MS Strategy for Reliable Detection of 10-ppm Level

Oct 22, 2018 - By continuing to use the site, you are accepting our use of cookies. Read the ACS privacy policy. CONTINUE. pubs logo. 1155 Sixteenth S...
0 downloads 0 Views 556KB Size
Subscriber access provided by The University of Texas at El Paso (UTEP)

Article

A 2D LC-MS/MS Strategy for Reliable Detection of 10-ppm Level Residual Host Cell Proteins in Therapeutic Antibodies Feng Yang, Donald E Walker, Jeannine Schoenfelder, Joseph Carver, Alice Zhang, Delia Li, Reed J. Harris, John Stults, X. Christopher Yu, and David A Michels Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03044 • Publication Date (Web): 22 Oct 2018 Downloaded from http://pubs.acs.org on October 22, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

A 2D LC-MS/MS Strategy for Reliable Detection of 10-ppm Level Residual Host Cell Proteins in Therapeutic Antibodies Feng Yang‡*, Donald E. Walker‡, Jeannine Schoenfelder, Joseph Carver, Alice Zhang, Delia Li, Reed Harris, John T. Stults, X. Christopher Yu and David A. Michels

Protein Analytical Chemistry, Genentech, A Member of the Roche Group, 1 DNA Way, South San Francisco, California 94080, United States ‡

Co-first authors

*Corresponding author: Email: [email protected]; Fax: +1 650 225 3554; Phone: +1 650 467 8190.

1 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Methodologies employing LC-MS/MS have been increasingly used for characterization and identification of residual host cell proteins (HCPs) in biopharmaceutical products to ensure their consistent product quality and safety for patients. To improve the sensitivity and reliability for HCP detection, we developed a high pH-low pH two-dimensional reversed phase LC-MS/MS approach in conjunction with offline fraction concatenation. Proof-of -concept was established using a model in which seven proteins spanning a size range of 29-78 kDa are spiked into a purified antibody product to simulate the presence of low-level HCPs. By incorporating a tandem column configuration and a shallow gradient through the second-dimension, all seven proteins were consistently identified at 10 ppm with 100% success rate following LC-MS/MS analysis of six concatenated fractions across multiple analysts, column lots and injection loads. Using the more complex Universal Proteomic Standard 1 (UPS-1) as an HCP model, positive identification was consistently achieved for 19 of the 22 proteins in 8-12 ppm range (10 ppm ± 20%). For the first time, we demonstrate an effective LC-MS/MS strategy that not only has high sensitivity but also high reliability for HCP detection. The method performance has high impact on pharmaceutical company practices in using advanced LC-MS/MS technology to ensure product quality and patient safety.

2 ACS Paragon Plus Environment

Page 2 of 27

Page 3 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

The production of monoclonal antibodies (mAbs) and other biotherapeutic proteins using genetically-modified prokaryotic or eukaryotic host cells with cell culture technology inevitably leads to their co-expression of host cell proteins (HCPs). Purification and clearance of HCPs from the active drug product during manufacturing is highly regulated by health authorities given the safety risk to patients1. While HCPs are effectively removed with an optimal purification process, residual HCPs remain present in the final drug product. For decades, multi-analyte enzyme-linked immunosorbent assay (ELISA) has been the gold standard for process development and quality control of biotherapeutics to measure an aggregate sum of HCPs2,3 for process development and quality control of biotherapeutics, but its limitations with HCP coverage and identification 4,5 has motivated researchers to explore alternative technologies. Peptide mapping by LC-MS/MS has been explored extensively as an orthogonal or complementary approach to ELISA for HCP quantitation. 2-5 The challenge of HCP detection is the very low levels of HCP peptides in a sample protein digest that is dominated by a 5-6 orders of excess of mAb peptides (plus low level mAb variant peptides). The identities of the HCP proteins are not known before the LC-MS/MS analysis. The only way to distinguish HCP from mAb is by identification of the parent protein from which each peptide is derived. Depending on the LC platform and MS instrument/methods used, LC-MS/MS offers the advantage of sensitive HCP identification down to 1-10 ppm.6-16 The advantages of LC-MS/MS have been demonstrated through identification of individual HCPs at the lowest spike level. For example, single column or 1-dimensional (1D) MS/MS with data dependent acquisition (DDA) has been described for HCP detection in biopharmaceutical products,6-9,17 but achieving higher sensitivity requires either an additional

3 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

affinity depletion step to remove large amounts of mAb (40-100 mg for example), HCP enrichment by affinity chromatography or additional data independent acquisition (DIA) analysis that relies on a comprehensive spectral library established from DDA data and complex data analysis. In contrast, dual column or 2-dimensional (2D) reversed phase liquid chromatography (RPLC) improves peak capacity and resolving power to separate such highly complex peptide samples with wide dynamic ranges. A common workflow applies online 2D-(high pH/low pH)RPLC to collect mass spectra with alternating low energy (for full MS) and elevated energy (MSE, for MS/MS) acquisition modes that enables simultaneous identification and quantitation of HCPs at 10-50 ppm with ~10-20 hours of LC-MS/MS analysis time.11-14 Doneanu et al recently identified peroxiredoxin-5 (21.8 kDa) in a purified mAb product at 1 ppm by incorporating high sample loading with a 3-dimensional system that utilized C18 chargedsurface hybrid (CSH) resin as the 2nd D separation coupled to the 3rd D traveling-wave ion mobility gas phase separation before MSE analysis.10 The use of semi-orthogonal 2D LC separation18-20 requires online analysis of up to 20 high-pH RPLC fractions to achieve good protein coverage.11-14 Fraction concatenation strategies have also been successful, which involves pooling early, middle and late high pH-RPLC fractions from the 1st D separation into a single fraction to increase the number of proteins identified in complex proteomics samples. 20-25 Some of these studies showed that in addition to reducing the number of peptide fractions in the 1st D without sacrificing protein identifications, concatenation also improved the orthogonality of the 2D RPLC-RPLC separations.20,22 To our best knowledge, the benefits of the concatenation approach in HCP applications have not been demonstrated in previous work. While current LC-MS/MS methods can provide sensitive HCP identification down to 110 ppm for specific proteins, at such low limits, the detection reliability by LC-MS/MS for any

