What are antibody drug conjugates (ADC) characterization?

ADCs are a novel and impressive clinical development. ADC characterization allows scientists and drug manufacturers to understand the physical and chemical properties of ADCs, improving their creation methods and development. 

What is ADC in immunology?

Antibody-drug conjugates (ADCs) are biopharmaceutical drug products designed for the treatment of various forms of cancer. The molecular buildup of an ADC is complex, consisting of a monoclonal antibody (mab) bound by a covalent bond to a potent cytotoxic agent. 

ADC drug distribution through the body can be targeted directly at the area of treatment. ADCs single out diseased cells and terminate them while avoiding causing harm to healthy cells. This feature gives ADCs the potential to be more effective and safer than other chemotherapy methods.

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What is ADC drug development?

ADC drug development is the use of the latest advancements in medical science to create novel therapeutic antibodies. The molecular structure of ADCs takes advantage of the lethal potential of cytotoxic drugs and the accurate delivery method provided by monoclonal antibodies. 

How are antibody-drug conjugates made?

Antibody-drug conjugates are made by covalently linking an antibody to a cytotoxic agent. The chosen antibody is designed to track and attach itself to antigens found in tumor cells. Once together, a chemical reaction will take place and the tumor cell will absorb the cytotoxin, killing it from the inside.

How do you characterize antibody-drug conjugates?

The characterization of antibody-drug conjugates is performed by researching the drug’s attributes. This is achieved with the use of high-resolution tools and intricate techniques. 

The complexities of biological manufacturing make the measurement of the quality of a biological drug harder than its traditional counterparts. While conventional analytical methods can be used to gauge core properties like identity and molecular mass, high-resolution analytical techniques are needed to answer questions about the compound’s charge, size, glycan structure, or post-translational modifications.

Antibody-drug conjugate characterization can help gather very valuable data about monoclonal antibodies. For instance, the drug-antibody ratio (DAR), or the average number of drugs attached to antibodies, can only be acquired via characterization. The DAR value is fundamental to understanding the potential efficacy of a drug, as well as the likelihood of it causing side effects.

Other essential information that can be gathered by characterization includes the potential of the antibodies to bind to their targets and the capacity of the drug for complementing the effects of the cytotoxic agent by sparking an immune system response. 

Investing in powerful analytics during the early stages of drug development can help biologic developers mitigate risks frequently encountered during the life cycle of a pharmaceutical product. Moreover, proper characterization eliminates many unknowns and prevents the development of future issues. 

What are monoclonal antibodies?

Methods for characterization of antibody-drug conjugates

High-Performance Liquid Chromatography (HPLC)

Previously known as high-pressure liquid chromatography, HPLC is an analytical chemistry technique that can be used for the identification, separation, and quantification of antibody-drug conjugates. 

HPLC is performed by using pumps to pass a liquid solvent that contains the sample mixture through a column filled with absorbent material. Once different flow rates are observed, these can be used to recognize particular components in the mixture. In addition, flowing through the column causes the separation of the chemicals constituting the mixture.

An HPLC system is commonly composed of a degasser, a sampler, pumps, and a detector. The role of the sampler is to bring the mixture into the mobile phase stream, which will guide it towards the absorbent column. A signal proportional to the amount of sample content passing through the column is generated by the detector. A quantitative analysis of the sample components is performed with this data.

Data analysis and operational control of the HPLC mechanism are achieved thanks to a digital microprocessor and user software. Some HPLC machines can generate a composition gradient in the mobile phase by mixing multiple solvents together. Commonly used detectors include UV/Vis and photodiode arrays (PDA).

The operational pressure of HPLC is much higher than that of traditional liquid chromatography, which is meant to use the force of gravity to pass the mixture through the absorbent column. HPLC columns are also constituted of smaller absorbent particles, giving them superior resolving power. When separating mixtures with HPLC, the ability to distinguish between different compounds is accentuated. 

Reversed-phase chromatography (RPC)

Also known as hydrophobic chromatography, this method uses a hydrophobic stationary phase. This technique gives particles a stronger affinity for less polar or hydrophobic compounds. Hydrophilic molecules are passed through the column and eluted first, separating them from their hydrophobic counterparts. The more hydrophobic the molecule, the more solvent will be needed in order to elute it. 

Ion-exchange chromatography (IEX)

Ion chromatography uses the surface charge of molecules to separate them. This method can successfully characterize almost any type of charged molecule, including amino acids, large proteins, and small nucleotides. 

Two types of ion-exchange chromatography exist: 

• Cation exchange chromatography: This method is used for the study of positively charged particles. The stationary face is negatively charged, causing positively charged molecules to be attracted to it. 
• Anion exchange chromatography: This approach works in the exact opposite way of cation-exchange chromatography. In this case, negative ions are attracted by a positive stationary face. Scientists use this method for protein purification, water analysis, and quality control.

