Deeper than the definition: redux edition

I’ve had the pleasure of writing for Bioanalysis Zone over the course of the past year, and when long-time columnist (and LC–MS whiz and book author!) Robert MacNeill came up with the clever idea of connecting our spring 2026 columns to The Bioanalysis Glossary, I was sold. This will be my first foray into column collaboration (not the liquid chromatography kind).

In project management lingo, there is the concept of explicit vs tacit knowledge. Explicit knowledge refers to concepts that are easily captured via a definition and transferred, whereas tacit knowledge refers to those insights that are harder to convey and are grounded in your experiences in a field. Indeed, after years in the bioanalytical lab, certain terms evoke a range of related images, concepts and considerations that go “deeper than the definition.”  In this follow-up column to Robert’s insightful piece on ADCs, UHPLC and superficially porous particle technology, I explore some of The Bioanalysis Glossary terms that evoke memories of ligand binding methods past.

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1. Acid dissociation

Bioanalysis Glossary definition: “Sample pretreatment step using a decrease in pH to dissociate an analyte from a protein (e.g., drug–antidrug antibody [ADA] complexes), often used in conjunction with various immunoassay/immunoextraction method formats to improve assay characteristics, such as an increase in tolerance of ADA detection to the presence of drug.”

As a self-described ADA assay aficionado, acid dissociation is a mainstay of the clinical immunogenicity playbook. Scientists first starting out in the ligand-binding space, especially those working on antibody drugs, are likely to be trained on a method that uses an acid dissociation step followed by a bridging step, in which antibodies to the drug bind to labeled drug that is captured onto a plate to generate an assay signal. The use of acid (typically in the range of 2–3 pH units, often acetic acid or glycine) dissociates the ADAs from the drug in the sample by disrupting protein-protein interactions, thus typically improving the tolerance of the assay to the presence of drug in the sample. Subsequent to acid treatment, the pH is neutralized via the addition of a basic solution (Tris base).  Acid dissociation also plays a role in ADA formats like ACE (affinity capture elution), PandA (precipitation and acid dissociation), and SPEAD (solid phase elution with acid dissociation). Each acidification step involves disruption of the ADA-drug interactions.

While acid dissociation is a well-established practice, methods still require some finessing to confirm appropriate conditions. The pH and type of acid treatment, as well as the ratio of acid to sample and the duration of acid treatment, can all be varied; for example, lower pH glycine solutions may be selected if acidic solutions at pH 3 are insufficient to achieve the necessary drug tolerance for the ADA assay. Consistent and thorough mixing of samples through the acid dissociation process is also essential. One common issue in method design is to progress too far along the path of method development experiments, only to discover that the format selected does not meet drug tolerance expectations. Instead, the impact of specific acid dissociation conditions can be readily tested early in development, incorporating different (contrived) sample types, including not just positive controls (PCs) but also PCs + drug. pH strips, while not suitable for regulated work or final verifications, can be a valuable tool in these experiments to quickly compare the pH of acidified samples and neutralized sample mixes across different combinations. Of note, drug development programs that face the issue of soluble target interference (i.e., soluble drug target in the sample causing either positive or negative interference with assay signal) may also benefit from the use of specific types of acids to dissociate noncovalent interactions between drug and target, which can also be tested with contrived samples in method development. Check out the recent paper by Ye et al. (2025) in Bioanalysis for a detailed case study of the impact of a panel of acid types on reduction of assay interference by a soluble dimeric target.

Despite its flexibility and utility, however, acid dissociation comes with caveats. Not all antibodies take kindly to being dipped in acid, and some loss of signal can occur as a result. The impact of acid treatment can be assessed with PC-spiked samples in development, but ADA developers face the age-old (well, perhaps decades-old) problem that a positive control antibody that is used in method development and validation will not necessarily recapitulate the performance of all the different clinical ADAs. This issue is elegantly evaluated by Kavita et al. (2017) in an exploration of acid dissociation on the behavior of ADAs of high vs low affinity for drug, in which the authors discuss the merits of considering the performance of antibodies with different affinities during assay development.

2. Biological matrix

Bioanalysis Glossary definition: “A material of biological origin that can be sampled for the measurement of specific analytes; matrices include blood, serum, plasma, urine, feces, cerebrospinal fluid, saliva, sputum and various tissues.”

I was drawn to this term primarily because, at face value, it appears so simple… paired with the fact that sample matrix considerations is a thread weaving through all aspects of bioanalytical sample, assay and analysis planning, taking us on a journey from assay conceptualization through to result reporting. Let’s briefly explore that thread below.

First, matrix selection is a key strategy question early on in program planning, as translational scientists and clinical pharmacologists work together with the bioanalytical team to determine the necessary matrices to sample. For biomarkers, the fluids (or even tissues) in which the marker is expected to be detected and, as relevant, change over time, must be considered, as well as the feasibility of sampling. Pharmacokinetic considerations drive needs for sampling as well, such as the inclusion of urine measurements of drug concentration or the need for tissue or cerebrospinal fluid (CSF) PK assessments.

Next, constraints associated with a specific matrix type is a key element during clinical trial setup by operational experts. Limitation of blood contamination of CSF, prevention of hemolysis and stability of the analyte during collection and handling are all key considerations. Bioanalytical teams must work with clinical teams to outline the volume needed per analytical test and determine total blood (or CSF, etc.) volumes that are reasonable per collection, as well as to prepare specific collection instructions. For matrices with limiting volumes, like pediatric CSF or tissue biopsies, the project team will likely need to rank the priority of the bioanalytical measurements and consider whether collected amounts are sufficient for repeat testing. Matrices with low protein content, such as CSF and urine, need mitigation strategies to reduce nonspecific binding, such as the selection of low-binding plastics or the use of anti-absorptive urine additives. See Ji et al. (2010) for further reading.

Third, as assay development commences, lab leads must consider the impact of the matrix type on design and processing. Complex matrices like tissue and sputum will typically require pre-processing, which also needs to be accounted for in budgeting time and cost for overall method development. Bioanalysis Zone previously covered the consideration of matrix effects on assay performance from a LC–MS perspective. Similarly, for ligand-binding assays, interferences from matrix must be assessed and mitigated as needed, via options like dilution and/or sample purification. Procedures for sample liquid handling must also be consistent with the viscosity and characteristics of the matrix type, especially when using automation for aspiration and dispensing. Please do not forget to double and triple-check the clinical trial laboratory manual and lab specifications documents against the assay validation plan. Many panicked moments in the halls of bioanalytical labs around the world have arisen when a miscommunication about plasma vs serum, the plasma anticoagulant type, or the requisite sample volumes has been discovered far too late to right the ship. Check early and often!

And finally, bioanalytical method developers and validators should become adept at sourcing and evaluating matrices. Blank matrices are needed for essential activities such as ADA negative control pool preparation, ADA cut point determination, LBA selectivity sample preparation, dilution of high-concentration PK samples and process evaluation during method development. This process may require comparison of different matrix vendors, discussion of pre-analytical collection and storage conditions, and review of donor characteristics. For example, when sourcing disease-state blank matrix for biosimilar programs, medication lists from patient sources can prevent the use of samples that have the comparator drug present. Custom collections may even be needed for unstable biomarkers or unusual matrix sources. Do not underestimate the importance of high-quality matrix sources and proper screening of lots for use in matrix pools!

Disclaimer: the opinions expressed are solely that of the author and do not express the views or opinions of their employers, Bioanalysis Zone or Taylor & Francis Group.