(W1030-09-51) Optimizing ADC Separations: Advantages of Bio-Compatible Versus Stainless Steel UHPLC Systems

Purpose: Protein biopharmaceuticals such as recombinant monoclonal antibodies (mAbs) and Antibody Drug conjugates (ADCs) are currently in widespread use for the treatment of various life-threatening diseases including cancer and autoimmune diseases ¹. In contrast to small molecule drugs, mAbs are large (150 kDa) and heterogeneous and ADCs are monoclonal antibodies (mAbs) to which a cytotoxic small molecule drug is chemically linked.² Compared to their corresponding antibodies, ADC structure is more complex and heterogeneous. As a result of the biosynthetic process and subsequent manufacturing and storage, hundreds of different variants may co-exist, differing in aspects such as N-glycosylation, N- and C-terminal processing, deamidation, oxidation, amino acid sequence, and disulfide bridges. Due to the structural complexity, a variety of tests are required to monitor Critical Quality Attributes (CQAs) throughout the development and manufacturing process, hence, unraveling this complexity represents an analytical challenge.  Many of these analytical tests require the use of buffer salts in the mobile phase, some at very high concentrations.

Methods: Hydrophobic interaction chromatography (HIC) is a method of performing separations of biomolecules with increasing hydrophobicity. This makes it a technique that is well suited to the analysis of ADCs, as these complex molecules are often more hydrophobic than the unmodified antibody. The technique can provide high resolution capable of separating species with minor differences in hydrophobicity, including those ADCs that have differing of drug-to-antibody ratio (DAR). Cysteine-linked ADCs are generated after the reduction of the interchain disulfides, resulting in free sulfhydryl groups that can be conjugated to specific maleimide linkers. The number of free sulfhydryl groups limits the number of defined positions for the drug to be conjugated, resulting in a mixture of zero (D0), two (D2), four (D4), six (D6), and eight (D8) drugs per antibody.³ A benefit of HIC comes from the mild conditions used for elution, which ensures that the biomolecule remains in an undenatured state. This can be advantageous for the analysis of cysteine-linked ADCs.⁴ Due to variations in the manufacturing process, size variants may occur and need to be well characterized. Immunogenic responses and differences in pharmacokinetics or potency could arise due to the existence of these species in the drug product. SEC is a standard technique to analyze these size variants from mAbs and ADCs. SEC analysis of ADCs may be challenging due to the presence of hydrophobic payloads⁵.

Results: Chromatographic data from a Bio-compatible UHPLC system shows improved resolution, reproducibility, and precision over a stainless-steel system.  The resulting DAR calculation of ADCs using HIC has less variation when a bio-compatible system is utilized.

Conclusion: For this reason, the Infinity II Bio LC portfolio is optimally suited to the conditions used in bio chromatography, with the high concentrations of corrosive salts typically used in HIC, to avoid potential corrosive damage to the system.

References: 1. 3. Walsh, G. Biopharmaceutical benchmarks 2018. Nat. Biotechnol. 2018, 36, 1136–1145.
2. McCombs, J. R.; Owen, S. C. Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry. J. Amer. Assoc. Pharm. Sci. 2015, 17(2), 339–51.
3. Schneider S., Agilent Technologies, Convenient and Reliable Analysis of Antibody Drug Conjugates, Application Note 2021.
4. Coffey A., Agilent Technologies, An AdvanceBio HIC Column for Drug to-Antibody Ratio (DAR) Analysis of Antibody Drug Conjugates (ADCs), Application Note 2020.
5. Qin V., Agilent Technologies, Size Exclusion Chromatography Analysis of a mAb and Antibody Drug Conjugate, Application Note 2019.

6 Replicate injections of high salt HIC separation of Sigma ADC Mimic on 1290 Bio Flex System



6 Replicate injections of high salt HIC separation of Sigma ADC Mimic on 1290 SS Flex System



DAR Calculations from both 1290 Bio and Stainless Steel Systems