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Isolation and Characterization of Monoclonal Antibody Charge Variants by Free Flow Isoelectric Focusing Brian D. Hosken, Charlene Li, Berny Mullappally, Carl Co, and Boyan Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03946 • Publication Date (Web): 09 May 2016 Downloaded from http://pubs.acs.org on May 14, 2016

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Analytical Chemistry

Isolation and Characterization of Monoclonal Antibody Charge Variants by Free Flow Isoelectric Focusing Brian D. Hosken1*, Charlene Li1, Berny Mullappally1†, Carl Co2‡, Boyan Zhang1○

1

Department of Protein Analytical Chemistry, 2 Department of Biological Technologies, Genentech, 1 DNA Way, South San Francisco CA 94080 *Corresponding Author: [email protected], Fax # 650-491-0120

ABSTRACT Capillary isoelectric focusing (cIEF) is widely used in the biopharmaceutical industry to measure the charge distribution of therapeutic proteins. The implementation of this technology has created a new challenge. Capillary volumes are on the order of hundreds of nanoliters and cannot be scaled up for the preparative collection of charge variants. This makes it difficult to identify the charge variants in a cIEF electropherogram. Therefore, preparative IEF methods are needed to fractionate charge variants for characterization. We used free flow electrophoresis (FFE) to isolate monoclonal antibody charge variants observed in a cIEF electropherogram. The same antibody was also fractionated using the Rotofor® and Offgel instruments for comparison. A strategy for purifying the fractionated charge variants and downstream characterization is described. Acidic and basic variants were identified and related back to the analytical cIEF charge profile. This study establishes free flow isoelectric focusing as a valuable tool for characterizing therapeutic proteins.

INTRODUCTION The manufacturing of a therapeutic antibody using a mammalian cell culture process produces a heterogeneous product that is highly dependent on the process.1 These product-related variants can be attributed to differences in primary, secondary, tertiary or quaternary structure. Post translational modifications also contribute to the heterogeneity of a recombinant, humanized monoclonal antibody (rhumAb). Each of these variants can alter the charge state of a rhumAb relative to its predominant form. The modification of an acidic or basic amino acid side chain directly impacts the net charge of a protein. Charge profiling methods can detect charge variants caused by many types of modifications making them an integral component of quality control systems and characterization/comparability studies. Ion-exchange chromatography (IEC)2-4 and capillary isoelectric focusing (cIEF)5-8 are the two charge profiling methods most commonly used in the biopharmaceutical industry. These methods and common post translational modifications have been reviewed with respect to the analysis of rhumAbs.9 IEC methods are popular because they are robust, provide good analytical performance and use commonly available instrumentation. A recently described salt mediated pH gradient IEC method is faster than the traditional salt gradient methods and shown to be suitable for multiproduct use.4 One advantage of analytical IEC methods is that they can easily be scaled up for the preparative separation of charge variants. There are several good examples of variant isolation and characterization using IEC.2,10,11 cIEF is a popular alternative to the IEC methods traditionally used for charge profiling. In some cases, cIEF is the only option because IEC is not compatible with molecules that have a strong charge patch12 or hydrophobic antibody drug conju-

gates. cIEF typically provides higher resolution than chromatography methods due to the focusing effect of the pH gradient. cIEF separations can also be suitable for multiple products; allowing a single method to be used for many rhumAbs. cIEF can be performed using a traditional CE instrument with mobilization of the focused protein past a detector13 or using a specialized instrument capable of whole capillary imaging.7,8 Both formats have one significant limitation. The nanoliter volumes that enable rapid, automated and high resolution separations also prevent capillary methods from being used for variant isolation and characterization. This is a significant limitation that could prevent the implementation of cIEF during later stages of clinical development unless a complimentary, preparative method is available. One solution is to use preparative IEC to fractionate charge variants and then relate them to the peaks in the analytical cIEF profile.8 This approach may or may not be successful because IEC and cIEF methods employ fundamentally different separation mechanisms. Ideally, a preparative IEF separation would be used to isolate and characterize the variants observed in cIEF charge profiles. For this purpose, the preparative IEF method should have the following characteristics: 1) It should be capable of resolving the same species observed in the analytical method. 2) It needs high resolution for separating rhumAb isoforms with minor pI differences. 3) It should be capable of fractionating large amounts of protein for downstream characterization. 4) The separation and recovery steps should not degrade the protein or create artifacts. Preserving the native state is desirable so that higher-order structure and bioactivity can be evaluated. 5) The pH gradient should be customizable so that the preparative method is a good surrogate for the capillary format.

