Bioanalytical Services

The initial heterogeneity of an ADC may evolve further in vivo due to biotransformation, undesirable de-conjugation of payload from the ADC ³ or differences in the clearance rate of DAR species.⁴ Due to its structural complexities and heterogenous and dynamic nature, an ADC requires a very distinct bioanalytical strategy for characterization and quantification. Three different analytes including total antibody, antibody conjugated drug and free drug are measured to correlate with efficacy or toxicity of ADCs. Their multicomponent nature and complex structure combining large and small molecule characteristics necessitates the need for more complex or multidisciplinary bioanalytical approaches.⁵ Large molecules have a well-defined tertiary structure that is suited for ligand binding assays (LBAs) or MS approaches, whereas bioanalysis of small molecule drugs is predominantly performed by LC – MS/MS.² Moreover, the assay format requires constant adaptation during the different stages of drug development. The extent of endogenous target cross reactivity, nature of payload and linker, bioconjugation technique, the required dynamic range and throughput of the assays will determine the bioanalytical strategy employed. Strategically envisioning which bioanalytic assay would be appropriate early in the program would streamline the work and minimize challenges later in the process.

Early phase bioanalytical assays are modified from platform methods and are devised mainly to help guide the research and interpret early in vivo results. Some cleavable linkers attached to specific areas of the mAb can lack stability in mouse but be stable in human plasma.⁶ Measuring this can allow modification of the linker slightly to increase its stability in mouse plasma and obtain more relevant data.  This data can be generated by in vitro and in vivo studies and the program can evolve by changing the cleavable sequence.  The complexities of an ADC molecule require diverse analytical methods. The most common bioanalytic platforms used to examine biotherapeutics include ligand-binding assays (LBA)⁷ and enzyme-linked immunosorbent assays (ELISA).⁸

What are the factors to consider while choosing a bioanalytical method?

The major analytical challenges associated with developing or studying ADCs revolve around characterization and quantification of the dynamically changing mixture of ADC species as they undergo biotransformation. While choosing the optimum bioanalytical method for ADCs, there are many components to contemplate over. These include:

1. Antibody concentration
2. Amount of drug loaded over time
3. Any modification of the linker-payload or the concentration of free payload

At an early stage for stability and PK studies, knowing the total amount of mAb and amount of conjugated payload is probably the most crucial factor to get pertinent information.

Since each bioconjugate is unique, having a company that understands the bioconjugate field from both the chemistry and biological perspective is important during early drug discovery. This knowledge especially comes into play when the drug or the linker is cleaved, metabolized, or modified in vivo.

What is NJ Bio’s expertise related to bioanalytical services?

Considering all these factors, NJ Bio offers specialized bioanalytical services for early research and has invested in acquiring state-of-the art instrumentations. Depending on the information sought (such as ADC stability, clearance, exposure, safety etc.) at a given phase of the drug development, we use Ligand Binding Assays (LBA), LC-MS and Hybrid capture LC-MS for analyzing diverse ADC analytes. Herein, we consider the various components of ADCs such as warheads, linkers and conjugation strategies that affect the ADME properties of an ADC. We realize that sophisticated bioanalytical assays are required for assessment of intact ADCs, total antibody, released warheads and relevant metabolites. At NJ Bio, we are committed to providing detailed bioanalytical testing services. Our expertise also spans other biological services such as in vitro stability, in vitro testing and biological services which are critical for design and subsequent development of a clinically successful ADC. These services are pivotal to move your ADC program forward. We can help you select the most reliable analytical methods with quality standard operating procedures (SOPs) in order to meet critical deadlines and make more confident research decisions.

