meets a liquid. Every partially filled medication bottle already contains one such interface at the top of the solution. Proteins may interact with that surface, even when the bottle remains undisturbed.

A bubble creates another air–liquid interface within the medication.

As a bubble moves through the liquid, its curved surface is surrounded by the medication. Protein molecules may move toward and adsorb at that surface. Because different regions of a protein interact differently with water and air, adsorption may cause some molecules to reorient or undergo structural change.

Under certain conditions, proteins at the interface may interact with one another and form films or associated structures. Expansion, compression, movement, merging, or rupture of the interface may then disturb those structures and release altered protein material into the surrounding solution. Those protein species may remain soluble, associate with other proteins, or contribute to submicron, subvisible, or visible particles.

This does not mean that every protein contacting air will unfold or aggregate. Susceptibility varies according to the medication, formulation, protein concentration, temperature, surfactant system, exposure time, and intensity of the physical stress.

The established principle is more precise:

Air–liquid interfaces can create conditions that contribute to instability and aggregation in susceptible therapeutic proteins.

What Bubbling Studies Have Found

Several studies help explain why bubbles deserve attention.

In 2021, Sreenivasan and colleagues passed air through solutions containing therapeutic monoclonal antibodies. Under accelerated experimental conditions, bubbling was associated with increasing turbidity and the formation of visible and subvisible aggregates. Aggregate-rich samples also demonstrated changes in protein structure and biological activity. The degree of aggregation varied with protein concentration, sample volume, temperature, airflow rate, surfactant presence, and silicone oil exposure.

These experiments used controlled stress conditions and did not reproduce routine clinical infusion. Their most pronounced findings should not be interpreted as the amount of protein change expected during normal medication administration. The study nevertheless demonstrated something important: bubbling can function as a meaningful combination of air–liquid interfacial exposure and mechanical stress.

A 2024 study by van Haaren and colleagues examined immunoglobulin G at the air–liquid interface of a bubble under flow. Using ATR-FTIR spectroscopic imaging, the researchers directly observed IgG accumulation around an injected air bubble. Under defined experimental conditions, they also detected changes in protein secondary structure associated with aggregation.

Temperature influenced the results, and the protective effect of surfactant varied according to the experimental conditions. The study reinforces that protein behavior around a bubble is measurable, condition-dependent, and more complex than simple exposure to air.

A 2026 study by Cohrs and colleagues evaluated the agitation stability of ten approved monoclonal antibodies. The products did not all respond in the same way. For bevacizumab and nivolumab, experimentally eliminating the air–liquid interface reduced aggregate counts by more than tenfold under the conditions studied.

That result should not be generalized to every antibody or medication. Its broader significance is that the air–liquid interface may be highly consequential for some therapeutic proteins and less consequential for others.

Together, these studies support three important conclusions:

• Proteins can accumulate and undergo structural change at air–liquid interfaces.

• Bubbling can create meaningful interfacial and mechanical stress under experimental conditions.

• The magnitude of the effect depends heavily on the medication, formulation, and conditions involved.

Why a Bubble Is Different From the Bottle’s Natural Surface

The presence of air alone is not the central issue. A partially filled bottle naturally contains an air–liquid interface at the top of the medication, and I2F does not remove that interface.

The distinction involves the number, location, and movement of the interfaces being created. A relatively undisturbed surface remains in one general location. A bubble forms within the medication, travels through it, exposes changing portions of the solution to its surface, and eventually merges with or ruptures at the headspace.

A stream of bubbles repeatedly creates additional interfaces within the medication.

Those bubbles may:

• Expand or contract

• Merge with other bubbles

• Carry proteins at or near their surfaces

• Create localized fluid movement

• Disrupt protein films when they rupture

• Contribute to foam formation

• Continually expose new portions of the medication to a moving surface

Foam can increase the interfacial area further because it consists of many gas-filled structures separated by thin liquid films.

The issue is therefore not simply whether medication is exposed to air. It is how much interfacial area is created, how rapidly that interface changes, and how a specific protein responds to those conditions.

Connecting the Research to Rigid-Bottle Infusion

Rigid medication bottles require replacement air because they cannot collapse as their contents leave. Without adequate incoming air, negative pressure develops inside the bottle, and flow may become inconsistent, slow, or stop.

Vented equipment provides a way for air to enter. This is an established part of direct-bottle medication administration. For example, the FDA-approved prescribing information for GLASSIA, an alpha-1 proteinase inhibitor, specifically permits direct-vial infusion and directs the use of a vented spike.

In an inverted rigid bottle, a conventional vent may terminate near the stopper and below the medication’s surface. Incoming air must then rise through the liquid to reach the headspace.