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There are three platforms for preparative IEF that meet these criteria to varying degrees: the Biorad Rotofor® system,14,15 the Agilent Offgel16,17 and the free-flow electrophoresis (FFE) instrument18 which has been sold by several vendors in recent years. These preparative methods have been reviewed with respect to their more common applications in proteomics.19 Preparative IEF has been widely used in proteomics studies to reduce the complexity of a lysate sample or to eliminate very abundant proteins such as albumin and immunoglobulins thereby allowing the identification of minor proteins. The biopharmaceutical industry has a more challenging application; to fractionate minor isoforms of the same protein in quantities sufficient for downstream characterization. There are few examples of this application. Fractionation and characterization of antibody charge variants using the Offgel instrument was recently described.20,21 Two isoforms of aldose reductase were isolated using the Rotofor®.22 Three charge variants of a therapeutic antibody were isolated by FFE and attributed to deamidation.23 While this study demonstrated the usefulness of FFE for isolating charge variants, it did not discuss the critical separation parameters and optimization of the pH gradient. This study also did not relate the preparative FFE separation back to the analytical charge profiling method. FFE has the greatest potential for matching the preparative IEF characteristics stated above. It is a continuous, solution phase separation that uses carrier ampholytes to establish a pH gradient in a thin layer between two plates. The pH gradient and focused proteins are collected directly into 96-well plates. This format allows very high resolution when narrow range ampholytes are used. The separation buffers can also be customized to approximate the capillary sample matrix. FFE instrumentation has improved from earlier models.24 The optimized design and operation for commercially available instruments was described in 1996.25 FFE separation protocols for proteomics applications have been published. 18,26,27 FFE has also been featured in several reviews.19,28-30 In this study we systematically optimized a FFE separation for rhumAbs with pI values in a broad range (7.2 to 9.3). A high throughput method for recovering the rhumAb isoforms from the separation media was developed. The fractionated isoforms were related back to the analytical cIEF profile. The separation efficiency of FFE was also compared to the Rotofor® and Offgel systems. The charge variants isolated by FFE were characterized by analytical and biological methods resulting in the identification of many post translational modifications. The experiments described here can be broadly applied to other rhumAbs and therapeutic proteins. EXPERIMENTAL Materials: The three recombinant humanized monoclonal antibodies (rhumAbs) used in this study were all IgGs produced in CHO cells at Genentech. Antibodies with different pI values were selected for this study: 8.2 for rhumAb1, 7.2 for rhumAb2 and 9.3 for rhumAb3. The reagents and vendors used in this work are listed in their respective experimental sections. Imaged Capillary Isoelectric Focusing: iCIEF was performed using an ICE280 instrument and coated capillary cartridge (5cm × 100µm ID) from Convergent Bioscience (now Protein Simple). Charge variants were buffer exchanged into 10mM phosphate buffer and then diluted to 0.5mg/mL. Equal volumes of the sample and iCIEF separation buffer were combined so that the final concentration of each component was:

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0.25mg/mL protein, 0.3% methyl cellulose, 2M urea, 1% 3-10 ampholytes and 0.15% of each pI marker. The anolyte was 80mM phosphoric acid in 0.1% methyl cellulose. The catholyte was 100mM sodium hydroxide in 0.1% methyl cellulose. The anolyte, catholyte, 1% methyl cellulose, and pI markers were purchased from Convergent Bioscience. Pharmalyte® 310 was obtained from GE Healthcare. Samples were focused for 1 minute at 1500 V followed by 7 minutes at 3000 V. After imaging, the pixel location was converted to a pH scale using 7.05 and 9.22 pI markers. Free flow isoelectric focusing: FFE was performed on a system purchased from Beckton Dickinson but now supported by FFE Service, Gmbh, Germany. We optimized the conditions to provide a linear gradient from pH 6-10.5. The separation buffer compositions and inlet positions are summarized in Table 1. A similar version of the FFE instrument and its operation were described in detail by Weber and Bocek.25 Table1. Separation Media and Inlet Positions for the FFE. Separation Media

