Gyrolab® SpinBlog

How to be confident in immunoassay determination of free versus bound target or drug

It’s all about maintaining equilibrium

Immunoassays must be able to generate accurate and precise quantitative measurements of analytes such as drug candidates and their targets, and many studies also demand the accurate determination of levels of free or bound drug, or target. Achieving this is a challenge and success depends on many factors, including taking steps to maintain the equilibrium formed in vivo between various molecular forms of the analyte. This can be achieved with care in sample preparation, and by using an immunoassay platform that requires minimal sample dilution, and delivers results quickly before the equilibrium can shift.

The need to measure free or bound

Determining the concentration of biotherapeutics is vital during preclinical and clinical studies aimed at assessing:

  1. relationships between drug concentrations in circulation and drug exposure,
  2. pharmacokinetics (PK) of the drug candidates,
  3. dosing for human Phase I–III studies,
  4. PK/pharmacodynamics (PD) modeling, and
  5. relationships between drug exposure and efficacy/safety.

Ligand binding assays (LBAs) such as ELISA have been the most common analytical method for determining biotherapeutic drug concentrations. The development of methods and the interpretation of the results have required an understanding of how the design and format of an LBA can affect the generation of data on apparent drug and/or target concentrations, and how this can affect the interpretation of PK/PD and safety data. This is especially the case with monoclonal antibody (mAb)-based therapeutics. At the AAPS National Biotechnology Conference in 2008, a hot topic session on free versus total PK/PD assays stimulated the formation of a discussion group by the AAPS LBA Bioanalytical Focus Group (LBABFG) that resulted in a consensus-based ‘AAPS White Paper’ that resulted from discussions of the LBABFG (1,2). Some years later, the annual 2017 11th Workshop on Recent Issues in Bioanalysis handled a key question: What assay should be developed: free, total or active drug PK assays? (3).

The art of maintaining balance – the law of mass action

The choice of bioanalytical strategy will depend on which drug or target form needs to be determined. For example, measuring active drug can be very important in understanding and interpreting PK and PD data due to the importance of free/active drug exposure. Alternatively, true total drug data might be required to gain an understanding of the drug clearance. Whatever the need, a major challenge is maintaining the equilibrium achieved in vivo between free and bound drug and target that results from the law of mass action:

Free drug + Target  Drug:Target

Anything that changes the concentration of one or other of the component species will disturb this equilibrium, e.g. sample handling, dilution, and the very act of determining the concentration of one of the component species. For example, attempting to measure free target using a therapeutic drug as capture reagent can lead to a shift in equilibrium that results in back dissociation and overestimation of free target. This need to maintain equilibrium creates a significant challenge in designing and developing assays that measure only one molecular species. For example, which matrix should be used to prepare a standard curve and QC sample? Is there dilutional linearity when diluting samples into the assay working range? And is it feasible to develop drug-specific reagents capable of binding with high affinity to both drug-free- and drug:target complex to get data on total target?

How to perturb the equilibrium as little as possible

The most important factors that will improve the chance of successfully measuring the true levels of free or bound drug, or target, e.g. generating in vitro results that are more representative of in vivo conditions, are:

  • Minimizing dilutions of drug/target mixes i.e. the MRD of the assay
  • Minimizing analytical times, for example when the therapeutic is used as capture reagent

As Yang and Quarmby pointed out in their editorial on free versus total ligand-binding assays in 2011, ”This discussion on free versus total LBAs is also expected to motivate analytical technology vendors to work more closely with their drug-development customers” (4). As we will see below, one pharmaceutical company has a technology that fulfills the requirements for measuring free target or drug.

The technology of choice when quantifying free target

A recent study on the quantitation of free complement protein C5a in human plasma illustrates both the challenges of measuring free target and a solution (5). Complement protein C5a is an important component of the alternative complement cascade and plays a role in many conditions, including renal injury, asthma, and sepsis, which makes it a potential target for biotherapeutics. For example, Alexion Pharmaceuticals have developed a monoclonal antibody drug candidate, ALXN1007, that targets C5a and quantitation of free C5a is important in understanding the pharmacodynamics.

Measuring C5a is particularly challenging, since it is the product of cleavage of C5 and both residual C5 and C5a share a common neoepitope. There are no known immunoassay reagents that are absolutely specific for C5a—all bind C5 to some extent. ALXN1007 has a high affinity for C5a but a low affinity for C5. In an immunoassay using ALXN1007 as capture reagent, Dysinger and Ma solved this problem by pretreatment to deplete samples of C5, followed by dilution with high salt buffer that favors the high affinity binding of C5a. They also chose Gyrolab system since its flow-through technology enables quantitation of free target analyte with minimal sample dilution and rapid sample incubations, which minimizes non-specific interference and unwanted dissociations. The authors concluded that, “Due to the advantages that it provides, Gyrolab has become our default technology of choice for quantitation of free target.”

Find out more about determining free versus bound target using Gyrolab system by viewing the webinar, “A Gyrolab Assay for the Quantitation of Free Complement Protein C5a in Human Plasma

View the webinar

Submit your questions/comments to and Mark Dysinger will reply as soon as possible.



  1. Therapeutic monoclonal antibody concentration monitoring: free or total? Kuang B, et al. Bioanalysis 2(6), 1125–1140 (2010). 
  2. Bioanalytical approaches to quantify ‘total’ and ‘free’ therapeutic antibodies and their targets: technical challenges and PK/PD applications over the course of drug development. Lee JW, et al. AAPS J. 13(1), 99–110 (2011). 
  3. 2017 White Paper on recent issues in bioanalysis: a global perspective on immunogenicity guidelines & biomarker assay performance (Part 3 – LBA: immunogenicity, biomarkers and PK assays). Gupta, S et al, Bioanalysis (2017) 9(24), 1967–1996 
  4. Free versus total ligand-binding assays: points to consider in biotherapeutic drug development. Yang, J and Quarmby, V. Bioanalysis (2011) 3(11), 1163–1165
  5. A Gyrolab assay for the quantitation of free complement protein C5a in human plasma. Dysinger, M & Ma, M. AAPS J (2018) 20:106