This creates a clear mechanical connection between container function and the published protein research:

1. Medication leaves the rigid bottle.

2. Air must enter to replace the displaced volume.

3. Incoming air may be introduced below the liquid surface.

4. That air rises through the medication as bubbles.

5. Each bubble creates a mobile air–liquid interface.

Laboratory studies have not yet established the amount of protein change produced by this process during routine direct-bottle infusion. Clinical conditions may differ considerably from accelerated bubbling and agitation experiments.

However, the physical mechanism identified in the research—protein exposure to dynamic air–liquid interfaces—is present in the workflow. That makes the replacement-air pathway a legitimate and underexamined medication-handling variable.

IVIG as an Important Example

Intravenous immune globulin is particularly relevant to this discussion.

IVIG is a concentrated preparation composed primarily of immunoglobulin G (IgG). A patient’s prescribed dose may require multiple rigid bottles, and the medication may be pooled into another container or administered sequentially from its original bottles. These workflows can involve replacement-air entry, bottle changes, fluid transfer, bubble formation, foam, and repeated surface exposure.

The presence of bubbles or foam does not prove that clinically significant IVIG aggregation is occurring. The existing studies do not establish that conventional venting causes infusion reactions, reduced potency, or other clinical consequences during IVIG administration.

However, IVIG combines several factors that make the question worth studying:

• It is a concentrated therapeutic protein.

• Its primary component is IgG.

• Multiple rigid containers may be needed for one treatment.

• Replacement air may enter those containers during withdrawal or infusion.

• Bubbles and foam can be observed during medication handling.

• The medication is clinically and economically valuable.

The protein science is therefore directly relevant, even though the clinical magnitude remains to be measured.

What I2F Changes

Most venting systems are evaluated according to whether they allow enough air into a rigid bottle to maintain flow. I2F introduces another consideration: how replacement air is managed and where it is delivered.

Aeristasis™: Controlled Air Equilibrium at the Point of Vial Access

AII describes the design principle embodied by I2F as Aeristasis™—a design philosophy focused on improving medication-delivery stability through the management of air-pressure dynamics, equilibrium, and air interaction during medication preparation and infusion workflows.

I2F implements this principle by routing filtered replacement air directly to the bottle’s headspace through a pathway separate from the pathway used to withdraw medication. This actively equilibrates the vial headspace as fluid leaves the container without intentionally introducing replacement air below the medication’s surface.

Aeristasis represents a mechanical approach to stabilizing medication preparation and infusion without altering the drug formulation.

Side-by-side demonstration of replacement-air behavior during rigid-bottle infusion. The conventional venting configuration introduces replacement air through the medication, while I2F applies the Aeristasis principle by directing filtered replacement air to the bottle’s headspace. This demonstration illustrates the difference in air pathway and observed bubbling behavior; it is not a controlled study of protein stability or clinical outcomes.

The bottle’s natural air–liquid interface remains. What changes is the need for incoming replacement air to first travel through the medication as a stream of bubbles.

Based on its design and AII’s observations, this headspace-directed pathway may reduce:

• Replacement-air bubbling through the medication

• Foam formation

• Repeated creation of dynamic air–liquid interfaces

• Interfacial disturbance associated with bubble movement and rupture

These are meaningful physical changes to the medication-delivery process.

They should not yet be interpreted as proof that I2F reduces protein aggregation, subvisible particles, loss of biological activity, immunogenicity, infusion reactions, or other clinical outcomes. Those endpoints require controlled comparative testing.

However, the absence of completed clinical-outcome studies does not erase the significance of the mechanism. It defines what should be studied next.

What the Evidence Supports Today

The available evidence can be separated into four levels.

Established by Published Research

• Air–liquid interfaces can influence therapeutic protein stability.

• Proteins can adsorb, reorient, and undergo structural changes at those interfaces.

• Bubbling creates additional dynamic air–liquid interfaces.

• Dynamic interfacial stress can contribute to aggregation and particle formation under experimental conditions.

• Different proteins and formulations vary substantially in their susceptibility.

Established by the Mechanics of Rigid Containers

• Rigid bottles cannot collapse as medication leaves.

• Air must enter to replace the displaced liquid.

• When replacement air enters below the medication’s surface, it travels through the liquid as bubbles.

• Those bubbles create additional mobile air–liquid interfaces.

Supported by I2F’s Design and AII’s Observations

• I2F routes filtered replacement air to the headspace.

• This changes the pathway by which replacement air enters the bottle.

• The design avoids intentionally introducing replacement air below the medication’s surface.

• AII has observed differences in bubbling and foaming behavior when replacement air is directed to the headspace.

Not Yet Established Through Controlled Comparative Research

• The amount of aggregation caused by conventional venting during routine infusion

• Whether headspace-directed replacement air reduces aggregate or particle counts

• Whether particular therapeutic proteins benefit more than others

• Whether I2F affects potency, immunogenicity, infusion reactions, or other patient outcomes

Presenting these distinctions clearly allows the existing science to remain meaningful without overstating what has been proven.