Inlet

Composition

Anode Stabilization Buffer*

1

100mM H2SO4

Separation Buffer 1*

2-3

1% w/v 3-5 & 6-8 ampholytes (1:5 ratio)


4

50:50 mixture of buffers 1 & 3


5-6

1% w/v 8-10.5 ampholytes

Cathode Stabilization Buffer*

7

100mM NaOH, 100mM LArginine

Counter Flow

CF

0.2% w/v HPMC, 25% Glycerol

Anolyte

-

100mM H2SO4

Catholyte

-

100mM NaOH

*Solutions were prepared from the same stock solution of counter-flow media.

Pharmalyte® 8-10.5 (GE Healthcare), Bio-Lyte® 3-5 and 6-8 (Biorad), hydroxypropyl methylcellulose, HPMC (Amresco), glycerol (JT Baker) and L-Arginine (Sigma) were used to prepare the separation media. The separation buffers were introduced into the chamber at a flow rate of 40mL/hour. The chamber was cooled to 10°C and 1500V was applied. The rhumAb1 sample was diluted to 2.5mg/mL with separation buffer 2 and introduced into the chamber at a flow rate of 0.8mL/hour through sample inlet port 2. A negatively charged dye, 2-(4-Sulfophenylazo) chromotropic acid trisodium salt (SPADNS) from Sigma, was added to the sample for the purpose of tracking its progress through the separation chamber. A total of 10mg of rhumAb1 was fractionated at a rate of 2 mg/hr. Fractions were collected into three 96 deep-well plates from VWR. The pH of each well, from the first and third plates, was measured with a Corning semi-micro probe. rhumAbs 2 and 3 (4mg each) were fractionated during a single run on another day. The samples were introduced into the chamber one after another with a short pause in between. The SPADNS dye, which runs along the interface of the anode stabilization buffer and separation buffer 1 was used as an indicator for starting and stopping fraction collection. Offgel Electrophoresis: rhumAb1 was fractionated on two 24cm, 6.3 – 8.3 IPG strips (Bio-Rad). The rhumAb1 sample was first buffer-exchanged into water using a NAP™-5 col-


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umn (GE Healthcare) then diluted with an ampholyte mixture to a final concentration of 1mg/mL protein, 1% 3-10 ampholytes (GE Healthcare), 12% glycerol and 0.2% polysorbate 20. The same ampholyte mixture diluted with an equivalent amount of water was used to rehydrate the IPG strips for 15 minutes. A 150µL aliquot of the sample was loaded into each of the 24 wells (3.6mg per strip). Focusing was performed at constant current until 64 kV hours were reached (about 37 hours). The chamber was maintained at 10°C. Protein containing fractions were identified by their absorbance at 280nm. Fractionated material was desalted with PD Spin Trap™ G-25 columns (GE Healthcare) prior to iCIEF analysis. Rotofor® Electrophoresis: The Bio-Rad Rotofor® was used with the Mini focusing chamber. It has a volume of 18mL and a membrane core that divides the chamber into 20 compartments. The anolyte was 0.25M 2-(NMorpholino)ethanesulfonic acid (MES) and the catholyte was 0.1M sodium hydroxide. Bio-Lyte 7-9 ampholytes were diluted to 2% w/v and prefocused in the chamber for 1 hour. Then 5mg of rhumAb1 was loaded into compartments 7-9 and focused for 5.5 hours at a constant power of 12W. Fractions were harvested, then buffer-exchanged and concentrated using Amicon® centrifugal filters (10kD MWCO) from Millipore, prior to iCIEF analysis. Recovery of rhumAb1 from FFE fractions: A schematic of the recovery process is shown in the Supporting Information. Protein containing fractions were identified by combining equal amounts (50µL) of Coomassie Plus™ Protein Assay reagent (Pierce) and fractionated material in a standard 96 well plate. Absorbance was measured at 595nm after two hours. The fractionated rhumAb1 variants were then recovered from the FFE separation buffer by protein A affinity chromatography in solid phase extraction (SPE) cartridges. Empty 3mL SPE cartridges (Isolute) were packed with 300µL mAbSelect SuRe™ resin (GE Healthcare). The resin was supported by two 20µm frits (also from Isolute) placed above and below the resin cake. Cartridges were placed in a vacuum manifold and equilibrated with 3mL phosphate buffered saline (PBS), pH 7.4. Corresponding fractions from the three plates were pooled and loaded onto the SPE cartridges. The columns were washed with 3mL PBS. The rhumAb1 variants were then eluted with 1mL of 0.1M acetic acid and collected directly into 4mL Amicon® centrifugal filters (30kD MWCO) from Millipore. Samples were immediately buffer exchanged into 10mM phosphate buffer (pH 7.4) and concentrated to at least 1mg/mL. The fractionated rhumAb1 material was stored at 28°C. Downstream characterization experiments were completed within 2 weeks of fractionation. RESULTS & DISCUSSION Optimization of FFE Separation: Free flow electrophoresis has many parameters that can be optimized to create the desired pH gradient. Our goal was to create a linear gradient from pH 6 to 10; a range that covers nearly all of the antibodies in our pipeline. We began by evaluating various combinations of narrow range ampholytes from Bio-rad (Biolyte®), Serva (Servalyt™) and GE Healthcare (Pharmalyte®). For the pH gradients shown in Figure 1A, a single separation buffer containing 1% w/v ampholytes (alone or mixed 1:1) was introduced into inlets 2 - 6. The anodic and cathodic stabilization buffers were 100mM H2SO4 and 100mM NaOH. The other separation media, flow rate, voltage and temperature were identical to those described in the Experimental section.