Ligand Biding Assays (LBA) for antibody drug conjugates

In recent times, ELISA is one of the most commonly used LBA for detection and quantification of specific proteins in a complex mixture. Recent advances have enabled the development of highly sensitive LBA for quantification studies.⁹ It is a powerful method to determine concentration of an analyte in in vivo samples. There are a variety of methods that can be employed for this purpose but considering the complexities and nature of ADCs the most common method is to immobilize a capture reagent to a plate such as an Anti-idiotypic (anti-ID) antibody, the protein of interest or a more general capture reagent. Majority of PK determinations and immunogenicity assessments for biotherapeutics are generally done using LBA.⁹ The capture reagent is coated onto a polystyrene plate. The analyte is diluted and incubated in order to bind to the capture reagent. Unbound analyte is removed from the plate by a washing step. Once well captured, a detection antibody is added and a signal by fluorescence or calorimetry is observed.¹⁰

One striking advantage of ELISA over LC–MS is that there is little investment in equipment. It measures the bioconjugate function and can report a decrease in antibody capture. However, over time the levels of monoclonal antibodies decrease in serum. An anti-payload would in general detect the presence of a monoclonal antibody with a linker payload but cannot be used for quantification purposes. Another drawback of ELISA is that detection and capture reagents may not be commercially available and thus would need to be generated and standardized appropriately.

Fig. 1: ADC bioanalysis using ligand-binding assay

Recent developments with respect to pre-enrichment of analytes in low abundance in biological matrices and signal amplification have contributed to increasing the efficiency and sensitivity of LBA methods in quantitative analysis.^11–14 One such superior approach to measure bioanalytes is the use of a highly sensitive MSD instrument. Electro-chemiluminescent detection technology using SULFO-TAG label as detection reagent works efficiently to improve ELISA sensitivity. In this approach, the bottom of the MSD multi-well plates is equipped with electrodes, which conduct electrical energy from the MSD instrument to generate light emission from the SULFO-TAG labels. The assay signal can be amplified by multiple excitation cycles. MSD platform also gives the added advantage of low background noise as only labels near the electrode surface can be detected. Thus, MSD can provide great assay sensitivity (pg/mL).¹⁰

Fig. 2 MSD technology

Based on binding properties of the analyte and the assay reagents used, LBA can quantify total antibody concentration (antibody with or without drugs) or conjugated antibody concentration (antibody with one or more drugs attached).

Plotting a concentration vs. time profile graph is used to calculate PK parameters for the therapeutic under study and form a potential relationship with its safety and efficacy.

Fig. 3:  Example of a mouse PK study of an ADC using ELISA assay

As easy as this may sound, there are many factors to consider when choosing LBA as your preferred bioanalytical assay. Being an immunoreactive assay, minor possible changes during reagent manufacturing, can potentially affect their selectivity and binding efficiency.¹⁵ Thus, devising the bioanalytical technique carefully is extremely crucial to obtain reproduceable results. Nevertheless, limitations of LBA can generally be overcome when the capture and the detection antibodies are finely selected.⁵ Having expertise in this area makes it possible to strategically plan the assay in such a manner that the complications and limitations of this approach can be overcome and reproduceable and accurate results can be obtained.

Liquid Chromatography-Mass Spectrometry (LC-MS) Assay

Liquid chromatography mass spectrometry (LC-MS) has been routinely used to characterize and quantify ADCs in the drug discovery process.¹ With recent advancements, the use of LC-MS for bioanalysis has been massive. Compared to the traditional methods, LC-MS can be broadly used to analyze small molecules, intact proteins, digested proteins and specific domains of proteins. This gives LC-MS an edge over other analytical tools. LC-MS thus became indispensable for use in ADC characterization.¹⁶ In ADC research, LC-MS offers the added advantage of analysis of antibody concentration, DAR and conjugated payload in contrast to ELISA that can only analyze the presence of linker-payload on the antibody without providing any quantifiable data of the same. LC-MS can also help determine any modification of the linker-payload when attached to the antibody. It can also provide valuable information regarding biotransformation, catabolism, plasma/tissue exposure, target engagement for better prediction of PK/PD, and successful designing of therapeutics.

