What does the FDA’s new biosimilars guidance actually require?

A BLI characterization checklist for meeting the FDA’s Biosimilar CAA requirements and potentially eliminating the need for Phase 3 trials.

A new era in biosimilars regulation

As the original inventors of biolayer interferometry (BLI) technology, Gator Bio has worked with dozens of customers that are navigating the biosimilar approval process. That experience means that we’re experts on many of the analytical methods that the FDA is looking for and what it takes to generate data that meets that bar. This guide walks through what the Comparative Analytical Assessment (CAA) process looks like and highlights the innovations that make Gator Bio a strong partner in your regulatory process. We will link relevant application notes here as they are published.

Are phase 3 clinical trials still required for biosimilars?

Probably not. The FDA’s October 2025 guidance explicitly states that comparative efficacy studies are not required when a strong comparative analytical assessment (CAA) is in place.

The FDA’s October 2025 draft guidance codified a significant shift in how the agency evaluates biosimilarity: a rigorous comparative analytical assessment is now recognized as often more sensitive than a comparative efficacy study for detecting clinically meaningful differences between a biosimilar and its reference product. For well-characterized biologics that meet specific criteria, this means large-scale comparative efficacy trials are no longer the default requirement.

That’s huge. However, this exception still requires that the molecules be:

• Derived from a clonal cell line
• Highly purified and fully characterized
• Have a known structure-activity relationship, with quality attributes evaluable by CAA assays
• Has completed a human PK similarity and immunogenicity study against the reference product

The one clinical requirement that remains non-negotiable is the human PK similarity study and immunogenicity assessment. As the guidance states: comparative efficacy studies are not required, but PK similarity and immunogenicity data must still be generated against the reference product^[1].

What changed in March’s 2026 biosimilar guidance?

The March 2026 guidance allows internationally-developed biosimilars to use non-US-licensed comparator products in place of the US reference product.

Building on the October 2025 draft guidance, the FDA released a formal fourth revision to its biosimilar Q&A guidance in March 2026, allowing non-US-licensed comparator products to substitute for the US-approved reference product in clinical studies, provided there is adequate data supporting their similarity.

Previously, sponsors wishing to use a non-US comparator had to conduct a full three-way PK bridging study comparing the proposed biosimilar, the US reference product, and the non-US comparator, with all three pairwise comparisons required to meet pre-specified similarity criteria. Under the new guidance, you no longer need to import a US version of the product for comparative pharmacokinetic (PK) testing as long as the international substitute is scientifically justified and the “totality of the evidence supports a demonstration of biosimilarity”^[1].

However, to unlock this route , which represents up to $20 million in PK study cost savings per program, your characterization needs to be airtight.

What is an FDA (CAA), and what must it include?

A CAA is the complete analytical evidence package demonstrating that a biosimilar is highly similar to the reference product.

Requirement	Typical Analytical methods	Role of BLI
Primary Sequence and post-translational modifications	MS	–
Higher-order structure	CD, HDX-MS, FTIR	–
Size/charge variants	SEC, AUC, cIEF	–
Target antigen binding: Affinity (KD) and kinetics (koff and kon)	BLI, SPR	Primary method
Full Fc receptor panel (FcRn, FcγRI/IIa/IIb/IIIa/IIIb)	BLI	Primary method
Cell-based functional assays (ADCC, ADCP, CDC)	Varies	Supports via receptor binding data
Glycan Profiling linked to Fc receptor results	LC-MS	Orthogonal validation

Click here to enlarge table

At a minimum, it covers primary sequence, higher-order structure, size and charge variants, target antigen binding kinetics, a full Fc receptor panel, cell-based functional assays, and Glycan profiling. BLI is one of the primary methods for several of these important checkpoints.

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What specific roles does BLI fill in the CAA?

A complete BLI panel for a mAb biosimilar CAA covers seven distinct binding measurements: antigen affinity/ kinetics, FcRn binding and release, the full activating and inhibitory FcγR panel, complement initiation via C1q, and epitope confirmation.

The table below summarizes the complete BLI panel, including the recommended capture chemistry for each assay alongside references for the various protocols.

ASSAY	CAPTURE AND LIGAND	LIGAND	ANALYTE	KEY PARAMETER REPORTED	REFERENCES
Antibody-antigen kinetics	Human Fc Gen II	Biosimilar, Reference	Antigen	Affinity (KD), kinetics (koff, kon)
FcRn pH 6.0 binding	ProL/SA	Biosimilar/Reference (Biotinylated if SA)	FcRn	kon, response at end of association	[2] [3] [4]
FcRn pH 7.2 Release	ProL/SA (same tip and assay as above)	Biosimilar/Reference (Biotinylated if SA)	FcRn	koff
FcγRIIIa (CD16a) affinity	HIS XT	FcγRIIIa-His	Biosimilar, Reference	Relative binding response; KD	[4]
FcγRIIa (CD32a) affinity	HIS XT	FcγRIIa-His	Biosimilar, Reference	Relative binding response; KD	[4] [5]
FcγRI (CD64) affinity	HIS XT	FcγRI-His	Biosimilar, Reference	Relative binding response; KD	[4]
C1q	ProL (direct capture), SA (antigen capture then Ab)	Biosimilar, Reference	C1q	Response	[6, 7, 8]
Epitope Binning	ProA or HFC Gen II (Sandwich); HIS XT (Tandem)	Sandwich: Ab -> Antigen -> Ab Tandem: Antigen -> Ab -> Ab		Blockage %	[Epitope Binning Guidebook]