A combination of Biolyte® 6-8 and Pharmalyte® 8-10.5 was chosen because it provided the most linear gradient in the desired range. FIGURE 1. pH gradient optimization. Combinations of narrow range ampholytes were evaluated (A). The cathodic end of the gradient was stabilized by adding 100mM arginine to the cathode stabilization buffer (B). The anodic side of the gradient was stabilized by adding 3-5 ampholytes to separation buffer 1 (C).

We also evaluated the impact of using a single homogenous separation buffer containing both narrow range ampholytes versus a three buffer system containing the individual ampholytes. The three buffer system required additional media preparation but it created an initial step gradient when introduced



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into the chamber through separate inlet ports (refer to Table 1 for the buffer composition and inlet ports). Under applied voltage, the resulting pH gradient was more linear than the one achieved using a single homogenous buffer (data not shown). The current also stabilized sooner and was lower indicating that a stable pH gradient formed quicker with the three buffer system. The basic end of the pH gradient was not reproducible from run-to-run. We observed variability in the slope with significant dips in pH. This was attributed to cathodic isotachoporesis (ITP), a common problem in isolectric focusing that leads to the loss of the most basic ampholytes.31 Arginine (pI 10.8) was added to the cathode stabilization buffer to serve as a sacrificial ampholyte. The pH gradients in Figure 1B were obtained using the conditions described in the Experimental section with or without the addition of 100mM arginine. The addition of arginine improved the pH range and slope of the basic portion of the pH gradient by accumulating between the cathode stabilization buffer and the most basic carrier ampholyte. The anodic side of the gradient did not exhibit the same variability observed on the cathodic side however acidic carrier ampholytes were still added to separation buffer 1 to provide sacrificial ampholytes to mitigate anodic ITP. The pH gradients in Figure 1C were obtained using the conditions described in the Experimental section with or without the addition of 20% Biolyte® 3-5. The 3-5 ampholytes created some buffer between the anode and pH 6 ampholytes however they also occupied valuable space in the separation chamber. Adding glutamic acid or iminodiacetic acid to the anode stabilization buffer (similar to what was done for the cathode stabilization buffer) may have mitigated anodic ITP without giving up 10 fractions. Separation media flow rates of 40, 60 and 80mL/hour were evaluated. The pH gradient was similar for all three flow rates (data not shown) so we chose 40mL/hour for the final method. This increased the protein’s residence time in the separation chamber to about 22 minutes. It also consumed less buffers and reduced the sample dilution. Voltages of 1200, 1500 & 1800V were evaluated. The resulting pH gradients were similar (not shown) so we chose 1500V. Introducing the sample into different inlet ports (acidic, neutral or basic) had no effect on the pH gradient and little effect on the protein distribution (not shown). Antibodies were fractionated at a rate of 2mg/hr for this study however loads of 4 and 8mg/hr were also evaluated. Higher loads gave satisfactory results but there was some loss in resolution of the basic variants at higher loads (data not shown). When challenged with isolating minor isoforms it is best to perform a longer run than risk overloading the gradient and losing resolution. Applicability of FFE Separation to rhumAbs with a Wide Range of pI Values: After optimizing the initial solution conditions and electrophoretic parameters (described in the Experimental section), a linear pH gradient from 6.5 – 10, with a slope of 0.05pH units per fraction, was achieved. This gradient covers nearly all of the antibodies in our pipeline. We demonstrate the generic nature of this protocol by fractionating rhumAbs with pI values of 7.2, 8.2 and 9.3. The pH gradient and protein distributions for all three molecules are shown in Figure 2.