4 ACS Paragon Plus Environment

Page 4 of 27

Page 5 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

HCP independent of molecular size or sequence when present at or above a defined ppm level has not yet been adequately addressed. 8-14 i.e, if an HCP is found, is it detected every time the analysis is done? If there are multiple HCPs all present at certain level, how many will one find using their method of choice? Therefore, there is opportunity to develop a fast and reliable LCMS/MS method for sensitive HCP identification with simple workflow (DDA only) and data analysis (direct database search). In this study, we present a concatenated 2D LC-MS/MS workflow that enhances reliable low-level HCP identification (92-100% success rate) at or above 10 ppm using a set of off-the-shelf protein standards as HCP surrogates spiked into a mAb drug product. To the best of our knowledge, we demonstrate for the first time the application of an offline 1st D fractionation concatenation (high-pH RPLC) coupled with an online 2nd D CSH (low-pH) LC-MS/MS and DDA for a reliable and sensitive HCP detection method with simple data analysis.

EXPERIMENTAL SECTION Materials. The full length mAb-1 (IgG1) drug product was produced in-house (Genentech, South San Francisco, CA). A mixture of seven protein standards (7-STD) was composed of four commercially available proteins- bovine carbonic anhydrase 2 (CA2, Sigma-Aldrich #C2522), yeast enolase (ENO1 Sigma-Aldrich #E6126), yeast alcohol dehydrogenase (ADH1, SigmaAldrich #A7011) and human lactotransferrin (LTF, BioRad #PHP239), and three proteins produced at Genentech – putative phospholipase B-like 2 (PLBL2), glutathione synthetase (GS) from Chinese Ovary Hamster and FKBP-type peptidyl-prolyl cis-trans isomerase (FkpA) from E.coli. Universal Proteomic Standard 1 (UPS-1) was purchased (Sigma-Aldrich) and contains a mixture of 48 recombinant proteins of known equimolar concentration and MW ranging from 6.3

5 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

to 82.9 kDa. Recombinant porcine trypsin (proteomics grade) used for protein digestion was from Roche (Product Number 03708985001). Sample Preparation. To simulate the presence of low-level HCPs in drug product, mAb-1 was artificially spiked with different mixture of protein standards, either a 7-STD mixture or the UPS-1 (48 proteins) standard. All ppm values of spiked proteins were calculated by use of the respective weights of the individual proteins. The 1000 ppm 7-STD mixture stock solution (5 mg/mL final mAb-1 concentration) was made by adding aliquots of each protein standard solution (corresponding to 15 µg of each protein after protein purity adjustment) to 300 µL of 50 mg/mL mAb-1and adding mAb-1 formulation buffer (pH 6.0) to a final volume of 3 mLs. The 7-STD mixture at 100 ppm level was prepared by mixing the 1000 ppm 7-STD-spiked sample with non-spiked mAb-1 (5 mg/mL) sample. Similarly, 7-STD mixture at 0, 5, 10, 20, 50, and 100 ppm levels were prepared by co-mixing the 100 ppm 7-STD-spiked sample with non-spiked mAb-1 sample; each level was prepared in triplicate to assess repeatability. The 7-STD-spiked mAb-1 was used to determine intermediate precision of the 2D LC-MS/MS method at the 10 ppm level. To further verify method reliability in detecting ≥10 ppm HCPs in purified mAb products, UPS-1 was spiked into mAb-1 (5 mg/mL) with the 48 protein levels ranging from 3 ppm to 44 ppm. Each sample was digested with trypsin following a previously published protocol.8 Tryptic digest (from 0.8 mg of protein, 1.5 mL) was loaded onto the first dimension high pH reversed phase high performance liquid chromatography (RP-HPLC) fractionation. 1st Dimensional Offline High-pH RPLC Fractionation and Concatenation. Reversed-phase HPLC separation was performed on a Waters XBridge BEH C18 column (5 µm, 130 Å, 4.6 x 250 mm) using an Agilent 1200 Infinity HPLC system. Mobile phases consisted of 5 mM ammonium formate (pH 10) in water (Mobile Phase A) or 90 % acetonitrile in mobile phase A

