Affinity profiling of monoclonal antibody and antibody-drug-conjugate preparations by coupled liquid chromatography-surface plasmon resonance biosensing

Abstract

Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are highly potent biopharmaceuticals designed for targeted cancer therapies. mAbs and ADCs can undergo modifications during production and storage which may affect binding to target receptors, potentially altering drug efficacy. In this work, liquid chromatography was coupled online to surface plasmon resonance (LC-SPR) to allow label-free affinity evaluation of mAb and ADC sample constituents (size and charge variants), under near-native conditions. Trastuzumab and its ADC trastuzumab emtansine (T-DM1) were used as a test sample and were analyzed by aqueous size-exclusion chromatography (SEC)-SPR before and after exposure to aggregate-inducing conditions. SEC-SPR allowed separation of the formed aggregates and measurement of their affinity towards the ligand-binding domain of the human epidermal growth factor receptor 2 (HER2) receptor immobilized on the surface of the SPR sensor chip. The monomer and aggregates of the mAb and ADC were shown to have similar antigen affinity. Conjugation of drugs to trastuzumab appeared to accelerate the aggregate formation. In addition, cation-exchange chromatography (CEX) was coupled to SPR enabling monitoring the maximum ligand-analyte binding capacity (R_max) of individual charge variants present in mAbs. Deamidated species and lysine variants in trastuzumab sample were separated but did not show different binding affinities to the immobilized HER2-binding domain. In order to allow protein variant assignment, parallel MS detection was added to the LC-SPR setup using a column effluent split. The feasibility of the LC-MS/SPR system was demonstrated by analysis of trastuzumab and T-DM1 providing information on antibody glycoforms and/or determination of the drug-to-antibody ratio (DAR), while simultaneously monitoring binding of eluting species to HER2. Graphical abstract ᅟ.

Keywords: Biopharmaceutical antibody-drug conjugates; Cation exchange chromatography; Online coupling; Size exclusion chromatography; Surface plasmon resonance; Trastuzumab.

Graphical abstract

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Fig. 1

Schematic representation of the online LC-SPR system. LC separation of biomacromolecules by, e.g., SEC or CEX is followed by successive online UV absorbance and SPR detection. An external switch valve 1 and pump is used for optional heart-cutting analysis. The SPR sensor is regenerated in between the affinity analysis, employing external switch valve 2 and syringe pump. Optional parallel MS detection can be performed using a post-column split

Fig. 2

SEC-UV-SPR of (1 mg/mL) trastuzumab (i) and T-DM1 (ii). (a) UV chromatogram. (b) SPR sensorgram

Fig. 3

SEC-UV-SPR of stressed T-DM1 sample (1 mg/mL). a UV chromatogram, b SPR sensorgram, and c SPR sensorgrams obtained after heart-cutting of the HMW bands (19–26 min) (red) and the main peak (28–29 min) (black)

Fig. 4

SPR affinity curves of monomer (red) and aggregate (black) species observed during LC-UV-SPR of samples of stressed a trastuzumab and b T-DM1. For stressed trastuzumab samples, total sample concentrations of 10–60 μg/mL and 500–3000 μg/mL were injected for analysis of monomer and aggregate peaks, respectively. For stressed T-DM1 samples, total sample concentrations of 50–300 μg/mL and 500–3000 μg/mL were injected for analysis of monomer and aggregate peaks, respectively

Fig. 5

CEX-UV-SPR of trastuzumab. a UV chromatogram. b SPR sensorgram. c First derivative of SPR sensorgram

Fig 6

Heart-cutting CEX-SPR of trastuzumab (1–20 mg/mL injected in triplicate). Affinity curves of the CEX-separated heart-cutted peaks shown in the top UV chromatogram

Fig 7

The SEC-SPR-MS analysis of (a) trastuzumab and (b) T-DM1 sample. A single run provides (i) UV chromatogram, (ii) SPR sensorgram, (iii) the raw, and (iv) deconvoluted mass spectrum