Click here to enlarge table

Gator BLI is recognized as a biosimilar characterization method in multiple US Pharmacopoeia (USP) chapters: [1105] (Immunological Test Methods, covering surface plasmon resonance and related optical biosensor methods), [1106] (Immunogenicity Assays), and [1108] (Assays to Evaluate Fc-Mediated Effector Function). This inclusion in the USP indicates the method’s reliability, reproducibility, and suitability for the kinetic measurements, making it the primary tool for measuring target antigen binding and the full Fc receptor panel. Moreover, Gator BLI’s throughput and sensitivity make it well-suited to the comparative, multi-lot work that biosimilarity characterization demands.

Gator Bio has also released a variety of off-the-shelf capture options that make these experiments straightforward, with His XT offering an easy on-ramp for Fc receptor studies and anti-FAB biosensors for FcRn characterization. This means that Gator BLI isn’t just a discovery tool, but also an indispensable checkpoint for the CAA process.

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Why does FcRn binding matter for biosimilar approval, and how do you measure it?

FcRn results predict antibody serum half-life – a biosimilar with different FcRn binding kinetics will have a different half-life than the reference product.

Antibodies persist in the bloodstream for a remarkably long time relative to most other proteins. This extended half-life is driven by FcRn (the neonatal Fc receptor), which is expressed in the endosomes of macrophages and endothelial cells. As antibodies are internalized by these cells and trafficked toward the lysosome, FcRn binds the antibody Fc region at the acidic endosomal pH (~6.2) and diverts them away from degradation, recycling them back into circulation. At physiological blood pH (7.4), FcRn releases the antibody, completing the rescue cycle. Biophysical characterization of these pH-dependent binding events is mechanistically predictive of serum half-life.

BLI has already been shown to measure FcRn interactions reliably and reproducibly^[2]. Moreover, BLI has a lower risk of aggregate interference that might be seen in bead-based assays for FcRn^{[3, 9]}. In addition, BLI has been shown to have the sensitivity to detect oxidation of Met 252 and Met 428, making BLI a cost-effective formulation assessment option.

Some tips for FcRn receptor studies on BLI:

• Capture the pH change during the assay: Observe both association and dissociation at low pH (6.2) then transition to a higher pH (7.4) to observe the change in dissociation^{[3, 9, 2]}.
• Capture in a way that leaves the Fc domain exposed: Light chain capture via Protein L or Streptavidin capture of biotinylated Ab enable FcRn engagement (Anti-Fab is not recommended – the capture molecule contains an Fc domain).

How does Fcγ receptor binding data show biosimilarity?

FcγR binding kinetics are an important component of characterizing a biosimilar’s effector function profile, and deviations from the reference product will affect clinical activity.

Differences in Glycans on biosimilars affect Fc receptor function and have real consequences for effector function ^[10]. For instance, fucosylation affects FcγRIIIa (needed for ADCC/NK cell engagement)^[11], while galactosylation primarily influences FcγRIIa ADCP/phagocytic engagement (though it can also affect FcγRIIIa) ^{[12, 13]}. BLI is well-suited for characterizing these deviations, offering a direct measurement of Fc receptor binding kinetics that complements Mass Spectrometry Glycan profiling and avoids artifacts common to bead-based methods^[5].

Some tips for Fc receptor studies on BLI:

• Improve data capture for fast-on/fast-off receptors (e.g. CD32a/FcγRIIa): Set your data acquisition rate to 10 Hz, lower shaker speeds to 400 RPM and, when possible, cool the plate to 25ºC or lower (as it is on the Gator Pivot).
• Loading density is critical: Overloaded biosensors will lead to unusual and hard-to-characterize kinetic traces. For example, using a His XT biosensor, consider loading CD32a/FcγRIIa to less than 0.5 nm shift (<10% of maximum capacity), resulting in a flat plateau during antibody association.
• Block biosensors or use double-referencing when working with high Ab concentrations: Many antibodies interact with biosensor components at concentrations above 2 uM.
• Keep biosensors the same for both reference and biosimilar: This avoids variations in biosensor load density. Run both in immediate succession to avoid day-to-day variation.
• Run orthogonal glycoengineered controls: For example, run afucosylated IgG as a positive control for FcγRIIIa binding to confirm the activity of Fc receptors before the assay.
• Choose a biosensor with non-Fc capture chemistry: A common culprit of this kind of error is traditional anti-His biosensors, which use a rabbit or mouse antibody to capture his-tagged Fc receptors. Gator His XT uses an AI-designed novobody and doesn’t include an Fc domain (learn more here). Other examples of biosensors that do not contain Fc capture chemistry include Strep-Tactin XT, Streptavidin, and Ni-NTA.