6 ACS Paragon Plus Environment

Page 6 of 27

Page 7 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

(Mobile Phase B). The flow rate was 0.5 mL/min and column temperature was maintained at 25 °C. The total 90 minutes LC method with linear gradient included a 15 minutes wash with 0% B, 0-3% B in 3 minutes, 3-30% B in 20 minutes, 30-42.5% solvent B in 10 minutes, 42.555% B in 18 minutes, 55-100% B in 5 minutes, a column wash (5 minutes at 100% B), 100-0% B in 0.5 minute and column re-equilibration (14.5 minutes at 0% B) steps. A total of 60 fractions were collected into a 96 well plate containing 10 µL of 10% trifluoroacetic acid in each well, collected between the LC separation time 15 to 78 minutes. The 60 high pH RP fractions (0.525 mL each) were concatenated into 6 fractions by combining fractions 1, 7, 13, 19, 25, 31, 37, 43, 49, 55 for pool 1; 2, 8, 14, 20, 26, 32, 38, 44, 50, 56 for pool 2; and so on. The concatenated fractions were dried in the speed vacuum concentrator for approximately 8 h to ~50-100 µL. Each sample was then adjusted to 250 µL volume using LC-MS grade water and stored at −80°C until LC-MS/MS analysis. 2nd Dimensional Online Low pH RP- UHPLC- MS/MS. Online LC-MS/MS analysis was performed using a Thermo Scientific Vanquish  UHPLC system that was interfaced with a Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer. All separations were performed on a single Waters CSH130 C18 (1.7 µm, 2.1mm x 150mm) column or a tandem CSH130 C18 column composed of two CSH C18 columns connected by a 5-µL sample loop (Waters). Mobile phases consisted of 0.1% formic acid in water (Mobile Phase A, pH~2.7) or acetonitrile (Mobile Phase B). The flow rate was 0.2 mL/min and column temperature was maintained at 60 °C. Refer to our published work8 for the details of a shorter 65-minute LC gradient. A longer, 125-minute, LC gradient included a linear gradient of a 5 minutes wash with 0% B, 0-2% B in 1 minute, 2-5% B in 2 minutes, 5-15% solvent B in 30 minutes, 15-22% B in 42 minutes, 22-32% B in 26 minutes, 32-40% B in 2 minutes, 40-90% B in 0.5 minute, a column 7 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

wash (0.5 minutes at 90% B), 90-0% B in 0.5 minute and column re-equilibration (15.5 minutes at 0% B) steps. The sample load for each fraction pool was 25 µL with a tandem column and 12.5 µL with a single column. The Orbitrap Fusion Lumos mass spectrometer was operated in the data-dependent mode acquiring collision induced dissociation (CID) scans (ion trap detection, 1 × 104 target ions) after each full MS scan (R=120,000 at m/z of 200, 4 × 105 target ions) for the top 12 most abundant ions within the mass range of 380 to 1580 m/z. For MS/MS, an isolation window of 3 Th was used to isolate ions in ion trap prior to CID for the top 12 most abundant ions. All CID scans used a normalized collision energy of 30 and a maximum inject time of 100 ms. The dynamic exclusion time was set to 30 s and charge state screening was enabled to reject unassigned and singly charged ions. A system suitability sample (10 µg of unfractionated trypsin digest of mAb1 spiked with 7-STD each at 10 ppm) was analysed to verify the performance of the 2nd D LCMS/MS operation before and after running the fractionated samples. Refer to Figure S-1 and S-2 for the base peak profiles of system suitability sample and six concatenated fractions. Data Analysis. LC-MS/MS DDA data files were searched against a database containing sequences of all Genentech biotherapeutic products, plus those of all spiked protein standards, concatenated to the CHO Canonical and Isoform database from Uniprot.org (35,256 entries), using Thermo Proteome Discoverer software (version1.4) with SEQUEST search. A protein is required to have two unique peptides (at 5% false discovery rate for peptides, evaluated with a decoy database search) for positive identification. To pass the system suitability test, at least one of the 7-STD proteins needs to be positively identified. Refer to Table S-1 for a summary of system suitability test results.

8 ACS Paragon Plus Environment

Page 8 of 27

Page 9 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

RESULTS AND DISCUSSION High-pH RPLC Concatenation vs. Non-concatenation Fractionation. The overall workflow and strategy for concatenation and non-concatenated RPLC fractionation applied in our study is presented in Figure 1. To explore the benefit of fraction concatenation in HCP detection, 60 fractions from the first RPLC dimension (high-pH) were concatenated into six fractions by pooling every 6th collected fraction with equal time interval (10 total fractions) into one fraction pool. As a control, six non-concatenated fractions were prepared by combining 10 consecutive first dimension fractions into one in the conventional way. Five percent of each fraction pool (10 µg) was then analyzed by the second RPLC dimension (low-pH) online LC-MS/MS using a 15 cm long CSH column and a Fusion Lumos MS with a 65-min separation time. To simulate the presence of low-level HCPs, a seven-protein standard (7-STD) spanning 28.9 to 78.1 kDa was spiked into the mAb-1 at 10 ppm each and used to evaluate the method reliability in detecting low-levels of non-product related proteins. Using the non-concatenated pooling strategy, only four standards were identified at the 10 ppm level in mAb-1with ≥ 2 unique peptides, as shown in Figure 2A, and only one unique peptide was detected for each of the remaining three spiked standards. In contrast, the concatenated fractionation method identified all 7-STD proteins with a higher number of unique peptides, demonstrating the advantage of applying the concatenation strategy for identifying low-level proteins and improving protein sequence coverage, as shown by the increased number of peptide identifications. These results are consistent with previous findings using concatenation strategy for proteomics studies.21-25 In addition, this 2D LC-MS/MS approach with offline concatenation