Why Regulatory Authorities Care About Aggregation

Aggregation and subvisible particles are not merely academic formulation concerns.

FDA guidance identifies protein aggregates as a potential immunogenicity risk factor and recommends evaluating the range and levels of subvisible particles in therapeutic protein products. FDA also recognizes that the clinical relevance of an aggregate depends on factors such as its amount, type, and route of administration.

European Medicines Agency guidance similarly recognizes that protein denaturation and aggregation may potentially trigger immune responses and that clinical-use conditions may affect product quality. Its general therapeutic-protein guideline does not formally apply to heterogeneous plasma-derived immunoglobulin preparations such as IVIG, so it should not be treated as IVIG-specific guidance.

The broader regulatory principle remains important: aggregation and particle formation are recognized product-quality variables with potential implications for safety, immunogenicity, and therapeutic performance.

The presence of an aggregate does not establish that a patient reaction will occur. Clinical risk depends on the medication, aggregate characteristics, administered quantity, treatment pattern, route of administration, and patient population.

The Research Question That Comes Next

Published research has already answered the broad scientific question: dynamic air–liquid interfaces can affect therapeutic proteins.

The next question is more specific:

Does the type, location, and movement of replacement air during clinically representative rigid-bottle infusion create measurable differences in protein quality?

Controlled comparative research is needed to evaluate the effects of headspace-directed replacement air on bubbling, foaming, protein stability, particle formation, medication recovery, and biological activity under clinically representative conditions.

Conclusion

Published research shows that bubbles create dynamic air–liquid interfaces and that those interfaces can affect susceptible therapeutic proteins under certain conditions.

Rigid medication bottles require replacement air. When that air is introduced below the medication’s surface, it creates bubbles that travel through the solution before reaching the headspace.

I2F changes that pathway.

By applying the Aeristasis™ principle and routing filtered replacement air directly to the headspace, I2F addresses a physically meaningful part of rigid-bottle medication delivery that conventional venting has largely treated as unavoidable.

Controlled research is still needed to determine whether this change produces measurable differences in protein stability, particle formation, biological activity, or clinical outcomes. But the mechanism is supported by established protein science, the physics of rigid containers, and AII’s observations of medication preparation and infusion workflows.

Based on the published evidence, the physical mechanisms involved, and AII’s observations, we believe I2F has the potential to make a meaningful clinical impact on medication handling and delivery.

References

1. Sreenivasan S, Jiskoot W, Rathore AS. Rapid aggregation of therapeutic monoclonal antibodies by bubbling induced air/liquid interfacial and agitation stress at different conditions. European Journal of Pharmaceutics and Biopharmaceutics. 2021;168:97–109. doi:10.1016/j.ejpb.2021.08.010.

2. van Haaren C, Byrne B, Kazarian SG. Study of monoclonal antibody aggregation at the air–liquid interface under flow by ATR-FTIR spectroscopic imaging. Langmuir. 2024;40(11):5858–5868. doi:10.1021/acs.langmuir.3c03730.

3. Cohrs M, Pagureva N, Özbulak U, De Neve W, Braeckmans K, De Smedt S, Tcholakova S, Vinarov Z, Svilenov HL. Predicting agitation stability of monoclonal antibodies during developability assessment. Molecular Pharmaceutics. 2026;23(6):3421–3433. doi:10.1021/acs.molpharmaceut.6c00092.

4. Li J, Krause ME, Chen X, et al. Interfacial stress in the development of biologics: fundamental understanding, current practice, and future perspective. AAPS Journal. 2019;21(3):44. doi:10.1208/s12248-019-0312-3.

5. U.S. Food and Drug Administration. Immunogenicity Assessment for Therapeutic Protein Products: Guidance for Industry. August 2014. Direct PDF.

6. European Medicines Agency, Committee for Medicinal Products for Human Use. Guideline on Immunogenicity Assessment of Therapeutic Proteins. Revision 1. EMEA/CHMP/BMWP/14327/2006 Rev. 1. Adopted May 18, 2017; effective December 1, 2017.

7. U.S. Food and Drug Administration. GLASSIA [Alpha1-Proteinase Inhibitor (Human)]—Full Prescribing Information. Revised September 2023.

8. Hooper JA. Intravenous immunoglobulins: evolution of commercial IVIG preparations. Immunology and Allergy Clinics of North America. 2008;28(4):765–778, viii. doi:10.1016/j.iac.2008.06.002.

Human-Use Status: I2F has not been cleared or approved by the U.S. Food and Drug Administration for human clinical use and is not currently available for human use in the United States. References to potential human applications reflect scientific discussion and areas for future research; they should not be interpreted as established claims of safety, effectiveness, or clinical performance.