Characterizing Antibody-Drug Conjugates Using Micro Pillar Array Columns Combined with Mass Spectrometry (µpac -MS)

Transcription

1 Characterizing Antibody-Drug Conjugates Using Micro Pillar Array Columns Combined with Mass Spectrometry (µpac -MS) Abstract MARKET Pharma, Biotech KEYWORDS The use of micro pillar array columns (µpac ) for characterizing antibody-drug-conjugates is presented. (ado-trastuzumab emtansine) and, for comparison, Herceptin (trastuzumab) tryptic digests were analyzed on a cm µpac C8 column and peaks eluting were detected by ultraviolet (UV) spectroscopy and high-resolution mass spectrometry (MS). µpac -MS, peptide mapping, antibody-drug-conjugates, Trypsin Koen Sandra, Jonathan Vandenbussche, Isabel Vandenheede, Bo Claerebout, Jeff Op de Beeck, Paul Jacobs, ADYEKHK () Wim De Malsche, EAKVQWK () Gert Desmet, Pat Sandra VSNKALPAPIEK (). QAPGKGLEWVAR (). Research Institute for Chromatography (RIC), Kortrijk, Belgium, Ghent, Introduction Belgium Vrije Universiteit Brussel, Brussels, Belgium The successes of monoclonal antibodies (mabs) as therapeutics have triggered the development of various next generation formats. In oncology, antibody-drug conjugates (ADCs) are particularly promising, since they synergistically combine a specific mab linked to a biologically active cytotoxic drug via a stable linker. The promise of ADCs is that highly toxic drugs can selectively be delivered to tumor cells thereby substantially lowering side effects as typically experienced with classical chemotherapy. Currently two ADCs are marketed, namely brentuximab vedotin (Adcetris ) and ado-trastuzumab emtansine () and over are in clinical trials. From a structural point of view, ADCs possess an unsurpassed complexity since the heterogeneity of the initial antibody is

2 superimposed with the variability associated with the conjugation strategy. Conjugation typically takes place on the amino groups of lysine residues or on the sulphydryl groups of interchain cysteine residues as is the case in, respectively, ado-trastuzumab emtansine and brentuximab vedotin. With 8- lysine and only 8 interchain cysteine residues available, lysine conjugation yields a more heterogeneous mixture of species compared to cysteine conjugation this despite the similar overall level of drugs incorporated per antibody (between and 8 with an average of -). This application note demonstrates how micro pillar array columns (µpac ) in combination with ultraviolet (UV) spectroscopy and high-resolution mass spectrometry (MS) comes in as a very powerful tool to assess drug conjugation sites. The inherent high permeability and low on-column dispersion obtained by the perfect order of the separation bed makes µpac based chromatography unique in its kind and offers several advantages compared to conventional column technologies (packed beds and monoliths). The peak dispersion originating from heterogeneous flow paths in the separation bed is eliminated (no A-term contributions) and therefore components remain much more concentrated (sharp peaks) during separation. The freestanding nature of the pillars also leads to much lower backpressure allowing the use of very long columns. These properties result in excellent chromatographic performance with high-resolution and high sensitivity. Materials and methods Materials Water and acetonitrile were purchased from Biosolve (Valkenswaard, The Netherlands). Trifluoroacetic acid, dithiothreitol (DTT) and -iodoacetamide (IAA) were from Sigma-Aldrich (St. Louis, MO, USA). Tris-HCl ph 7. was purchased as a M solution from Thermo Fisher Scientific (Waltham, MA, USA). Porcine sequencing grade modified trypsin was acquired from Promega (Madison, WI, USA) and Rapigest from Waters (Milford, MA, USA). Herceptin and were obtained from Roche (Basel, Switzerland). Sample preparation To a volume corresponding to µg of protein, µl of.% Rapigest in mm Tris-HCl ph 7. was added followed by the addition of mm Tris-HCl ph 8 to a final volume of 9. µl. The sample was subsequently reduced at 6 C for min by the addition of mm DTT (. µl of mm DTT in mm Tris-HCl) and alkylated at 7 C for h by adding mm IAA ( µl of mm IAA in mm Tris-HCl). Digestion proceeded for 6 h at 7 C using trypsin as protease added at an enzyme to substrate ratio of / (w/w). Lyophilized trypsin ( µg) dissolved in mm Tris-HCl ( µl) was added in a volume of µl giving rise to a final sample volume of µl. LC-MS An Ultimate RSLCnano system (Thermo Fisher Scientific) was used for LC-UV-MS measurements. Tryptic digests were analyzed on a cm C8 µpac column () at C. Elution was carried out with a linear gradient of (A).% TFA in HO and (B).% TFA in ACN/HO (8/ v:v), from % B to 7% B in min. The flow rate was nl/min. An injection program allowed the introduction of nl sample in between two plugs of mobile phase A. Loop size was µl and samples were kept at C in the autosampler tray while waiting for injection. UV detection was carried out at nm using a nl detection cell.