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FIGURE 2. pH gradient and protein distribution for rhumAb 1 (pI 8.2) and rhumAbs 2 (pI 7.2) and 3 (pI 9.3). rhumAb1 was fractionated on one day (A). rhumAbs 2 and 3 were fractionated successively on another day (B).

Ten mg of rhumAb1 was fractionated in one day during a single, continuous run. The protein isoforms, collected successively in 3 deep well plates, were distributed across 18 wells (Figure 2A). The rhumAb1 isoforms were purified and then characterized by analytical and biological testing as described in the following sections. In a separate experiment, four mg of rhumAbs 2 and 3 were fractionated successively during the same run. Figure 2B shows the pH gradient and protein distributions. We verified that the pH gradient was stable during the course of these separations by measuring the pH of the first and last plates. Identical pH gradients were obtained from plates collected 10 hours apart (data not shown). Therefore, fractions from multiple plates can be pooled with confidence that the separated variants won’t be mixed. iCIEF analysis of the fractionated antibodies was performed to assess the separation efficiency. The electropherograms for rhumAb1 are shown in Figure 3 and the peak areas are summarized in Table 2. Refer to the supporting information for rhumAbs 2 and 3 data. The three rhumAbs showed a trend towards higher resolution (peak enrichment) at lower pI values and worse resolution at higher pI values. The isoforms of rhumAb2 (pI 7.2) were highly enriched despite minor differences in pI (≤ 0.04). Additionally, the rhumAb2 fractions did not contain more than two species in significant amounts. In contrast, the isoforms of rhumAb3 (pI 9.3) were not as enriched. These FFE fractions also contained three to four species. The poor resolution of rhumAb3 may be attributed to the relatively small differences in pI values across the heterogene-


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ous mixture of charge variants. The focusing kinetics (change in mobility with pH) for rhumAb3 isoforms are slower and require further optimization of the separation conditions. Reducing the flow rate to provide additional focusing time or increasing the separation voltage are expected to improve focusing. Changing the carrier ampholyte composition so that there are additional ampholyte species with pI values between the protein isoforms may also improve resolution. FIGURE 3. iCIEF electropherograms of rhumAb1 FFE fractions. Only the 7 most enriched fractions are shown. The relative areas of each charge variants are summarized in Table 2.

B1

B2

Main

A4 A3 A2 A1

pI Development of a High-Throughput Recovery Process for FFE Fractions: The ampholytes and viscous buffer components interfere with downstream analytical methods and must be removed prior to characterization and biological testing. An efficient rhumAb recovery process was designed around the use of Protein A affinity chromatography in solid phase extraction cartridges (depicted in Supporting Information). This purification scheme addressed several challenges including the need to process many fractions in parallel each containing large volumes of dilute protein in a matrix composed of methyl cellulose, glycerol and ampholytes. The protein A resin concentrated the rhumAb on the cartridge while the interfering buffer components were removed with wash buffer. The rhumAb1 isoforms were eluted in a small volume of dilute acetic acid directly into a centrifugal filter for rapid buffer exchange into a buffer compatible with analytical methods. After the fractionation and purification steps, the final recovery was 7.7mg (77%) of the initial 10mg protein load. The total protein recovered from fractions 52 - 68 was calculated from the final volume of each fraction and its respective protein concentration. All 18 fractions containing protein were analyzed by iCIEF so that fractions containing highly enriched charge variants could be identified. The electropherograms of the most enriched species are shown in Figure 3 and the peak areas are summarized in Table 2. The peaks were integrated using limits derived from the unfractionated rhumAb sample. The enrichment factor given for each peak in Table 2 is the %area of the enriched peak divided by the %area from the unfractionated sample.