Fig. 4:  Example of conjugated payload (total ADC) using an LC-MS method

LC-MS however comes with a high entry and maintenance cost and the requirement of expertise in mass spectrometry.

Table 1: Application of MS on characterization and quantification of ADC¹

Fig. 5: LC-MS Unit at NJ Bio

Table 2: Comparison of LBA and LC-MS¹

LBA and LC-MS Hybrid Approach

Providing the best of two worlds in the field of bioanalytical methods is the ligand-binding and LC–MS hybrid approach which combines the strength of both these remarkable technologies. This combination has improved selectivity and sensitivity of LC–MS applications in analyzing and characterizing ADCs. Even if not strictly required for analysis, using ligand binding as a first step, followed by LC-MS is a powerful bioanalytical approach. The hybrid approach allows capture of the sample and its concentration and provides excellent sensitivity. Further, depending on the instrument used and the bioanalytic technique, an accurate DAR can be obtained based on the conjugated payload and antibody concentration from different species and tissues.

Fig. 6: Affinity Capture for Biotransformation assessment of ADC

Hybrid LBA – LC-MS platform combines the following three promising features:

1. Immuno-affinity enrichment of analyte (LBA)
2. Separation of complex mixtures using LC
3. Detection of analyte(s) using MS

This hybrid approach can also be used for in vitro assays. It ensures highly selective and specific identification as well as accurate and precise quantification for bioanalysis of ADCs. Following initial development and subsequent growth, the hybrid LBA/LC-MS method has showed promising results for PK assays.⁷

LBA – LC-MS Hybrid Assay can work to the advantage of a program in the following manner:
✓ Sensitive quantitative analysis for complex biotherapeutics
✓ DAR (Drug-antibody-ratio) sensitive
✓ Sensitive to biotransformation
✓ Could be multiplexed

Fig. 7: Workflow for bioanalysis of ADCs using hybrid LBA – LC-MS approach¹⁵

Frequently Asked Questions

What are bioanalytical services?

Bioanalytical services are services that are provided by contract research organizations having expertise, instrumentation and technology to analyze complex biomolecules and provide reliable and accurate data to researchers involved in drug discovery and development programs.

Which bioanalytical studies are required for ADCs?

Bioanalytical studies of ADCs are difficult as they need to consider the complexity and heterogeneity of ADCs. Generally, ELISA, LC-MS or hybrid LC-MS approach is used to study ADCs and other bioconjugates. Employing the most appropriate bioanalytical strategy based on the study compound can ensure that accurate and reproduceable data is obtained.

Why is the data from PK/ PD analysis important during ADC development?

Pharmacokinetics (PK) studies involve bioanalysis of an ADC molecule once it enters the human body. It enables understanding the ADC stability and interaction with the body while helping determine its half-life in serum and plasma. On the other hand, Pharmacodynamics (PD) studies the effect of drug on the body and its chemical interactions. PK/PD data is thus crucial to explain the dose-response relationship and helps researchers maximize the therapeutic index of an ADC.

Which is the optimal platform to assess and obtain reproduceable PK analysis data for ADCs?

Several analytical strategies and technologies can be used for PK analysis and assessing PD profiles. Various ligand binding assays, LC-MS and LBA/ LC-MS hybrid platforms are generally the gold standard for obtaining reproduceable bioanalytical data. NJ Bio helps you select the most appropriate platform based on sensitivity and other crucial parameters essential for your program. Our extensive knowledge in bioanalytical studies and bioconjugations enables us to devise the best strategy for you and provide precise data in crucial timelines required for your drug discovery program

What is the most common method of bioassay?

Enzyme-linked immunosorbent assays (ELISA) are one of the most common methods for ligand binding assays. At NJ Bio, we provide both standard and MSD-ELISA platforms for increased sensitivity. Our bioanalytical service team is well adept with all the bioassay strategies and can provide the best analytical support for your program.