9 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

provides higher throughput (6 hours of LC-MS/MS analysis time) than on-line 2D LC-MS/MS methods that often required 10-20 hours of analysis time to achieve 10-50 ppm sensitivity. 11-14

Selection of Gradient Length and Column Format for 2nd Dimension Online LC-MS/MS. While online LC-MS/MS analyses using a single CSH column and a 65-min gradient identified 2-3 unique peptides in all 7-STD proteins spiked at 10 ppm level (Figure 2A), the variability in sample preparation, separation, and MS detection conditions may differ for each experimental session, and proteins with 2 or 3 peptide counts may become challenging to consistently identify using the criteria of ≥ 2 unique peptides. Thus, a method providing higher number of unique peptides per protein is desired and improves the overall reliability in confirming the presence of an HCP. The overall number of unique peptides identified depends on the balance between the protein size (molarity), peptide sequence (ionization efficiency), and method sensitivity at a given ppm level. To achieve a higher peptide count, we first evaluated peptide identification as a function of column gradient using a single CSH column. As shown in Figure 2B, the 125-mins gradient increased the overall number of peptide identifications for the 7-STD proteins, except for PLBL2 (3 peptides identified), compared to a 65-min gradient. We next investigated the impact of method sensitivity on number of peptide identifications. As sample loading capacity is proportional to the column length, we explored the use of a tandem column (two single 15-cm columns connected sequentially via a 5-µL adaptor) by loading twice the amount of protein in an effort to increase the method sensitivity and number of unique peptide identifications without compromising separation efficiency. For the same mAb-1 sample spiked with 10 ppm 7-STD, the tandem column format identified a total of 43

10 ACS Paragon Plus Environment

Page 10 of 27

Page 11 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

peptides with a 65-min gradient and 62 total peptides with a 125-min gradient. For each spiked protein standard, the method identified at least 5 unique peptides, thus improving identification reliability and the method’s potential for HCP detection application at 10 ppm levels. Furthermore, the use of twice the sample load coupled with the tandem LC-MS/MS analysis caused only marginal peak broadening (Table S-2), as characterized by the full-peak-width-athalf-maximum (FWHM) for the top five mAb-1 peptides (10.9 vs. 9.7 sec). In contrast, when increasing the sample load by 2-fold on a single column, the average FWHM increased 1.8-fold (17.0 vs. 9.7 sec). For characterization of complex proteomics, longer columns have been shown to improve resolution and peak capacities at the same load and can therefore potentially increase the dynamic range of LC-MS detection. 26,27 In contrast, for detection of HCPs in drug products, the dynamic range between protein abundance and residual HCP levels is high but the sample complexity is relatively low. Therefore, our studies focus on using the higher sample load capacity with longer columns to increase sensitivity while maintaining similar peak resolution. A previous study10 used a CSH column to increase sensitivity of HCP analysis by using a high sample load. In addition to using the CSH column, our study shows the initial evidence that a longer column at higher sample load is effective to increase both the sensitivity and reliability of HCP analysis. At present, commercial CSH C18 columns are only 15 cm long; therefore, connecting two 15-cm long CSH columns in tandem with a small (5 µL) dead volume connector effectively created a longer column with marginal impact to peak resolution even at the high sample load. Figure 2B also highlights the likelihood of identifying peptides at the 10 ppm level, shown by the number of peptides identified, for each of the 7-STD proteins. In all test

11 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

conditions, CA2 was consistently identified with the highest number of peptides, regardless of column format or gradient length. The longer gradient greatly improved the peptide counts for the low responders, such as proteins FkpA and PLBL2, which were initially identified based on either 2 or 3 peptides with the 65-min gradient. PLBL2 was the least likely to be identified by the 65-min gradient, but was robustly detected with the 125-min gradient tandem column method. Moreover, our results show that, at 10 ppm spike level, all 7-STD proteins (28.9-78.1 kDa MW range) were identified with similar peptide counts using the method with a tandem column with 125-min LC-gradient (Figure 2B). Based on these results, a tandem column configuration with 125-min LC gradient and 10 µg target load was selected for subsequent experiments. Sensitivity and Repeatability. Figure 3 shows the number of peptides identified for the 7-STD at spike levels ranging from 5–50 ppm relative to mAb-1. At the 5 ppm spike level, PLBL2 was detected with ≥ 2 peptides in two out of three process replicate analyses; all other 7-STD proteins were identified in all three replicates. At 10 ppm or higher spike levels, all the 7-STD proteins were consistently identified with ≥ 4 peptides for all process replicates. Previous methods9,10,11-14 described the sensitivity based on the lowest level of spiked protein detected, but did not demonstrate comprehensiveness or consistency of detection at a given level for different proteins. In our study, we defined the detection limit as the ability of the assay to consistently detect all seven spiked proteins with ≥ 2 peptides per protein per sample (n=3), at and above a specific spike level. Therefore, 10 ppm was considered the detection limit for reliable HCP detection in the mAb product. The repeatability of the end-to-end process, including sample preparation (enzyme digestion and 1st D fraction collection and concatenation) and LC-MS/MS analysis are shown in

