Editors' Highlight pubs.acs.org/ac
Inline Protein A Mass Spectrometry for Characterization of Monoclonal Antibodies Kenneth M. Prentice, Alison Wallace, and Catherine M. Eakin*,† Department of Analytical Sciences, Amgen Inc., 1201 Amgen Court West, Seattle, Washington 98119, United States ABSTRACT: Purification of antibodies is an important first step to produce material for in depth characterization of biotherapeutics. To reduce the resource burden incurred by protein purification, we developed a high throughput protein A affinity capture step coupled to inline mass spectrometry (PrA-MS). Our method enables both UV quantitation of antibodies and product characterization of an intact molecule with microgram quantities of material. When purification and analysis are coupled along with the low material demand, PrA-MS is widely applicable to protein characterization and is uniquely advantageous for moieties that rely on molecular stoichiometry. Two model systems were studied using PrA-MS and are presented here: (a) bispecific antibodies (bsAb) and (b) glycan engineered antibodies. In the bsAb samples, hetero- and homodimer species, along with partial molecule, were readily identified and quantified directly from harvested cell culture fluid (HCCF). In the glycan engineered antibodies, fully afucosylated, as well as asymmetrically and symmetrically fucosylated, glycans were identified from HCCF in experiments that utilized a small molecule inhibitor of fucosyltrasferase. The PrA-MS method represents a high throughput alternative to offline purification and product characterization that may be leveraged across the product lifecycle of engineered antibodies to enable rapid development and production of important therapeutics.
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increase efficiency.13−15 Online microscale fractionation has gained popularity and facilitates high resolution characterization platforms with high throughput capabilities.16,17 Results from each of these techniques are utilized for critical understanding of the mAb characteristics prior to in vivo studies. For many of these techniques, accurate measurement and identification of attributes central to the biotherapeutic require purification prior to analysis to remove interfering culture media components. Offline affinity purification alone, or in conjunction with a single polishing step, is usually sufficient to allow measurement of standard mAb product related attributes.14 While this purification is often based on platform technologies well-known within the industry, it creates an additional step between expression of the mAb and product characterization.18,19 Material demands, throughput limitations, data timeliness, and equipment or raw material cost can all be significant barriers to offline purification, especially during the discovery phase of development. Time intensive offline purification also creates a challenge to process analytical technologies which require real time analysis of critical attributes to enable process control during manufacturing.20 To effectively analyze and characterize proteins without prior purification, we developed a high throughput protein A affinity capture step linked to inline mass spectrometry (PrA-MS). Protein A affinity is well established for mAb purification and was leveraged by applying mass spectrometry (MS) compatible
onoclonal antibodies (mAbs) engineered as therapeutic proteins are able to activate or block biochemical pathways through variable domain binding of target antigens. Although there is an enormous body of data supporting in vitro binding of therapeutic mAbs to clinically relevant antigens, in vivo efficacy and clinical success continues to be challenging.1,2 Diverse population polymorphism,3 drug pharmacokinetics,4 and antidrug antibodies5 are all factors which may affect overall drug efficacy. One strategy to increase the success of therapeutic proteins is to engineer beneficial mechanisms in addition to those activated by target antigen binding. Techniques such as antibody drug conjugates (ADC),6 dendritic or T-cell activation,7,8 and enhanced antibody dependent cellular cytotoxicity (ADCC)9 combine mAb specificity with highly potent secondary mechanisms. Characterization of this diverse group of specifically engineered therapeutic mAbs during development requires a host of analytical techniques.10 Methods such as size exclusion, ion-exchange, or hydrophobic interaction chromatography interrogate the protein in a native state and provide an understanding of the relative purity of the molecule.11 Denatured assays such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or capillary electrophoresis-SDS (CE-SDS) allow for further elucidation of product and process related impurities.11 Mass spectrometry is utilized for protein characterization at both the domain and residue specific level via intact, reduced, limited proteolysis and peptide mapping methods.12 Enrichment strategies leveraging classical offline purification enable targeted characterization of minor species and often incorporate robotic workstations to © 2015 American Chemical Society
Received: December 3, 2014 Accepted: February 3, 2015 Published: February 3, 2015 2023
DOI: 10.1021/ac504502e Anal. Chem. 2015, 87, 2023−2028
Analytical Chemistry
Editors' Highlight
solvents to introduce pure samples for direct intact analysis. This allows confirmation of critical attributes without downstream processing or high protein amounts. We designed the PrA-MS system with high throughput capabilities enabling efficient analysis and rapid communication of experimental outcomes. Here, we present two case studies that highlight the ability of PrA-MS to rapidly identify critical mAb product impurities directly from cell culture samples.