TABLE 2. Relative areas of rhumAb1 from iCIEF profiles shown in Fig 3. % Peak Area A1

A2

A3

A4

Main

B1

B2

rhuMAb1

1

6

18

3

54

14

4

Fraction 52

78

13

5

-

4

-

-

Fraction 56

-

79

15

-

6

-

-

Fraction 59

-

-

73

3

24

-

-

Fraction 61

-

-

23

11

65

-

-

Fraction 63

-

-

2.4

1.3

95

2

-

Fraction 65

-

-

-

-

7

92

1

Fraction 68

-

-

-

-

-

8

92

Comparison of the Preparative IEF Methods: rhumAb1 was also fractionated by the Rotofor® and Offgel systems for comparison. The fractionated isoforms were purified by the same protein A chromatography step and then analyzed by iCIEF. Table 3 summarizes the percentage of each charge variant in its most enriched fraction. FFE provided the highest practical resolution (∆pH/fraction) because it had the shallowest gradient and the most fractions. The ∆pH/fraction for these separation protocols was 0.05 for FFE, 0.09 for the Offgel, and 0.10 for the Rotofor®. FFE also had the highest enrichment of individual charge variants followed by the Offgel. rhumAb1 did not focus well in the Rotofor® for unknown reasons. TABLE 3. Comparison of preparative IEF methods. The % area is given for the most abundant fraction. Enrichment factors relative to the starting material are shown in parenthesis. %Area (Enrichment) A1

A2

A3

A4

Main

B1

B2

rhuMAb1

1

6

18

3

54

14

4

Rotofor®

27 (27x)

25 (4x)

34 (2x)

4 (1x)

76 (1x)

37 (3x)

19 (4x)

Offgel

50 (50x)

70 (12x)

78 (4x)

9 (3x)

81 (2x)

57 (4x)

*

FFE

78 (78x)

79 (14x)

73 (4x)

11 (4x)

95 (2x)

92 (7x)

92 (23x)

* The pI of this charge variant exceeded the pH range of the IPG strip (6.3-8.3)

Each platform has its advantages and disadvantages. For example, the Rotofor® is capable of fractionating large amounts of protein in the shortest time. It is the simplest to assemble and run however its resolution is somewhat limited by the number of fractions. In theory, the resolution can be improved by re-fractionating the focused proteins with narrower range ampholytes although this approach wasn't successful for rhumAb1. The Offgel system offers good resolution when narrow range IPG strips are appropriately chosen. The pI of the most basic variant in rhumAb1 exceeded the pH of the IPG strip used in this study. The protein load is lower and the separation takes longer than the other two methods. The gel also retains some of the protein which lowers recovery compared to the solution based methods. FFE has the highest resolution due to its 96 fractions. It is also capable of fractionating the most material because it is a continuous separation. Protein recov-



