High-Resolution Capillary Zone Electrophoresis with Mass

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High-Resolution CZE-MS Peptide Mapping of Therapeutic Proteins: Peptide Recovery and PTM Analysis in Monoclonal Antibodies and Antibody-Drug Conjugates Oluwatosin O. Dada, Yimeng Zhao, Nomalie Jaya, and Oscar Salas-Solano Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03643 • Publication Date (Web): 06 Oct 2017 Downloaded from http://pubs.acs.org on October 7, 2017

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Analytical Chemistry

High-Resolution CZE-MS Peptide Mapping of Therapeutic Proteins: Peptide Recovery and PTM Analysis in Monoclonal Antibodies and Antibody-Drug Conjugates Oluwatosin O. Dada!, Yimeng Zhao#, Nomalie Jaya!, and Oscar Salas-Solano! Department of Analytical Sciences, Seattle Genetics, Inc. 21823 30th Drive SE, Bothell, WA 98021, USA

Abstract RPLC-MS peptide mapping is routinely used for interrogating molecular and structural attributes such as amino acid composition, sequence variants, and post translational modifications (PTMs) in antibody derived therapeutics. RPLC has some limitations that often impact the analysis of certain peptides including large hydrophobic peptides, hydrophilic di/tri-peptides and glycopeptides. Capillary zone electrophoresis with mass spectrometry (CZE-MS) has great potential for peptide mapping due to high efficiency and outstanding sensitivity. In this report we demonstrate the utility of CZE-MS as an orthogonal and complementary technique to RPLC-MS for peptide mapping analyses of antibody-drug conjugates (ADCs) and their parent antibodies. This work is based on high-resolution CZE-MS separation recently developed in our group, where mixed aqueous-organic solvent system containing N,N–dimethylacetamide (DMA) or N,N–dimethylformamide (DMF) was used to improve the separation selectivity. The results described here show several advantages of CZE-MS for the analysis of small hydrophilic di/tripeptides, large hydrophobic peptides, glycopeptides, and hydrophobic drug-linked peptides.

!

Department of Analytical Sciences, Seattle Genetics, Inc. 21823 30th Drive SE, Bothell, WA

98021, USA #

Current Address: Weil Cornell Medicine, 1300 York Avenue, NY 10065, USA

1 ACS Paragon Plus Environment


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INTRODUCTION Among antibody-derived therapeutics, antibody-drug conjugates (ADCs) are in the clinical front line for several oncologic diseases.1 An ADC chemical framework is composed of an IgG protein backbone conjugated to a drug warhead with a linker.2,3 ADC’s take advantage of the high binding specificity and favorable pharmacokinetics of the antibody for target delivery of the drug to tumor cells.3 The specificity to target tumor antigen provided by the antibody limits exposure of the healthy cells to the drug warhead. The ability to characterize ADCs at various stages of production and storage is crucial for understanding their safety and efficacy. A widely-used technique for interrogating the primary structure of antibodies and ADCs is peptide mapping. In peptide mapping, proteins are enzymatically digested to smaller peptide fragments. The peptides generated are then analyzed, commonly by a chromatographic separation followed by mass spectrometry.4 Peptide mapping is a powerful tool for understanding the primary structure of a therapeutic protein, including amino acid composition, sequence variants, glycosylation heterogeneity, and post translational modifications (PTMs).5 Reversed phase liquid chromatography with mass spectrometry (RPLC-MS) is an established method for peptide mapping but it has some limitations for achieving 100% primary antibody sequence coverage in certain situations. As a work around, multi-enzyme digestion workflow is often used to improve sequence coverage in RPLC-MS, which requires the use of multiple proteolytic enzymes and increased MS analysis time. The incomplete sequence coverage with RPLC-MS is mainly due to the poor recovery or even loss of certain peptides that are either too hydrophilic or too hydrophobic for the reversed phase column interaction. For instance, poorly retained di/tri-peptides can go through the column undetected in the void volume, and highly hydrophobic peptides that tend to interact strongly with the column may be poorly recovered.6,7 Also, separation of glycopeptides is less efficient due to insufficient difference in their interaction with the column. Separation of glycopeptides can be improved with focused RPLC gradient as was recently reported8 but that approach severely impact the resolution of other peptides. For ADC modalities, utilizing a hydrophobic drug-linker, the conjugated peptide to which the drug warhead is attached, can be difficult to analyze by RPLC-MS due to the hydrophobicity of the drug molecule. Strong adherence of the conjugated peptide to the RPLC column can even cause no elution within the solvent gradient. An orthogonal and complementary technique that can overcome the limitations of RPLC-MS is capillary zone electrophoresis with mass spectrometry (CZE-MS). In CZE-MS analytes are 2 ACS Paragon Plus Environment