12 ACS Paragon Plus Environment

Page 12 of 27

Page 13 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Table S-3 and S-4, respectively. The relative standard deviation (RSD) of the total number of 7STD peptides identified was shown to be < 13% when present at 10 ppm or above, indicating good repeatability of the sample preparation. For total 7-STD peptides identified at target spike level (10 ppm), the RSD (3.9%, Table S-4) of the LC-MS/MS injection replicates was half that of the process replicates (7.8%, Table S-3), demonstrating excellent repeatability for LC-MS/MS analysis. Intermediate Precision. To evaluate intermediate precision in identifying 10 ppm 7-STD proteins spiked in the mAb1, six independent process replicates were prepared by two analysts using two 1st D columns, and analyzed by LC-MS/MS with two 2nd D RPLC columns on different days. To assess the intermediate precision of the sample preparation procedure including enzyme digestion and 1st D separation, five high abundance peptide peaks were selected based on their 214 nm absorbance across the LC gradient. Shown in Table S-5, RSDs are < 3% for the retention times and < 18% for peak areas of these peptide peaks from six independent sample preparation process replicates, thus demonstrating good method precision of the sample preparation process. Table S-6 shows that while there are differences (especially for PLBL2) between the minimal numbers of peptides identified for each STD protein, all the 7-STD proteins were consistently identified from LC-MS/MS analyses of all the six process replicates, and the RSD of total peptide counts is < 9%. These results show that the whole 2D LC-MS/MS workflow is precise in detecting proteins present at 10 ppm under various conditions (different analysts, sample preparation, 1st D and 2nd D LC columns) tested in different sessions.

13 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

To evaluate the effect of potential sample load variations (< 30% as shown in Table S-5) due to sample preparation on HCP detection, the sample load was varied by injecting 50% (tested as the worst scenario), 100%, and 130% of target sample volume (25 µL, or 10 µg) per sample set run. While the number of total and minimal peptide counts for identified 10 ppmspiked 7-STD proteins decreased at the 50% sample load, all 7-STD proteins were confidently identified at all sample load conditions (Table S-7). Therefore, potential variations from sample load are unlikely to affect the detection limit of 10 ppm and this method is sufficiently reliable. These studies also demonstrate the reliability of the DDA method in consistent detection of low level HCPs at 10 ppm or above. Compared to DIA, which often involves complex data analysis workflow and additional software, DDA data analysis is direct and simple, and can be easily performed by protein database searches. Reliability of the DDA method is expected for all advanced MS instruments; however, the defined analysis sensitivities may differ depending on specific instruments. The intent is to reliably detect all HCPs at a suitably sensitive level, avoiding false negative (undetected impurity) results. The method’s detection limit should be defined by its reliability ensured by replicate analysis (n=3 in our study) to consistently detect all spiked proteins, instead of reporting the most sensitive (lowest) detected protein level. Qualification of Method Reliability in Detecting Low-Level UPS-1 Proteins. To further assess and demonstrate the method reliability in detecting unknown residual HCPs in biopharmaceutical products, analyses were performed under the optimal conditions (six concatenated fractions, 30 cm-long tandem column and 125-min gradient) for mAb-1 sample spiked with known levels of 48 UPS-1 proteins with MW ranging from 6.3 to 82.9 kDa to simulate trace level HCPs in a product. These UPS-1 proteins have the same molarity; thus their

14 ACS Paragon Plus Environment

Page 14 of 27

Page 15 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

spike levels ranged from 3 to 44 ppm (26 proteins present at ≥ 10 ppm level and 22 proteins at < 10 ppm level). The spike ppm level and number of peptides identified for each UPS-1 protein at 10 µg target sample load are shown in Figure 4. In total, ≥ 35 of the 48 UPS-1 proteins were confidently identified at all levels of sample load (50, 100 and 130% of target, Table S-8). The results also demonstrate that positive identification (≥ 2 unique peptides, the criteria used throughout this study) was achieved with consistency (n=3) and with a success rate of 92.3% (24 out of 26) in detecting UPS-1 proteins at ≥ 10 ppm spike level (Table S-9) and ≥ 50% of the proteins at =2 peptide requirement for detection. In contrast, the six smaller proteins have a lower number of potentially detectable tryptic peptides. Furthermore, multiple sequence-derived peptide properties (hydrophobicity, basicity, etc.) can influence the ionization efficiency and hence the number of detectable peptides at low levels. Zero peptide detection of these 6 UPS-1 proteins in this study was probably due to the combination of above factors. This rationale is consistent with the different number of peptides found for each of the 10 ppm-spiked 7-STD sample.

15 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Our work, for the first time, has demonstrated an effective LC-MS/MS strategy that not only has high sensitivity but also high reliability for detecting all HCPs that might be present at or above a defined ppm level. This area has not been sufficiently addressed by previous work. Moreover, we proposed a new strategy to define the detection limit of the LC-MS/MS analysis as the ability of the assay to consistently detect all seven spiked proteins with ≥ 2 peptides per protein per sample (n=3), at and above a specific spike level. We strongly believe that the strategies and results presented in this work will have high impact on pharmaceutical company practices of using LC-MS for detecting low level product impurities and minor variants that might affect patient safety. We addressed the critical question on reliability of HCP detection by novel applications of two analytical strategies: fractionation concatenation and a dual column. While fraction concatenation strategies are already known,21,22 the benefits (increased sensitivity and coverage of low abundance HCPs) of the concatenation approach in HCP applications have not been demonstrated in previous work. HCP identification in purified antibody drug substance presents a unique challenge (>5 to 6 orders of magnitude of dynamic range, required to detect all HCPs at a defined low level in the presence of a single high abundance protein) compared to proteomics studies where the fraction concatenation strategies were first applied (to increase the number of proteins identified). In addition, while most studies26,27 used longer column to increase peak capacities at the same sample load to increase the coverage of complex proteomes, our study focused on using the higher sample load capacity with longer columns to increase sensitivity of low level HCP detection. The strategies we have applied to the HCP detection can also be broadly applied to any other LC-MS/MS analysis that has dynamic range and sensitivity challenges.