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ery is very good and the pH gradient can be customized due to the use of carrier ampholytes and a liquid medium. The protein only spends about 20 minutes in the separation chamber at 10°C so protein degradation is minimized. Plates can also be frozen immediately to limit degradation after the separation. Analytical Characterization of rhumAb1 Charge Variants: The seven fractions representing the most highly enriched charge variants were analyzed by SEC, MCE-SDS, Reduced and Intact LC/MS, and LC/MS/MS tryptic peptide mapping. Representative characterization data is shown for acidic peak 2 in Figure 4. The biological activity of each fraction was also determined with an antigen binding assay. Acidic peak 1, which accounts for only 1% of the unfractionated rhumAb1 sample, was enriched to 78% in fraction 52. A low molecular weight fragment was detected by SEC and MCE-SDS exclusively in this fraction. The mass of this fragment (96,398Da) was determined by intact LC/MS analysis and found to be consistent with a clip in the hinge region. This is a common modification which has been previously described for monoclonal antibodies.32,33 No other modifications were observed in acidic peak 1. Acidic peak 2 was enriched to 79% in fraction 56. It contained a mixture of species present at low levels. Glycopeptides containing sialic acid were detected in the peptide map (Figure 5A) of fraction 56 but not in fraction 63 (main peak). Figure 5B compares the averaged, full MS scan (from 29.0 – 29.5 min) of the coeluting glycopeptides peak. Masses consistent with the expected heavy chain peptide containing the N-linked glycans, G2+SA (+2060.8) and G2+2SA (+2352.1) were identified in fraction 56 but not in fraction 63. RP-LC/MS analysis of the intact rhumAb also revealed the presence of disulfide heterogeneity. Fig 5C compares the deconvoluted mass spectrum of the free light chain peak from fractions 56 and 63. The light chain from fraction 56 contained elevated levels of cysteinylation and glutathionylation relative to fraction 63. Capping of Cys 212 with glutathione or cysteine prevents the formation of the LC-HC intermolecular disulfide bond. These modifications are expected in the acidic region because glutathione adds two carboxylic acid groups and cysteine contributes one. Glycation was also observed in the LC/MS analysis of the intact and deglycosylated antibody from fraction 56. The addition of a hexose molecule adds 162 Da to the mass of the antibody and converts a Lys side chain from a primary amine to a secondary amine making it less basic. Glycation can occur at a hot spot34 however it is more likely to be spread across many sites making it difficult to detect at the peptide level.35 Acidic Peak 3 was enriched to 73% in fraction 59. It also contained a mixture of charge variants. Elevated levels of deamidation at Asparagine 388 were observed in fraction 59 by peptide mapping. The deamidated peptide eluted 1 minute earlier than the native one and had a mass difference of +1 Da in the high-resolution, full MS scan. The fragmentation patterns of the low resolution MS/MS spectra for the native and deamidated peptide were similar confirming their identity as HC375-396. Deamidation is expected in the acidic region because a neutral Asn is converted to an acidic Asp.36,37 Glycation was also identified by RP-LC/MS analysis of the deglycosylated antibody from fraction 59. FIGURE 4. Selected characterization data for rhumAb1. Fraction 56 (acidic peak 2) is compared to fraction 63 (main peak) in each

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panel. The UV trace from the peptide map showed no differences in the coeluting glycopeptides peak for HC293-301 (A). The averaged, full ms scan of HC293-301 (29.0-29.5min) showed differences in the N-linked glycoforms (B). Sialylated glycans were observed in fraction 56 but not in fraction 63. The deconvoluted LC/MS spectra of the free light chain peaks from the intact mass analysis were significantly different (C). The light chain in fraction 56 was predominantly capped with glutathione (GSH) and cysteine.

Acidic Peak 4 was enriched to 11% in fraction 61. This fraction contained high levels of the cysteinylated rhumAb. The expected disulfide bond between HC and LC didn’t form because cysteine 212 on the LC and Cys 224 on the HC were capped with a free cysteine during cell culture. Noncovalent interactions prevented the fragments from being separated under native SEC conditions but they were resolved by the denaturing MCE-SDS separation. RP-LC/MS analysis of frac-