An advantage of IEX over other chromatography methods is that only one interaction is necessary during the separation procedure. Furthermore, IEX improves the predictability of elution patterns. During cation exchange chromatography, cations will elude last, while negatively charged molecules will elude first in anion exchange chromatography.

Size exclusion chromatography (SEC)

Sometimes referred to as molecular sieve chromatography, this method separates molecules based on their size or weight. Naturally, this approach is more useful when working with large molecules or macromolecular structures like proteins and certain polymers. 

When using this method, the biological activity of the separated particles can be preserved. Furthermore, the filtration process will not be hindered by applying various solutions at the same time. Because solutes do not interact with the stationary phase, this method doesn’t cause any sample loss. 

Affinity chromatography

By using a highly specific macromolecular binding interaction, this method can extrude a biomolecular particle from a mixture. Depending on the type of biomolecule being worked on, the nature of the binding interaction changes. Compared to other chromatographic methods, affinity chromatography offers higher selectivity, resolution, and capacity in many protein purification procedures. 

Affinity chromatography takes advantage of the interaction between an analyte and the molecule it is bound to. This means that this method doesn’t necessarily require knowledge of molecular weight, charge, hydrophobic tendencies, or other physical properties of the analyte. However, knowledge of these factors benefits the procedure. In the case of ADCs, the molecules that facilitate chromatic affinity are the antibody and the antigen. 

Capillary Electrophoresis (CE)

Capillary electrophoresis (CE) is an analytical separation technique that is based on the different displacement speed of the different proteins within a liquid medium, contained in a capillary tube, when subjected to the action of an electric field.

Capillary electrophoresis (CE-SDS) can be used to analyzed reduced or non-reduced species of the ADC to provide quantitative information on purity, impurities, or degradation products. Imaged Capillary IsoElectric Focusing (iCIEF) is used to separate charged variants to understand the changes in such species between product lots upon release, or changes upon stability.  

Some of the main characteristics of this technique are:

• High speed of analysis: The separation times are generally quick (several minutes).
• High separation efficiency: Efficiencies around 10,000-100,000 theoretical plates per meter of column are usually obtained in protein analyses, depending on the protein to be analyzed and the separation conditions.
• Small sample volumes: A few (10 – 50) nanoliters of sample are used in each separation.
• Analysis automation: Capillary electrophoresis instruments allow analyses to be carried out without the constant attention of the operator.

Mass Spectrometry (MS)

Mass spectrometry is an analytical tool that is used for the measurement of the mass-to-charge ratio of ions. Besides being used to characterize ADCs and other molecular structures, mass spectrometry also has applications in other fields. Mass spectrometry can be performed on solid, liquid, or gaseous materials.

There are three main components in a mass spectrometer: an ion source, a mass analyzer, and a detector. The ion source is responsible for converting a portion of a sample into ions. Various methods have been created for this procedure and different ionizing mechanisms work better on different materials. The mass analyzer sorts the ions, which are then sent into the detector.

A mass spectrometry procedure consists of a sample being ionized. This can be done, for example, by bombarding the mixture with a beam of electrons. An electron bombardment can cause the breakup of some of the sample’s molecules into positive ions. Alternatively, the whole sample may become positively charged without being fragmented. 

The resulting ionized material can then be separated according to its mass-to-charge ratio. Methods used for this procedure include inducing their acceleration or subjecting them to electricity. Ions can also be separated by a magnetic field, as ions with the same mass-to-charge ratio will be affected by deflection equally. Once ions have been separated, they are moved into the detector.

An electron multiplier or a similar machine is commonly used to detect ions. The results are displayed as spectra of the signal intensity in relation to the mass-to-charge ratio of the ions. The contents of the sample can then be identified by correlating them to known masses or by recognizing characteristic patterns in their fragmentation.  

Liquid Chromatography-Mass Spectrometry (LC-MS)

This analytical chemistry technique uses the competencies of liquid chromatography coupled with those of mass spectrometry. This kind of approach is popular among chemical analysts due to the enhanced synergy provided by the individual characteristics of each technique. 

Liquid chromatography causes the physical separation of mixtures into multiple components and mass spectrometry can provide spectral information to help identify every separated component. The devices that perform these procedures are fundamentally incompatible, making an interface necessary for their cooperation. Extensively used LC-MS interfaces include electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI). 

Besides being extremely useful in the field of biotechnology, LC-MS is also used in a wide variety of sectors including food processing, environment monitoring, and the manufacturing of agrochemical and cosmetic products.

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