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EXPERIMENTAL SECTION Protein Stocks. The mAbs used in these studies were produced in Chinese hamster ovary (CHO) cells using conventional fed batch techniques and clarified using centrifugation. In order to generate a cell culture fluid control devoid of therapeutic protein, null vector cells were grown in bioreactors using the same conventional fed batch and clarification techniques as above. Bispecific antibodies were generated using charge pair mutations previously described.21 Glycan remodeled antibodies were generated by addition of a small molecule inhibitor of fucose at varying levels on day 0 of bioreactor operations. Drug substance standards were generated using conventional 3-column purification techniques.22 Deglycosylation of Bispecific Antibodies. Bispecific antibodies (bsAb) were deglycosylated in the presence of cell culture fluid prior to PrA-MS analysis using Peptide-NGlycosidase F (PNGaseF, New England Biolabs, Ipswich, MA). Briefly, sample pH was adjusted to 7.5, and the sample was incubated for 2 h at 37 °C at a ratio of 1 μg of protein to 5 U of PNGaseF. Samples were analyzed immediately after incubation. EndoF2 Treatment of Glycan Remodeled Antibodies. Glycan remodeled mAbs were enzymatically reduced to core Nacetylglucosamine (GlcNac) and associated fucose using endoglycosidase F2 (EndoF2, New England Biolabs, Ipswich, MA). Briefly, sample pH was adjusted to 4.5, and the samples were incubated for 1 h at 37 °C at a ratio of 1 μg of protein to 2 mU of EndoF2. Samples were analyzed immediately after incubation. PrA-MS Separation. Neat cell culture samples were injected onto an Applied Biosystems Poros A20 analytical column (Life Technologies, Grand Island, NY) running at 3 mL/min on an Agilent 1200 HPLC with UV detection (Agilent, Santa Clara, CA). The samples were loaded and washed in 20 mM ammonium acetate pH 6.8 for 3.75 column volumes and then eluted with a gradient of 0 to 100% of 0.1% acetic acid in 0.25 min. Total runtime, including column reequilibration, was 2.5 min. The flow passed through a UV detector and was split 1:10 with the low flow directed to an Agilent 6224 time of flight (TOF) MS to accommodate electrospray ionization (ESI). Organic modifier was added to the low flow, postsplit, for final composition of 25% acetonitrile (ACN), 0.75% acetic acid, and 0.25% formic acid (Figure 1). A standard curve from 5 to 200 μg was generated using a purified mAb drug substance for UV concentration determination. mAb drug substance was also spiked into the null vector spent cell culture media from 50% to 150% nominal load (100 μg) to check for assay specificity, linearity, precision, accuracy, and range. Agilent MassHunter (version B06.00) was used to control the system and analyze the UV data. Mass Spectral Analysis. Mass spectra were acquired using an Agilent 6224 TOF in positive electrospray ionization 20 000 m/z (1 GHz) mode. Profile mass spectra were collected from 100 to 7000 m/z after the unbound species had eluted (∼0.5−2
Figure 1. Schematic of the PrA-MS instrument configuration with annotated flow rates. Flow from the HPLC is directed over the affinity column, through a UV detector, and then split between the time-offlight mass spectrometer (TOF) low flow stream and a high flow offline collection/waste stream. Additional mobile phase is added to the low flow spit through an isocratic pump prior to the TOF.
min). A drying gas temperature of 365 °C was utilized; nebulizer and dry gas flow rates were set at 12 L/h and 30 psig, respectively. Capillary, fragmentor, and octopole RF energetics were set at 5000, 415, and 750 V, respectively. Mass spectra were averaged across the elution profile of the analyte and deconvoluted to zero charge species using the maximum entropy (MaxEnt) algorithm within the MassHunter software. Deconvolution settings were adjusted for survey analysis over a large mass range (∼20−160 kDa) for partial and intact molecule identification as well as targeted analysis (theoretical mass ±20 kDa) for accurate mass analysis and relative quantification. The relative abundance of each species was determined from the MaxEnt calculated peak areas using eq 1.
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peak area of individual species × 100% sum of peak areas of identified species
(1)
RESULTS AND DISCUSSION To facilitate online purification and analysis, a high throughput PrA-MS method was developed. Rapid analysis was facilitated by a flow rate of 3 mL/min over an 800 μL column with fast gradient for protein release. To reduce flow into the ESI source for mass analysis, flow was split postcolumn with acidified ACN added to the low flow stream (Figure 1). Organic modifier was added to the mobile phase postcolumn rather than precolumn to prolong column lifetime while retaining a composition appropriate for ESI ionization. The presence of the UV detector inline prior to the split flow allowed one to calculate the protein concentration from the harvested cell culture fluid (HCCF) by utilizing a standard curve generated from purified material. During method development, a mAb control was spiked into the null cell culture and analyzed using the above instrument configuration at loads varying from 10 to 200 μg with concentrations as low as 50 μg/mL for PrA-MS. The mAb peak was well resolved from the unbound host cell proteins (HCP) and other non-Fc containing matrix components (Figure 2A). Analysis of UV280 nm data gave a linear response (r2 > 0.999) with good recovery (>98%) and a limit of quantification (LOQ) of ∼10 μg, confirming the method is fit for titer measurement. Mass analysis revealed multiple charge states within 2000−4500 m/z, and deconvoluted mass spectra of the mAb peak identified major species at 146 406 Da with minor peaks at 146 260 and 146 568 Da representing A2G0F, A2G0, and A2G1F glycoforms, respectively. A mass accuracy of