16 ACS Paragon Plus Environment

Page 16 of 27

Page 17 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

CONCLUSIONS A sensitive and reliable 2D LC-MS/MS method was developed to detect low level ( 10 ppm) HCPs with ≥ 90% success rate in mAb drug products using simplified and direct database searching (DDA only) and data analysis. Quantitation of the identified HCPs can then be further performed using other established methods such as DIA, targeted MS/MS and protein-specific ELISA. This technique utilizes the concatenation strategy for the 1st D high-pH RPLC fractions, and a tandem CSH column for the 2nd D low-pH RPLC separation to achieve reliable detection of HCPs at 10 ppm sensitivity. The performance of this new technique was demonstrated from two models consisting of spiked protein standards with known ppm levels and having a broad range of MWs to represent HCPs in a drug product mAb-1. We found that without fraction concatenation, only four out of seven model proteins spiked in mAb-1 at 10 ppm level were identified, while concatenation enabled identification of all seven proteins. LC-MS/MS analysis of six concatenated fractions using a tandem column with a 125-min gradient demonstrated high method reliability in identifying all the 10 ppm-spiked model proteins and low-level UPS-1 proteins. Consistent results were achieved for multiple analysts, columns, and varying injection loads, demonstrating the reliability gained from fraction concatenation and tandem column. While the method performance demonstration is for a single mAb, the findings are applicable to other mAbs that are expected to have similar purity and complexity.

ASSOCIATED CONTENT Supporting Information. Additional information as noted in the text.

17 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACKNOWLEDGMENTS We thank Benjamin Barnhill from University of Virginia for his help with the initial setup and testing of the Orbitrap Fusion Lumos mass spectrometer.

REFERENCES (1) ICH Harmonised Tripartite Guideline. Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products Q6B. 1999. (2) Shukla, A. A.; Jiang, C.; Ma, J.; Rubacha, M.; Flansburg, L.; Lee, S. S. Demonstration of robust host cell protein clearance in biopharmaceutical downstream processes. Biotechnol. Prog. 2008, 24, 615-622. (3) Tscheliessnig, A. L.; Konrath, J.; Bates, R.; Jungbauer, A. Host cell protein analysis in therapeutic protein bioprocessing - methods and applications. Biotechnol. J. 2013, 8, 655-670. (4) Zhu-Shimoni, J.; Yu, C.; Nishihara, J.; Wong, R. M.; Gunawan, F.; Lin, M.; Krawitz, D.; Liu, P.; Sandoval, W.; Vanderlaan, M. Host cell protein testing by ELISAs and the use of orthogonal methods. Biotechnol. Bioeng. 2014, 111, 2367-2379. (5) Vanderlaan, M., Sandoval, W., Liu, P., Nishihara, J., Tsui, G., Lin, M., Gunawan, F., Parker, S., Wong, R. M., Low, J., Wang, X., ,Yang, J., Veeravalli, K., McKay, P., Yu, C., O'Connell, L., Tran, B., Vij, R., Fong, C., Francissen, K.,Zhu-Shimoni, J., , Quarmby V., and Krawitz, D. Hamster Phospholipase B-Like 2 (PLBL2): A Host Cell Proten Impurity in Therapeutic Monoclonal Antibodies Derive from Chinese Hamster Ovary Cells. BioProcess Int. 2015, 13, 18-22.

18 ACS Paragon Plus Environment

Page 18 of 27

Page 19 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

(6) Reisinger, V.; Toll, H.; Mayer, R. E.; Visser, J.; Wolschin, F. A mass spectrometry-based approach to host cell protein identification and its application in a comparability exercise. Anal. Biochem. 2014, 463, 1-6. (7) Madsen, J. A.; Farutin, V.; Carbeau, T.; Wudyka, S.; Yin, Y.; Smith, S.; Anderson, J.; Capila, I. Toward the complete characterization of host cell proteins in biotherapeutics via affinity depletions, LC-MS/MS, and multivariate analysis. mAbs 2015, 7, 1128-1137. (8) Walker, D. E., Yang, F., Carver, J., Joe, K., Michels, D. A., and Yu, X. C. . A modular and adaptive mass spectrometry-based platform for support of bioprocess development toward optimal host cell protein clearance mAbs 2017, 9, 654-663. (9) Kreimer, S.; Gao, Y.; Ray, S.; Jin, M.; Tan, Z.; Mussa, N. A.; Tao, L.; Li, Z.; Ivanov, A. R.; Karger, B. L. Host Cell Protein Profiling by Targeted and Untargeted Analysis of Data Independent Acquisition Mass Spectrometry Data with Parallel Reaction Monitoring Verification. Anal. Chem. 2017, 89, 5294-5302. (10) Doneanu, C. E.; Anderson, M.; Williams, B. J.; Lauber, M. A.; Chakraborty, A.; Chen, W. Enhanced Detection of Low-Abundance Host Cell Protein Impurities in High-Purity Monoclonal Antibodies Down to 1 ppm Using Ion Mobility Mass Spectrometry Coupled with Multidimensional Liquid Chromatography. Anal. Chem. 2015, 87, 10283-10291. (11) Schenauer, M. R.; Flynn, G. C.; Goetze, A. M. Identification and quantification of host cell protein impurities in biotherapeutics using mass spectrometry. Anal. Biochem. 2012, 428, 150157. (12) Doneanu, C. E.; Xenopoulos, A.; Fadgen, K.; Murphy, J.; Skilton, S. J.; Prentice, H.; Stapels, M.; Chen, W. Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry. mAbs 2012, 4, 24-44.