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tion 61 confirmed the identities of the two fragments as cysteinylated-heavy chain-heavy chain-light chain (Cys-HC-HCLC) and cysteinylated-light chain (Cys-LC). This variant was previously isolated using the Offgel system and identified by colleagues.38 Basic peaks 1 (fraction 65) and 2 (fraction 68) were both enriched to 92% purity. The predominant species in basic peaks 1 and 2 were related to the presence of one or two C-terminal lysine residues on the heavy chain. This is a common basic variant in recombinant antibodies.39 The C-terminal lysines were detected by peptide mapping and intact LC/MS analysis. The basic peaks also contained low levels of the cyclized Nterminus. The heavy chain N-terminal peptide with pyroglutamate (-18 Da) was identified in the peptide map. The cyclization reaction results in the loss of a carboxylic acid group creating a basic variant.40 Chemical Nature of rhumAb1 Charge Profile: The post translational modifications identified in the rhumAb1 iCIEF profile are summarized in Figure 5. This study revealed that individual peaks from the iCIEF profile are actually quite heterogeneous. Most of the isolated peaks contained more than one modification and the same modifications were also observed across multiple peaks. Despite this heterogeneity, all the isoforms still focused into discrete peaks with consistent spacing (~0.2pH units apart). We propose the following explanation. All of the PTMs we identified result in the addition or neutralization of either a carboxylic acid or primary amine at various locations on the antibody. These modifications cause a net change of +/- 1, 2 or 3 charge units from the unmodified rhumAb (main peak). Therefore, a rhumAb with a G2+2SA glycan or a rhumAb with a glutathione capped cysteine or a rhumAb containing both a deamidation and a glycation site would all have a shift of +2 charge units and the same pI. We note that it is statistically unlikely for more than one low level modification to occur on the same rhumAb in a measurable amount. Not all of the peaks in the rhumAb1 profile can be explained so simply. Acidic peak 4, containing the cysteinylated rhumAb, only has a pI shift of 0.1pH units from the main peak. Perhaps this modification resides in an environment where the pKa has shifted to an intermediate value. Biological Characterization of rhumAb1 Charge Variants: An antigen binding assay, that probes both the Protein A-Fc binding and antigen binding activity of rhumAb1 charge variants, was employed to assess the biological activity of the isolated charge variants. As shown in Table 4, all charge variants exhibited 74 to 107% activity relative to the unfractionated rhumAb1 sample. The difference between duplicate measurements ranged from 4 to 20%. All fractions are considered equally active. None of the identified modifications are expected to alter the biological activity of this binding assay. For example, the C-terminal Lys, N-terminal pyroGlu or disulfide heterogeneity does not alter the Fc binding domain. The other PTMs such as deamidation (Asn338 is located in CH2 domain of heavy chain) or glycation were not observed in the CDR and therefore would not alter antigen binding. The hinge fragment in acidic variant 1 is missing one of its Fab domains but still has one intact Fab and Fc domain needed to bridge the donor bead (with immobilized antigen) and the acceptor bead (with immobilized protein A).

FIGURE 5. Summary of modifications identified in rhumAb1 charge variants.

pI

TABLE 4. Biological activity of rhumAb1 FFE fractions. The difference between duplicate measurements is listed in parenthesis. Relative Antigen Binding A1

A2

A3

A4

Main

B1

B2

Fract 52

Fract 56

Fract 59

Fract 61

Fract 63

Fract 65

Fract 68

83% (8%)

74% (4%)

100% (20%)

75% (6%)

107% (5%)

87% (16%)

85% (5%)

These biological activity results demonstrate that the FFE separation and recovery processes did not alter the native state of the antibody variants. This is a critical attribute for preparative fractionation techniques because it enables the types of biological and functional testing required to characterize product related variants and assign critical quality attributes (CQAs). CONCLUSIONS A free flow isoelectric focusing method was developed to isolate the acidic and basic variants observed in an imaged cIEF electropherogram. Using this method, individual peaks from the charge profile of rhumAb1 were highly enriched in quantities sufficient for downstream characterization. We identified many post-translational modifications and demonstrated that the fractionated material retained its native state through a binding assay. Individual peaks were also shown to contain multiple charge variants ascribed to those modifications causing the same shift in pI values. We demonstrated the generic nature of free flow isoelectric focusing by fractionating antibodies with low and high pI values. The charge variants of rhumAbs 1(pI 8.2) and 2 (pI 7.2) were highly enriched in discrete FFE fractions. rhumAb3, with a pI of 9.3, did not focus as well although fractions were significantly enriched compared to the starting material. We also used the Rotofor® and Offgel instruments to fractionate rhumAb1 and assess their suitability for isolating



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charge variants. FFE was superior in that it provided the highest resolution and the pH gradient can be customized to match the analytical cIEF method. Protein isoforms can be separated continuously with excellent recovery of the fractionated material.

ASSOCIATED CONTENT Supporting Information Scheme for isolating charge variants from FFE fractions. Experimental information for physio-chemical and biological characterization. Examples of FFE fractionation for rhumAb2 and rhumAb3. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected] , Stemcentrx, 450 East Jamie Ct, South San Francisco, CA 94080. Fax # 650-491-0120

Present Addresses †Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, New York 10591 ‡ Biogen IDEC, 225 Binney Street, Cambridge MA 02142 ○ Beijing Mabworks Biotech Co. Ltd, Building 3, Huilongsen Sci & Tech Park, Kechuang 14th Street, Beijing E-town, China 101111

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT The authors thank David Michels and Will McElroy for providing the Offgel fractionation protocol and training. Robert Wildgruber of FFE Service, GmbH was a valuable resource during our gradient optimization experiments. We are also grateful to Patricia Rancatore for her technical review and scientific discussion.

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