See also Mass Spectrometry in Diagnostic Techniques

Companies: Refeyn (mass photometry)

Definitions

Amide: includes a derivative form of carboxylic acid in which the hydroxyl group has been replaced by an amine or ammonia. Due to the existence of strong electronegative atoms, oxygen adn nitrogen, next to carbon, dipole moment is produced and the molcule with amide group presents polarity or hydrophilicity.  

Analytics for Antibody Drug Conjugates (ADCs): 

Analytics for Drug to Antibody Ration (DAR):

A variety of techniques have been used to emasure the DAR or payload that can be delviered to a tumor cell using an ADC. The simplest techniqued relies on a UV/VIS spectroscopic analysis of the ADC. Depending on teh chemisty used to attach the drug to the antibdoy, alternative methods for determining the DAR such as HIC can be used. Reduction of itner-chain disulfides to produce free sulfydryl groups allows conjugation at specific residues using maleimide-containing linekrs and genereates conjugates with a mixture of 0, 2, 4 and 6-8 drugs per antibody. As a result of the significantly reduced heterogeneity of this linkage chemistry relative to lysine-linked conjugates, the mixture has proven to be amenable to anaysis to HIC. HIC is performed under non-denaturing conditions at neutral pH with a gradient from high salt to low sat and often incldues a low concentraiton of an organic modifier in the low salt mobile phages to improve the elution of the mAb laoded with hydrophobic drugs. As a result, even though some of the itner-chain disulfides are disrupted due to the conjugation reaction, the combination of covalent binding and strong non-covalent forces between the chains are sufficient to keep the mAb intact during analysis. Thus, each peak observed correspodns to an intact mAb species with an increasing nubmer of bound drugs. (Jacobson, “Analytical methods for physicochemical characterization of antibody drug conjugates” mAbs 3:2, 161-172, 2011).

Size Variant Analysis of Conjugates:

The presence of HMW variants (i.e., aggregates) in an ADC has the potentail to elicit the production of the anti-therapeutic antibody (ATA) response. Many of the drugs that are conjugated to antibodies to produce ADCs are relatively hydrophobic and can increase the likelihood of aggregate formation during manufacturing and storage. Most published methdos of ADCs use the same size-exclusion chromatography analysis that are used for teh parent monoclonal antibodies. SEC is perforemd under non-denaturing conditions. CD-SDS has emrged as a preferred technique that offers clear advantages over SDS-PAGE in terms of speed, reroducibiity, resolution, robustness and ease of automation for non-conjugated antibodies. One of the earliest applications to ADCs was the analysis of a BR96-DOX conjugated formed by linkage of drug to inter-cahin suolfhydryls. The addition of SDS to the sampel dissocaites antibody chains taht are not covalently linked by intact disulfides due to partial reeduction and reaciton with linekr-drug, and provdies information about the sites of conjugation along with a comparison to traditional SDS-PAGE. (Jacobson, “Analytical methods for physicochemical characterization of antibody drug conjugates” mAbs 3:2, 161-172, 2011).

Charge-Based Separations:

Analysis of charge variants such as those resulting from deamidation or formation of N-temrinal pyroglutamates may be an important quality attribute of a mAb, expecially if the changes affect binding or biologcial activity. Depending on the characteristics of the drug, the linker and the conjguation site, methods that can be applied to the aprent mAb may not be applicable to the ADC or may give significantly different informaiton. Attachmetn of an uncharged linker and drug thorugh lysine residues decreases the net positive charge by one for each bound drug-liner. In this case, spearation based on charge, such as ion exchange chromatography IEC) or iso-electric focusing (IEF) resutls in profiels taht characterize the drug leoad, rather than proving information about the udnerslying mAb. (Jacobson, “Analytical methods for physicochemical characterization of antibody drug conjugates” mAbs 3:2, 161-172, 2011).

Analysis of Unconjugated Drug:

Another improtant quality attribute shared by all aDCs is the quantity of free (unconjguated) drug, which poses concerns for differential toxicity and potentail safety issues. ELISA, capillary electrophoresis (CD) and high performance liquid chromatography (HPLC) are methods which ahve been sued to determine free drug levels in various ADCs. (Jacobson, “Analytical methods for physicochemical characterization of antibody drug conjugates” mAbs 3:2, 161-172, 2011).