19 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(13) Farrell, A.; Mittermayr, S.; Morrissey, B.; Mc Loughlin, N.; Navas Iglesias, N.; Marison, I. W.; Bones, J. Quantitative Host Cell Protein Analysis Using Two Dimensional Data Independent LC-MS(E). Anal. Chem. 2015, 87, 9186-9193. (14) Zhang, Q.; Goetze, A. M.; Cui, H.; Wylie, J.; Trimble, S.; Hewig, A.; Flynn, G. C. Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry. mAbs 2014, 6, 659-670. (15) Husson, G.; Delangle, A.; O'Hara, J.; Cianferani, S.; Gervais, A.; Van Dorsselaer, A.; Bracewell, D.; Carapito, C. Dual Data-Independent Acquisition Approach Combining Global HCP Profiling and Absolute Quantification of Key Impurities during Bioprocess Development. Anal. Chem. 2018, 90, 1241-1247. (16) Huang, L.; Wang, N.; Mitchell, C. E.; Brownlee, T.; Maple, S. R.; De Felippis, M. R. A Novel Sample Preparation for Shotgun Proteomics Characterization of HCPs in Antibodies. Anal. Chem. 2017, 89, 5436-5444. (17) Bomans, K.; Lang, A.; Roedl, V.; Adolf, L.; Kyriosoglou, K.; Diepold, K.; Eberl, G.; Molhoj, M.; Strauss, U.; Schmalz, C.; Vogel, R.; Reusch, D.; Wegele, H.; Wiedmann, M.; Bulau, P. Identification and monitoring of host cell proteins by mass spectrometry combined with high performance immunochemistry testing. PLoS One 2013, 8, e81639. (18) Gilar, M.; Olivova, P.; Daly, A. E.; Gebler, J. C. Orthogonality of separation in twodimensional liquid chromatography. Anal. Chem. 2005, 77, 6426-6434. (19) Delmotte, N.; Lasaosa, M.; Tholey, A.; Heinzle, E.; Huber, C. G. Two-dimensional reversed-phase x ion-pair reversed-phase HPLC: an alternative approach to high-resolution peptide separation for shotgun proteome analysis. J. Proteome Res. 2007, 6, 4363-4373.

20 ACS Paragon Plus Environment

Page 20 of 27

Page 21 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

(20) Dwivedi, R. C.; Spicer, V.; Harder, M.; Antonovici, M.; Ens, W.; Standing, K. G.; Wilkins, J. A.; Krokhin, O. V. Practical implementation of 2D HPLC scheme with accurate peptide retention prediction in both dimensions for high-throughput bottom-up proteomics. Anal. Chem.2008, 80, 7036-7042. (21) Wang, Y.; Yang, F.; Gritsenko, M. A.; Wang, Y.; Clauss, T.; Liu, T.; Shen, Y.; Monroe, M. E.; Lopez-Ferrer, D.; Reno, T.; Moore, R. J.; Klemke, R. L.; Camp, D. G., 2nd; Smith, R. D. Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells. Proteomics 2011, 11, 2019-2026. (22) Yang, F.; Shen, Y.; Camp, D. G., 2nd; Smith, R. D. High-pH reversed-phase chromatography with fraction concatenation for 2D proteomic analysis. Expert Rev. Proteomics 2012, 9, 129-134. (23) Zhang, H.; Liu, T.; Zhang, Z.; Payne, S. H.; Zhang, B.; McDermott, J. E.; Zhou, J. Y.; Petyuk, V. A.; Chen, L.; Ray, D.; Sun, S.; Yang, F.; Chen, L.; Wang, J.; Shah, P.; Cha, S. W.; Aiyetan, P.; Woo, S.; Tian, Y.; Gritsenko, M. A.; Clauss, T. R.; Choi, C.; Monroe, M. E.; Thomas, S.; Nie, S.; Wu, C.; Moore, R. J.; Yu, K. H.; Tabb, D. L.; Fenyo, D.; Bafna, V.; Wang, Y.; Rodriguez, H.; Boja, E. S.; Hiltke, T.; Rivers, R. C.; Sokoll, L.; Zhu, H.; Shih Ie, M.; Cope, L.; Pandey, A.; Zhang, B.; Snyder, M. P.; Levine, D. A.; Smith, R. D.; Chan, D. W.; Rodland, K. D.; Investigators, C. Integrated Proteogenomic Characterization of Human High-Grade Serous Ovarian Cancer. Cell 2016, 166, 755-765. (24) Mertins, P.; Yang, F.; Liu, T.; Mani, D. R.; Petyuk, V. A.; Gillette, M. A.; Clauser, K. R.; Qiao, J. W.; Gritsenko, M. A.; Moore, R. J.; Levine, D. A.; Townsend, R.; Erdmann-Gilmore, P.; Snider, J. E.; Davies, S. R.; Ruggles, K. V.; Fenyo, D.; Kitchens, R. T.; Li, S.; Olvera, N.; Dao, F.; Rodriguez, H.; Chan, D. W.; Liebler, D.; White, F.; Rodland, K. D.; Mills, G. B.; Smith, R.