Pictured: FcγRIIa/CD32a (R167)-antibody interaction measured in-house. CD32a was loaded to 0.5 nm on HIS XT biosensor followed by Ab from 2500 to 78 nM following guidelines given above. K_D: 9.62*10^-7, k_on: 1.26*10⁵, k_off: 1.21*10^-1 aligning with literature.

For a more direct assay of glycosylation, Gator results had a linear relationship with FPLC results for quantifying sialylation in highly-glycosylated species. This method, detailed in this application note, can be beneficial when developing a biosimilar to a heavily-engineered reference product.

What is a C1q assay, and why does it matter?

C1q assays predict activation of the immune system

The Complement-Dependent Cytotoxicity (CDC) pathway is one of the key effector mechanisms through which IgG antibody therapeutics can mediate target cell killing. This starts with the antibody interacting with C1q which triggers a proteolytic cascade that leads to membrane attack complex (MAC) formation and target cell lysis. When validating a biosimilar, C1q interaction activity is a vital step in showing the potency and activity of the antibody of interest, showing that the biosimilar mirrors the reference product’s CDC-potentiating activity.

Some tips for C1q studies on BLI:

1. Immobilize the antibody on the biosensor surface via the light chain. This ensures that the entire Fc domain is exposed and available for C1q interaction. The most common biosensor for this purpose is Protein L. Alternately, if the antibody-antigen interaction does not dissociate quickly, antibody can be immobilized through interaction with the antigen. His XT, Strep-Tactin XT, Streptavidin and Ni-NTA are all acceptable options for antigen-based capture.
   • Note: use a biosensor that does not contain an Fc. Anti-FAB is not recommended. Similarly, amine coupling of the antibody to an Amine Reactive (AR) biosensor can result in reduced C1q activity and is not recommended.
2. Target the optimal density: C1q is a multimer that relies on avidity for its interaction. A load of 2-4 nm on ProL sensors gives consistent C1q interaction results.

Pictured: C1q interaction between Cetuximab and C1q (Abcam) measured in-house using ProL biosensor loaded to 2 nm. K_D: 5.56*10^-9, k_on: 3.56*10⁶, k_off: 1.98*10^-2 aligning with literature

What is epitope binning, and why does the FDA expect to see it in a biosimilar CAA?

Epitope binning confirms that the biosimilar and reference product bind the same site on the target antigen.

Many therapeutic antibodies not only activate an immune response to the bound target, but they also affect the target’s conformation and modulate its activity. To mirror this effect, biosimilars need to not only mimic the reference product in target binding affinity – they need to bind the same site.

Gator BLI enables either sandwich- or tandem-format binning experiments at scale with a broad array of off-the-shelf capture options. Gator instruments are optimized for epitope binning workflow, with the Gator Pro 32 channel system’s four plate positions generating 32×32 binning data in a single assay. Even 8 channel instruments can perform high-throughput binning experiments, with the Gator Plus running up to a 16×16 format, bringing high throughput to startup biosimilars labs. Click here to learn more about how to perform an epitope binning experiment using a Gator instrument.

How does 21 CFR Part 11 compliance factor into BLI data for biosimilar Biological License Application (BLA)?

Data integrity is not a box to check at the end.

21 CFR Part 11 establishes the FDA’s requirements for electronic records and signatures. Instrument data, method files, and analysis outputs need user authentication and an unbroken audit trail that documents who did what and when.  

Gator Bio instruments offer an optional Part 11-compliant software package which restricts protocol modification, controls access permissions by role and ensures no possibility of after-the-fact editing. This ensures that your data is submission-ready from the moment it is acquired.

Preparing for the patent cliff

The patent cliff is real and accelerating; over $200 billion in biologic revenues face loss of exclusivity by 2030, and the FDA’s October 2025 and March 2026 guidance updates have removed two of the biggest cost barriers to biosimilar development. For programs that invest in rigorous analytical characterization from the start, the path to approval is shorter and cheaper than it has ever been.

Gator Bio instruments run the full BLI-based CAA panel: antigen binding kinetics, FcRn binding and release, the complete FcγR panel, C1q, and epitope confirmation. The platform offers dedicated-spectrometer data quality across all 32 channels, temperature control for Fc receptor assays, HIS XT biosensors that eliminate Fc background interference, and an optional 21 CFR Part 11-compliant software package for submission-ready data integrity.

If you are building a CAA program and want to work through your assay strategy with a biosimilar characterization expert, reach out to us.