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separated based on their charge-to-size ratios in a narrow bore silica capillary filled with a conductive background electrolyte (BGE) with no stationary phase.9 In principle, the charge-tosize separation mechanism, with no hydrophobicity contribution, should permit optimum recovery of all peptides including those that are difficult to analyze by RPLC-MS. There is growing interest in application of CZE-MS for characterizing biologics. CZE-MS has been applied for peptide mapping,7,10 intact mass analysis,11-13 glycoanalysis,14,15 and quantitation of host cell proteins.16,17 CZE-MS peptide mapping has been previously used to characterize an ADC consisting of an IgG1 conjugated to monomethyl auristatin E but with a less resolving electrophoretic separation compared to RPLC-MS.18 In the first part of this work, we reported an optimized CZE-MS separation using mixed aqueous - aprotic dipolar solvent system containing N,N– dimethylacetamide (DMA) or N,N–dimethylformamide (DMF), as the background electrolyte. The DMA modified acetic acid BGE enables separation of peptides with greatly improved resolution.19 Here we used the DMA modified BGE to perform CZE-MS peptide mapping of an ADC that consists of an IgG1 protein conjugated to a highly hydrophobic pyrrolobenzodiazepine (PBD) dimer. The complementarity of CZE-MS to RPLC-MS was demonstrated with efficient separation and excellent recovery of specific peptides that tend to be challenging for RPLC-MS. With a single enzymatic digestion, CZE-MS enables 100% sequence coverage with enhanced recovery for di/tri-peptides, hydrophobic peptides, glycopeptides, and conjugated peptides. In addition, the analysis of common post translation modifications was in agreement with RPLCMS results, proving the reliability of the CZE-MS peptide mapping method.

EXPERIMENTAL SECTION Materials and Reagents All reagents were purchased from Sigma Aldrich (St. Louis, MO) unless stated otherwise. The antibody drug conjugate, ADC1, used in this study consists of a recombinant IgG1 protein conjugated to a PBD dimer with a stable linker on engineered cysteine20 residue. The antibody portion of ADC1 was expressed in Chinese Hamster Ovary (CHO) cells and purified according to established practices.21 3 kDa MWCO filter was purchased from Millipore (Billerica, MA). HPLC grade water, acetonitrile, 2-propanol, and acetic acid were purchased from Fisher



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Scientific (Waltham, MA). Lys-C (Lysyl Endopeptidase) was purchased from Wako (Richmond, VA). Neutral LPA coated capillary was purchased from CMP Scientific (Brooklyn, NY).

Lys C Peptide Digestion Reductive alkylation of ADC1 was carried out under denaturing conditions by heating the samples with DTT in the presence of guanidine-HCl and subsequent alkylation with sodium iodoacetate (IAA). The reduced samples were buffer exchanged into 50 mM ammonium bicarbonate (pH 8) with 3 kDa MWCO filter. The final protein concentration was measured prior to digestion. The digestion was performed at 37oC for 4 hours. in silico digestion of ADC1 primary sequence was performed in GPMAW version 10.0 (Lighthouse data, Odense M, Denmark) with 0 miscleavage restriction.

Reversed Phase LC-MS (RPLC-MS) Approximately 20 µg of digested samples were injected onto a BEH C18 column (2.1 x 150 mm, 1.7 µm, 130 Å) heated to 55 °C. Separation was performed by reversed phase HPLC (Thermo Accela 1250 HPLC) where mobile phase A was composed of 0.1% formic acid (FA) in water. Mobile phase B was composed of 0.08% FA in acetonitrile. Peptides were eluted using a linear gradient to 33% B. After passing through the UV detector, the eluate was directed into an Orbitrap mass spectrometer (Thermo Q-Exactive MS) fitted with an electrospray ionization (ESI) source operating in positive ion mode. The capillary voltage was set at 4.5 kV. Desolvation was achieved at a temperature of 300°C and using sheath gas nitrogen at a flow rate of 10 µL/min. Data dependent MS/MS spectral data were generated from MS survey scans acquired in the m/z range of 200 - 2000 and analyzed using Thermo Xcalibur software. MS analysis was carried out in positive mode at a resolution of 70,000 for MS and 17,500 for MS/MS scans. The RAW data files were processed with Thermo Xcalibur version 2.2 for peptide identification based on accurate mass and MS/MS confirmation.

CZE-MS Analysis


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The condition for CZE-MS analysis is similar to the one reported in another report.19 Sciex CESI 8000 (Sciex, Farmingham, MA) system was coupled to a high mass accuracy/high resolution QExactive Orbitrap mass spectrometer (Thermo Fisher, Waltham, MA) with EMASS II CE-MS interface (CMP Scientific, Brooklyn, NY). The separation capillary was a 95 cm long and 50 µm ID LPA neutral coated capillary (CMP Scientific, Brooklyn, NY) etched at the outlet end. The ESI spray was delivered with a borosilicate glass emitter (0.75 mm ID, 5 cm long, and 30 µm tip) from CMP Scientific. The background electrolyte (BGE) was 20% acetic acid-15% DMA-1% mnitrobenzyl alcohol. The ESI sheath liquid was 0.1% formic acid in 10% methanol. The separation was performed with 3 psi/7s injection (approximately 25 ng of the sample), +30 kV separation voltage, and +2 kV ESI voltage. The MS data acquisition settings are similar to those for RPLC-MS above with the capillary voltage and the sheath gas flow rate set to 0.

Capillary Electrophoresis with Laser-Induced Fluorescence (CE-LIF) for N-Glycan Profile Samples were buffer exchanged into 50 mM succinate pH 5.5 and incubated overnight at 37°C with PNGase F (New England Biolabs, Ipswich, MA). The sample was heated to 95°C for 5 minutes and the precipitated protein was removed by centrifugation. The resulting supernatant was then dried in a vacuum centrifuge before being resuspended and fluorescently labeled with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) (Molecular Probes, Carlsbad, CA) according to the established method.22 Separation of the labeled glycans was achieved by capillary electrophoresis (SCIEX PA-800 Plus) using a N-CHO coated capillary cartridge (65 cm Length x 50 µm inner diameter).22 Detection was achieved using laser-induced fluorescence (LIF) with excitation at 488 nm and emission monitored at 520 nm.

RESULTS AND DISCUSSION ADC1 is derived from a humanized IgG1 antibody composed of two kappa light chains and two gamma heavy chains with cysteine residues engineered into the antibody backbone of each heavy chain.20 Since ADC1 inherits the post-translational heterogeneity and many of the physicochemical properties of the parent antibody, most of the results discussed here also cover the parent antibody primary structure.



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Commonly used enzymes for peptide mapping include trypsin and lysyl endopeptidase C (Lys C). In this work we used Lys C enzyme for digestion. In silico lys C digestion of ADC1 primary sequence generated 13 light chain peptides (L1 – L13) and 30 heavy chain peptides (H1 to H30). The Lys C peptides include several di/tri-peptides L11-[HK], H14b–[PK], H19-[CK], H23– [AK], H10–[VDK], and H18–[EYK] and three large peptides, L3–[L.(56).K], H6–[G.(55).K], and H9–[D.(61).K]. The parenthesized numbers denote the number of amino acids between the first and the last residues. Two of the large peptides contain CDR residues that are critical for target antigen binding. In addition, there is a peptide fragment carrying the drug-linker conjugated to a cysteine residue. Good recovery of these peptides is crucial to ensure full sequence coverage and understanding of their chemical modifications. Experimental Lys C digestion of ADC1 and the parent antibody was performed as described above. The digested samples were then analyzed by CZE-MS and also by RPLC-MS. Figure 1 shows the CZE-MS electropherogram of ADC1 in comparison with the parent antibody. For the most part, the peptide fingerprint of the ADC closely resembles that of the parent antibody except at around 77 min migration time (MT). The missing peak in the ADC profile is due to the change in migration behavior from unconjugated to conjugated peptide carrying the drug-linker. The drug-linker is a neutral species conjugated to an engineered cysteine with a stable linker. Upon conjugation, the peptide conjugated to the drug-linker acquires a different charge-to-size ratio due to additional mass of the drug-linker and therefore migrates differently. Multiple forms, around 78 and 86 min, were observed for the drug-linked peptides due to heterogeneity induced by the sample preparation artifacts.


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10

Parent Antibody 8 6 4

Relative Intensity (108)

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2 0 20

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130

10

Antibody-Drug Conjugate 8 6 4 2 0 20

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Minutes Figure 1: Base peak electropherogram of the Lys C peptides for the parent antibody and ADC1 analyzed by CZE-MS. The arrows point at the difference between the two profiles. The druglinked peptides migration shifted to the regions indicated with smaller arrows.

Sequence Coverage, Di/tri-Peptides, and Large Hydrophobic Peptides In RPLC-MS, 100% sequence coverage is rarely achieved with a single-enzyme digestion workflow because hydrophilic di/tri-peptides are often lost due to poor interaction with the column, and large hydrophobic peptides tends to overly adhere to the column and therefore poorly recovered. For ADCs, the attached drug-linker further increased the hydrophobicity of the conjugated peptide which can also impact their recovery with RPLC-MS. CZE-MS peptide mapping can provide good recovery for all peptides from single amino acid to large hydrophobic peptides and therefore permits 100% sequence coverage with a single-enzyme digestion. CZE7 ACS Paragon Plus Environment


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MS results shown in Figure 1 produced 100% sequence coverage with all of the 43 predicted Lys C peptides confidently identified and almost baseline resolved. Minor missed cleavage peptides were also identified. Peptide identification was performed by cross referencing the observed peptide m/z against the predicted m/z values of the most abundant isotope with MS/MS confirmation. As shown in Figure 2A, all di/tri-peptides, HK, PK, CK, AK, VDK, and EYK, which often elute in the void volume in RPLC-MS, were clearly recovered with distinct migration times. Although the detection mass range only captures the MS1 of these peptides except EYK, the peptides were easily identified as a result of high resolution separation with intense MS signal, Figure 2B. The single amino acid K-(H11) was not detected because it falls outside the detection mass range, but was detected in a miscleavage peptide [KVEPK].

A


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B Figure 2: (A) Extracted ion electropherogram of di/tri-peptides from CZE-MS Lys C peptide mapping of ADC1. (B) Full MS1 spectra of di/tripeptides in A with their m/z values and charge states.

The three large peptides L3, H6, and H9 were also identified with good MS intensity. Figure 3 shows the extracted ion electropherogram of L3(+5), H6(+6), and H9(+5) in comparison with L8(+3). Each large peptide migrates distinctly with good MS signal. To better understand the recovery performance of CZE-MS for large hydrophobic peptides, the recovery of L3, H6, H9 by CZE-MS were estimated and compared to RPLC-MS recovery. Relative recovery was defined as the ratio of the area counts of the most abundant ion to the area counts of the +3 ion of L8. L8 is a more hydrophilic, intermediate size peptide arbitrarily chosen for the comparison. It produces +4, +3, and +2 ions with the +3 ion being the second most abundant charge state in both RPLC-MS and CZE-MS. Chart 1 shows the sequence, MS ion distribution, GRAVY (grand average of hydropathy) value (hydrophobicity scale), and the calculated %recovery of the three large hydrophobic peptides. H9 peptide has a nonconsensus cleavage at [K206–P207] that produced two child fragments, H9a, [D.(55).K] and H9b, [PSNTK]. H9a is still relatively large and hydrophobic while H9b is small and hydrophilic. All H9 species were detected with good recovery and the calculated recovery for H9 and H9a are reported in Table 1. Overall, the %recovery for CZE-MS is twice more than that of RPLC-MS for all of the three large hydrophobic peptides. This observation is not surprising as CZE separation mechanism is only based on charge-to-size ratio with no hydrophobicity contribution. The absence of stationary phase interactions in CZE makes it more appropriate for analyzing large hydrophobic peptides especially with a neutral coated capillary surface where electroosmotic flow and capillary surface adsorption is minimal. It is also worth noting that the DMA modified BGE produced better recovery for large peptides than traditional aqueous acetic acid commonly used in CZEMS.19 The enhanced performance over the traditional acetic acid BGE is probably due to enhanced solubility of hydrophobic peptides during separation.



1.4

L3 (+5)

1.2


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1.0

H6 (+6)

0.8

H9a (+4)

0.6

L8 (+3) 0.4

H9 (+5)

0.2 0.0 10

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Minutes

Figure 3: Extracted ion electropherograms of L3, H6, H9, and H9a and L8(+3) ion from CZE-MS peptide map of ADC1.

Table 1: Calculated recovery of large hydrophobic peptides, L3, H6, and H9, based on the ratio of their most abundant ions to L8 (+3) ion. Sequence

GRAVY

Relative Recovery (peak

Value

area/L8 peak area) RPLC-MS

CZE-MS

-1.29

1.0

1.0

L3: [L.(56).K]

-0.11

0.2

3.5

H6: [G.(55).K]

-0.57

0.4

3.0

H9a: [D.(56).K]

0.14

1.0

2.0

H9: [D.(56).K/PSNTK]

-0.04

0.2

0.5

L8 [VDNALQSGNSQESVTEQDSK]

Conjugated Peptides In the ADC, the drug-linker is conjugated to an engineered cysteine residue. The in silico Lys C peptide from the fixed (Fc) region carrying the drug-linker is [THTCPPCPAPELLGGPCVFLFPPK], with the engineered cysteine underlined. Prior to conjugation, this peptide is relatively hydrophobic with a GRAVY value of +0.27. In RPLC-MS, 10 ACS Paragon Plus Environment

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elution of the conjugated peptide is only possible in the gradient wash with high organic solvent composition. Similar to large hydrophobic peptides, CZE-MS provides excellent recovery for the conjugated peptide. Figure 4 shows two populations of the conjugated peptides present with 1 (peak a, b, c) or 0 (peak d, e, f) IAA missed alkylation during the sample preparation. The occurrence of under-alkylated species is likely caused by the drug-linker steric hindrance. Multiple masses were also observed within each alkylation groups, which is caused by oxidation of the PBD dimer amine group under the harsh sample preparation condition. Changes to the drug leads to additional sub-population with +18 Da or +33 Da. 1.0

b

0.8


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0.6

a e

0.4

0.2

c

f

d 0.0 60

65

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100

Minutes P

P - 1Alkyl

1243.25 1243.59 1242.92

1223.92 1223.58

P - 1Alkyl + 18 Da

1224.25

P - 1Alkyl + 33 Da

1229.92 1229.59 1230.25

1224.58 1223.25 1224.92

1234.59 1234.26 1234.93

1230.59

1229.25

1230.92

1223

1224

1225

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1227

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1229

1230

1231

1232

1244.25

1234

1235

1236

1253.59

1249.92

1254.26 1254.93

1248.59

1255.59

1253.26 1250.26

1235.59

1233

1253.93

1248.921249.59

1242.58

1235.26

P + 33 Da

P +18 Da

1243.92

1242

1237

m/z

m/z

1243

1244

1245

1246

1247

1248

1249 m/z

1250

1251

1252

1253

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1256

1257

m/z

A



341.22 +1

100 95 90 85 80 75 70

Relative Abundance

Relative Abundance

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65 60

568.21 +1

55 50

244.17

45 40 35

239.11

30 25

488.29 +1

20 15

340.16

623.20 +2

726.30 +1

797.33 +1

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B Figure 4: (A) Extracted ion electropherogram of the conjugated peptide (P: [THTCPPCPAPELLGGPC(PBD)VFLFPPK]) and the full MS spectra observed masses of the conjugated peptide. (B) MS/MS fragment ions generated by the conjugated peptide. All different masses of the conjugated peptide showed identical MS/MS fragmentation.

All of the detected masses for the conjugated peptides are consistent with expected values. The different masses of the conjugated peptide migrated in two clusters; those that are mono alkylated (1 missed alkylation) and fully alkylated (0 missed alkylation) by IAA. MS/MS confirmation of the different conjugated peptide masses is shown in Figure 4b with the signature y2 (341.22) fragment ion from the peptide backbone and the fragment ions, 726.34(+1), 568.24(+1), and 797.38(+1) from the PBD dimer molecule. All observed masses showed identical MS/MS fragmentation. Similar to the large hydrophobic peptides, the recovery of the conjugated peptide was estimated as the ratio of the total area counts of all forms to the area counts of L8 (+3). The calculated recovery for the conjugated peptide with CZE-MS is 70% compared to

High-Resolution Capillary Zone Electrophoresis with Mass

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