21 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

D.; Paulovich, A. G.; Ellis, M.; Carr, S. A. Ischemia in tumors induces early and sustained phosphorylation changes in stress kinase pathways but does not affect global protein levels. Molecular & cellular proteomics : Mol. Cell. Proteomics 2014, 13, 1690-1704. (25) Kumar, A.; Baycin-Hizal, D.; Wolozny, D.; Pedersen, L. E.; Lewis, N. E.; Heffner, K.; Chaerkady, R.; Cole, R. N.; Shiloach, J.; Zhang, H.; Bowen, M. A.; Betenbaugh, M. J. Elucidation of the CHO Super-Ome (CHO-SO) by Proteoinformatics. J. Proteome Res. 2015, 14, 4687-4703. (26) Shen, Y.; Zhang, R.; Moore, R. J.; Kim, J.; Metz, T. O.; Hixson, K. K.; Zhao, R.; Livesay, E. A.; Udseth, H. R.; Smith, R. D. Automated 20 kpsi RPLC-MS and MS/MS with chromatographic peak capacities of 1000-1500 and capabilities in proteomics and metabolomics. Anal. Chem. 2005, 77, 3090-3100. (27) Shen, Y.; Tolic, N.; Piehowski, P. D.; Shukla, A. K.; Kim, S.; Zhao, R.; Qu, Y.; Robinson, E.; Smith, R. D.; Pasa-Tolic, L. High-resolution ultrahigh-pressure long column reversed-phase liquid chromatography for top-down proteomics. J. Chromatogr. A 2017.

22 ACS Paragon Plus Environment

Page 22 of 27

Page 23 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

1st D Offline High-pH RPLC, 60 fractions 1

7

13

19

25

31

37

43

49

55

1

10 20 30 40

50

Concatenation

11

21

31

41

51

60

Non-concatenation

6 Fraction Pools

1

2

3

4

5

6

2nd D Online Low-pH RPLC coupled to MS/MS Analysis: (CSH C18 column, commercially available, 1.7 µm, 2.1 x 150 mm)

Figure 1 Strategy of concatenation and non-concatenated fractionation applied to the high-pH RPLC dimension of the 2D LC-MS/MS analysis. Examples are shown to illustrate the fraction combination strategies- the specific numbers of 1st D fractions (numbers in the smaller colored circle) are combined into the corresponding 2nd D fraction pools (numbers in the bigger circle with the same color as combined fractions).

23 ACS Paragon Plus Environment

Analytical Chemistry

12

A Number of Peptides Identified

10 8 6 4 2 0

FkpA

CA2

ADH1

ENO1

GS

PLBL2

LTF

(28.9 kDa) (29.1 kDa) (36.8 kDa) (46.8 kDa) (52.2 kDa) (65.5 kDa) (78.1 kDa) Nonconcatenated

2

8

2

3

1

1

1

Concatenated

3

6

8

11

2

3

5

B

Number of Peptides Identified

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 27

14 12 10 8 6 4 2 0

FkpA

CA2

ADH1

ENO1

GS

PLBL2

Single Column, 65 min

2

10

7

7

3

3

LTF 4

Single Column, 125 min

4

11

8

11

6

3

13

Tandem Column, 65 min

3

11

6

10

7

2

4

Tandem Column, 125 min

7

11

12

11

6

5

10

Figure 2 Identification of 7-STD proteins (sorted from low to high MW) spiked at 10 ppm level in mAb1 by 2D LC-MS/MS: A) using single column, with and without concatenation strategy for 1st D fractionation (65 min 2nd D LC gradient), and B) using single or tandem column with different 2nd D LC gradient time, all with concatenation strategy.

24 ACS Paragon Plus Environment

Page 25 of 27

FkpA

CA2

ADH1

ENO1

GS

PLBL2

LTF

44 Average Number of Peptide Identified (N=3)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

40 36 32 28 24 20 16 12 8 4 0 5ppm

10ppm

20ppm

50ppm

Figure 3 Sensitivity of 2D LC-MS/MS analysis in detecting 7-STD (sorted from low to high MW) at different spike levels into mAb1. Three process replicates were performed. * PLBL2 was only identified with ≥ 2 peptides in replicate 2 and 3, with 1 peptide identified in replicate 1. Error bar shows the standard deviation of the number of peptides identified for each protein.

25 ACS Paragon Plus Environment

Analytical Chemistry

UPS-1 Protein Spike Level (ppm)

24

50

22

45

20

40

18 35

16 14

30

12

25

10

20

8

15

6 10

4

UPS-1 Proein Spike Level (ppm)

Peptide Counts Target Load

Peptide Counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 27

5

2 0

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

UPS-1 Protein Index

Figure 4 Detection of 48 UPS-1 proteins at different spike levels (44 to 3 ppm, sorted from high to low ppm) into mAb1 at target load. Blue bars indicate the number of peptides identified from UPS1 proteins and red diamonds indicate spike levels of UPS-1 proteins.

26 ACS Paragon Plus Environment

Page 27 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

For TOC only 1st D Offline High-pH RPLC & Concatenation 1

7

13

19

25

31

37

43

49

55

1

2

3

4

5

6

2nd D Online Low-pH RPLC/MS

27 ACS Paragon Plus Environment