The hidden coagulation risk in plasma-derived biologics

For development teams working on plasma-derived biotherapeutics or recombinant proteins, hemostatic risk is often treated as a late-stage concern, something to evaluate once a formulation is close to clinical entry. Dr. Ryan Dorfman, COO at Prolytix, urges that this timing is backwards. Trace concentrations of activated clotting factors can set the coagulation cascade in motion with profound clinical consequences, and the analytical methods needed to detect these contaminants at picomolar sensitivity are available now.

A cascade primed for amplification

The coagulation cascade is designed to respond fast and to amplify; a single activated enzyme recruits ten more, those ten recruit a hundred. Calibrated for life-saving speed when a bleeding event occurs, that same sensitivity makes it acutely responsive to trace contaminants in biologic formulations.

"If you think about the coagulation cascade from a physiological standpoint, it's primed and ready to go," says Dorfman. "It is very responsive to small amounts of activators or inhibitors. And so, the concern is that if these products have contaminants associated with them, they can cause those unwanted effects, both bleeding and clotting."

Intravenous immunoglobulin (IVIG) illustrates the problem precisely. Residual Factor XI (FXI) can co-purify with the immunoglobulin G (IgG) fraction during plasma fractionation because standard downstream processing does not reliably separate the two proteins. Subsequent process-related activation of FXI is believed to be the primary source of Factor XIa (FXIa) detected in IVIG products.

"Infinitesimal amounts, picomolar amounts, can cause clotting risk in patients taking IVIG," Dorfman explains. Severe adverse outcomes could potentially include myocardial infarction, stroke, and deep vein thrombosis.

A well-documented voluntary recall of IVIG products traced the elevated thrombotic risk directly to FXIa content in the drug product, establishing FXIa as the primary target for hemostatic risk monitoring in that therapeutic class.

Similar exposure dynamics apply to Factor XIIa and plasma kallikrein, both key components of the contact activation pathway. However, the clinical significance extends beyond activation of individual coagulation factors. Coagulation, platelet activation, and complement activation are highly integrated biological networks characterized by extensive bidirectional signaling and amplification. Consequently, activation of one pathway frequently propagates activation of the others, potentially resulting in a disproportionate biological response relative to the initiating stimulus.

This systems-level interdependence underscores the potential severity of process-related procoagulant impurities and reinforces the need for sensitive detection and control strategies during biotherapeutic manufacturing.

Two methods are better than one

Characterizing the full hemostatic risk of a drug product requires more than one analytical method. High-resolution mass spectrometry (HRMS) provides what Dorfman describes as a bird's eye view.

"It's not typically quantitative, but it gives you an inventory of what contaminants are in the drug product." From that map, teams can identify which proteins are present and prioritize which require closer scrutiny.

Functional assays then take over where mass spectrometry leaves off. As an example, non-activated partial thromboplastin time (NaPTT) is a broad screening assay that confirms whether active enzymes are present without identifying the specific culprit. It is, Dorfman says, a general-use assay that points to a problem rather than naming it.

Once the target enzyme is identified, more specific tools can be deployed. For IVIG, where FXIa is the established risk driver, Prolytix developed a thrombin generation assay (TGA) that exploits the enzyme's activity directly. "The FXIa in the drug product fundamentally rescues the deficient plasma, and then thrombin is generated," Dorfman explains. Paired with HRMS, the TGA provides a second, independent line of evidence for the same finding, the kind of orthogonal confirmation that analytical labs rely on.

Adding the platelet dimension

Plasma-based assays remain the backbone of most hemostatic risk programs, but they capture only part of the picture. Thromboelastography (TEG) extends the assessment to whole blood, where platelets are present and contribute to primary hemostasis. If drug product contaminants are inhibiting platelet activation or aggregation, plasma-only assays will generate a false-negative result.

"By doing TEG, you can assay whole blood," Dorfman says. "You're adding complexity to the assay. Are platelets being impacted by the contaminants? Are you inhibiting the platelets from assisting in the clotting process as the primary hemostatic plug?" TEG closes that gap and rounds out the functional coagulation profile.

The cost of waiting

In practice, Prolytix is most often engaged after an adverse hemostatic signal has appeared, after a clinical trial has been halted and a development team is working backward to identify the cause.

In oncology programs, where the underlying cancer itself can dysregulate coagulation through tissue factor expression on tumor cells, attributing a bleeding or thrombotic event to the investigational compound is a difficult and expensive analytical problem to resolve.

"The last thing you want to do is get to the clinic and find out you have a hemostatic risk problem," Dorfman says. "That's a tremendous waste of time, energy, and money. If you can pinpoint those problematic enzymes early on and advise a manufacturing strategy to rid the process of them, you're going to be much better off down the road."

Building the right assay panel

Prolytix's approach to hemostatic risk assessment follows a tiered logic: a broad assay such as non-activated partial thromboplastin time (NaPTT) screen establishes whether active enzymes are present, HRMS maps the specific contaminant population, and a bespoke functional assay validated to picomolar sensitivity becomes the cornerstone of ongoing release testing. Each tier narrows the question, and the final assay answers it.

That framework applies across therapeutic modalities including cell and gene therapy, where contaminant profiles are less well-characterized than in established modalities like IVIG, as well as monoclonal antibodies, enzyme replacement therapies, and rare bleeding disorder programs.

"We can really capture all the therapeutic areas with regard to hemostatic risk," Dorfman says. "Even though blood coagulation is our expertise, we are good protein biochemists and we like solving large-molecule problems fundamentally."

That breadth is only useful if it is brought in at the right point. The teams that integrate hemostatic risk testing during discovery and early development are the ones positioned to catch a problem while the process can still be changed, rather than working backwards from a halted trial to a cause they cannot cleanly identify.

Dive deeper into hemostatic risk assessment strategies for plasma‑derived biotherapeutics, download the Eliminating Hemostatic Risk whitepaper >>