3 High-resolution accurate mass measurements were obtained on an Agilent 6 Q-TOF mass spectrometer (Agilent Technologies) equipped with a dual nano ESI source operated in positive electrospray ionization mode. The µpac column was connected via a µm ID/8 µm OD fused silica capillary to a PicoTip emitter (8 µm tip ID New Objective, Woburn, MA, USA) via a true zero dead volume conductive union (UH-6 stainless steel adapter from IDEX, Washington, WA, USA). To connect the PicoTip emitter to the union, a F- blind fitting equipped with a M- perfluoroelastomer conductive ferrule was used (IDEX). Capillary voltage was set at +. kv, drying gas flow rate and temperature were set at L/min and C, fragmentor voltage at 7 V. The TOF was calibrated on a daily basis and subsequently operated at high accuracy (< ppm) without utilizing reference masses. Data were collected in centroid mode at a rate of spectrum per s in the extended dynamic range mode ( GHz) offering a resolution of 9, at m/z All-ions MS/MS experiments were performed at a collision energy of ev. LC data were acquired in Chromeleon (Thermo Fisher Scientific) and MS data in MassHunter (Agilent Technologies). Data analysis was performed in MassHunter Qualitative Analysis with BioConfirm add-on. Results and discussion The anatomy of (ado-trastuzumab emtansine) is shown in Figure. It combines the anti-her antibody trastuzumab (Herceptin ) with the cytotoxic microtubule-inhibiting maytansine derivative DM conjugated to lysine residues via a non-reducible thioether linker. DM is conjugated with an average of. drugs per antibody. Figures and show, respectively, the UV and MS total compound chromatograms of a Herceptin and tryptic digest. Since Herceptin and have the same amino acid sequence, the majority of the peptide map is identical. Differentiating peaks, corresponding to the DM conjugated peptides, are nevertheless observed and are located in the late eluting part of the chromatogram. Indeed, the conjugation of DM makes the peptides more hydrophobic explaining the elution behavior. Upon collision-induced dissociation (CID), DM conjugated peptides, give rise to specific fragments originating from the cytotoxic drug, e.g. at m/z 7.6. This ion can be used to selectively recognize DM conjugated peptides in the µpac -MS chromatogram. For that one can operate the Q-TOF MS system in the all-ions MS/MS mode which means that the quadrupole is operated in the RF only mode thereby transferring all peptides to the collision cell where CID takes place. As illustrated in Figure, when extracting from the data the specific fragment ion at m/z 7.6 at high mass accuracy, all conjugated peptides are revealed in the chromatogram of compared to Herceptin. The latter chromatogram is virtually empty illustrating the selectivity that is offered by this all-ions MS/MS functionality. Figure shows the extracted ion chromatograms associated with a selection of identified conjugated peptides. A striking observation is the appearance of isomeric DM conjugated peptides which can be explained by the existence of two stereochemical configurations (diastereomers) of the antibody-drug linkage through maleimide.

4 Conclusion The use of the µpac column in characterizing ADCs has been demonstrated. The resolving power offered by the µpac column allows an in-depth study of conjugation sites and accommodates the separation of isomeric conjugated peptides. In combination with all-ions MS/MS, conjugates peptides can selectively be recognized facilitating data interpretation. Figure : Anatomy of. DM (Maytansine derivative) Thioether linker [N-maleimidomethyl] cyclohexane--carboxylate (MCC) n =. GF GF Trastuzumab

5 9. mau Herceptin..! Conjugated peptides.. +. min Figure : μpac -UV nm peptide map of Herceptin and. x x Herceptin Conjugated peptides Figure : μpac -MS peptide map of Herceptin and.

6 6 x Herceptin. l -----K----- x Figure : All-ions MS/MS chromatograms of the Herceptin and tryptic digest. The ion at m/z 7.6 was extracted at high mass accuracy. x ADYEKHK () x EAKVQWK () x. x. VSNKALPAPIEK () QAPGKGLEWVAR () "#$ Figure : Extracted ion chromatograms of selected peptides showing the appearance of isomeric conjugated peptides. The location of the conjugation site in the mab structure is shown as well.

7 7 µpac driven separations Better by Design Conventionally LC columns are fabricated by stacking (packed beds) or depositing (monoliths) material into a capillary. µpac technology (micro Pillar Array Column) is unique in its kind as it is built upon the precise micromachining of designed chromatographic separation beds into silicon. This approach brings along three crucial and unique characteristics: Perfect Order. µpac beds are designed with a high degree of order, eliminating heterogeneous flow paths otherwise present in conventional columns (so called Eddy dispersion). Flow through µpac columns adds very little dispersion to the overall separation. As a result, peaks remain sharper and sensitivity is increased. High Permeability. µpac s operate at moderate pressures, typically lower than bar. Separation channels with exceptional length ( cm to cm) are therefore possible. These are folded onto a small footprint by a interconnecting concatenating bed segments. Solid Backbone. The micromachined backbone of the separation bed forms a rigid structure that is not influenced by pressure. There are no obstructions by touching surfaces, and there is no risk for perturbations by pressure fluctuations. Technologiepark-Zwijnaarde B-9 Ghent (Zwijnaarde) Belgium info@pharmafluidics.com Katrien Vanhonacker, MSc, MBA VP Business Development & Sales katrien.vanhonacker@pharmafluidics.com M: