THE JOURNAL OF BIOLOGICAL CHEMISIRV VOL 287. NO. 2, pp. 1381 —I 396, January 6, ZDI 2
`0 2m 2 ty The American Society for Biochemistry and Molecular Biology, Inc. Published in the USA
`
`Elucidation of Acid-induced Unfolding and Aggregation of
`Human lmmunoglobulin |gG1 and lgGZ FcIEI
`Received for publication, August 25, 201 1, and in revised form, November 1, 201 1 Published,JBC Papers in Press, November 14, 201 1, DOI 10.1074/ij1 1 1297697
`
`Ramil F. Latypov“, Sabine Hogan’, Hollis Lau*, Himanshu Gadgil“, and Dingjiang Liu§2
`From *Drug Product Development, Amgen Inc., Seattle, Washington 981 79 and §Drug Product Development, Amgen Inc.,
`Thousand Oaks, California 91320
`
`Background: Monoclonal antibodies and PC fusion proteins contain an IgG Fc moiety, which is associated with various
`degradation processes, including aggregation.
`Results: Fc unfolding is triggered by the protonation of acidic residues and depends on the IgG subclass and CH2 domain
`glycosylation.
`Conclusion: Fc aggregation in acidic conditions is determined by CH2 stability.
`Significance: Understanding PC aggregation is important for improving the quality of Fc-based therapeutics.
`
`
`
`no:'9:KmuomusKqfiJO'O‘ll'MMWFdflllwas
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`Popwlumoa
`
`rently approved therapeutic mAbs belong to the IgG class and
`have a structure schematically depicted in Fig. 1. Intact mAbs
`are composed of two identical light chains and two identical
`heavy chains, which are covalently linked via several inter- and
`intrachain disulfide bonds. The light chains and heavy chains
`form two (VL and CL) and four structurally homologous
`domains (VH, CH1, CH2, and CH3), respectively. The overall
`IgG structure consists oftwo identical Fab domains (VL, CL, VH,
`and CH1) and one PC3 domain (CH2 and CH3) that are con—
`nected by a flexible hinge region. The Fc portion harbors one
`conserved Asn-297 glycosylation site in each of its CH2
`domains. The Fab and PC regions of mAbs have different bio-
`logical functions. The Fab regions are responsible for binding to
`the antigen, whereas the Fc portion plays a role in modulating
`immune cell activity. In addition to mAbs, there are other
`classes of biotherapeutics, such as Fc fusion proteins, that also
`contain Fc. These molecules are composed of therapeutically
`active peptide or protein moieties that are attached to either the
`C termini or N termini of an IgG PC. In such cases, the presence
`of an IgG Fc moiety may result in improved physiological func—
`tion, ease of production, solubility, etc. However, the Fc region
`is also associated with a range of degradation processes, includ—
`ing oxidation (7) and aggregation (8). A detailed understanding
`of how certain structural changes within the Fc domain lead to
`aggregation represents an important step toward improving the
`quality of these therapeutic agents.
`Fc-based biologics offer significant manufacturing and phys—
`iological advantages. Their purification process is greatly sim—
`plified by the available selection of affinity resins targeting the
`Fc portion (9, 10). The presence of a relatively large (~50 kDa)
`and highly soluble Fc moiety confers increased solubility and
`half—life (11). In addition, the Fc region engages in specific bio-
`logically relevant interactions that may require CH2 glycosyla-
`tion (antibody—dependent, cell-mediated cytotoxicity; comple-
`ment activation; in viva clearance; etc.) (12—15). Uncovering
`
`Understanding the underlying mechanisms of Fc aggregation
`is an important prerequisite for developing stable and effica—
`cious antibody-based therapeutics. In our study, high resolution
`two—dimensional nuclear magnetic resonance (NMR) was
`employed to probe structural changes in the IgGl PC. A series of
`lH-‘SN heteronuclear single-quantum correlation NMR spectra
`were collected between pH 2.5 and 4.7 to assess whether unfold—
`ing of CH2 domains precedes that of CH3 domains. The same pH
`range was subsequently screened in PC aggregation experiments
`that utilized molecules of IgGl and IgG2 subclasses with varying
`levels of CH2 glycosylation. In addition, differential scanning
`calorimetry data were collected over a pH range of 3—7 to assess
`changes in CH2 and CH3 thermostability. As a result, compelling
`evidence was gathered that emphasizes the importance of CH2
`stability in determining the rate and extent of PC aggregation. In
`particular, we found that PC domains of the IgG! subclass have a
`lower propensity to aggregate compared with those of the lgGZ
`subclass. Our data for glycosylated, partially deglycosylated, and
`fully deglycosylated molecules further revealed the criticality of
`CH2 glycans in modulating Fc aggregation. These findings pro—
`vide important insights into the stability of Fc-based therapeu—
`tics and promote better understanding of their acid-induced
`aggregation process.
`
`In order to ensure the safety and efficacy of biotherapeutics.
`it is critical to understand and prevent protein degradation. The
`presence of aggregates in therapeutic proteins may jeopardize
`their safety and efficacy by eliciting unwanted immunogenic
`responses (1, 2). Mitigation of aggregation processes while
`maximizing biotherapeutic shelf—life remains one of the out-
`standing challenges in biotechnology.
`Monoclonal antibodies (mAbs) continue to represent the
`leading group of biopharmaceutical products (3— 6). All cur—
`
`El‘lhis article contains supplemental Figs. 51—55.
`‘To whom correspondence should be addressed: Drug Product Develop-
`ment, Amgen Inc, 1201 Amgen Ct. W., Seattle, WA 98119.Tel.: 206—265—
`8851; E—mail: rlatypov@amgen.com.
`2 Present address: Formulation Development, Regeneron Pharmaceuticals,
`Tarrytown, NY 10591.
`
`3The abbreviations used are: Fc, fragment crystallizable; CEX, cation-ex-
`change HPLC; DSC, differential scanning calorimetry; Fab, fragment anti—
`gen-binding; rCE-SDS, capillary electrophoresis under reducing/denatur-
`ing conditions; PNGase F. peptide-.N—glycosidase F; HSQC, heteronuclear
`singlequantum correlation.
`
`JANUARY 6, 2012-VOLUME 287-NUMBER 2 m
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`JOURNAL OF BIOLOGICAL CHEMISTRY 1 381
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`Popvolumoa
`
`The Escherichia colt—derived IgGl Fc. CHO-derived IgGl Fc,
`CHO—derived IgG2 PC, and the uniformly 2H,‘5N-labeled,
`E. coli-derived IgGl Fc were supplied by the Protein Sciences
`group at Amgen, Inc. Intact mAbs (IgGl—A, IgGl-B, IgG2-B,
`and IgGZ—C) were supplied by the Process Development group
`at Amgen, Inc. All purified proteins were verified greater than
`95% pure by SDSvPAGE and size exclusion HPLC. Other
`reagents and chemicals were of analytical grade or better. All
`solutions were filtered through a 0.22—um filter prior to use.
`Protein Preparation, Identification, and Characterization—
`The E. coli— and CHO—derived Fc were supplied in 10 mM
`sodium acetate with 9% (w/v) sucrose at pH 5.2. The purity and
`identity of the Fc were verified by reversed—phase HPLC and mass
`spectrometry (see supplemental Figs. 81—53 for details). The
`E. colt—derived IgGl Fc material was the most homogeneous. It
`contained only one minor impurity, a species presumably with an
`unpaired disulfide. The CHO—derived IgGl Fc contained fully gly—
`cosylated species of expected mass and three minor species: 1) a
`sineg oxidized species, 2) a species with an unpaired disulfide, and
`3) a partially glycosylated species. The CHO—derived IgG2 PC was
`more heterogeneous, containing some clips and host cell proteins.
`Its major fraction was composed of two fully glycosylated species
`that presumably differed in sulfation.
`The lgGl—B-derived Fc fragments with differing levels of
`CH2 glycosylation were prepared as follows. Two milliliters of
`IgGl—B at 6 mg/ml were incubated with 24 pl 0f PNGase F
`(New England Biolabs, Ipswich, MA) in 1 X G7 buffer for 45 min
`at 37 °C. Endoproteinase Lys-C (Roche Applied Science) was
`then added to the reaction mixture at a protein/enzyme weight
`ratio of 200:1. The sample was incubated at 37 °C for an addi—
`tional 15 min before quenching with 150 mM ammonium ace—
`
`FIGURE 1. Schematic diagram of an lgG molecule. As indicated by the
`dashed line, an lgG structure consists of one Fc and two identical Fab regions.
`Thin blue lines represent intra- and interchain disulfide bonds. The structure
`of a carbohydrate unit attached to Asn-297 of the CH2 domain is shown sep-
`arately. LC, light chain; HC, heavy chain; 6, galactose; GN, N—acetylglucos—
`amine; F, fucose; M, mannose.
`
`the various sources of Fc instability that are connected with
`particular CH2 glycoforms will enable production of biologics
`with enhanced pharmacological properties.
`In a typical purification process, mAbs and PC fusion proteins
`are exposed to acidic conditions during viral inactivation and
`elution from affinity resins (9, 16). It is well known that low pH
`conditions may result in protein denaturation and aggregation
`(17, 18). It was shown that acidic pH and high ionic strength can
`promote formation of nonnative protein structures. Some of
`the best
`studied, partially folded, acid—denatured states
`(A—states or molten globule states) are populated at low pH in
`the presence of salt. For example, an acid molten globule state
`of cytochrome c is formed at pH 2.0 —2.5 in the presence of
`0.5—1.5 M salt (19 —21). Apomyoglobin, filactamase, and staph—
`ylococcal nuclease also exhibit an acid— and salt—induced forma—
`tion of A-states at low pH (22—25). Monoclonal antibodies and
`their fragments are no exceptions to this rule. Buchner and
`co—workers (26 —28) demonstrated that
`intact mAbs, Fab
`regions, and even isolated CH3 domains form A—states at acidic
`pH and high ionic strength. Although the stability and structure
`of these states are highly dependent on the protein and experi—
`mental conditions, their common characteristic is a tendency
`to aggregate (17, 29, 30). Unlike small, single—domain proteins.
`mAbs are complex glycoproteins composed of several inde-
`pendently folded domains. Commercial mAb preparations are
`rather heterogeneous and may contain differentially processed,
`
`1 382 JOURNAL OF BIOLOGICAL CHEMISTRY
`
`m VOLUME 287- NUMBER 2 -JANUARY 6, 2012
`
`Fc Aggregation in Acidic Conditions Is Determined by CH2 Stability
`
`LC
`
`F |
`
`. — GN — GN — M /\ M — GN — G
`
`M — GN — G
`
`incompletely glycosylated, and covalently modified forms (31).
`The understanding of mAb aggregation is challenged by the
`intrinsic and extrinsic complexity (not to mention the storage
`history) of antibody preparations.
`Despite the aforementioned issues, significant progress has
`been made in understanding and preventing aggregation in bio—
`pharmaceuticals (for a recent review, see Ref. 32). It was recog—
`nized that lgG aggregation can be induced by various factors
`and proceed through different mechanisms (33—36); however,
`the role of individual antibody domains in aggregation
`remained poorly understood. Recently, we proposed that acid-
`induced aggregation of mAbs is controlled by the stability of
`CH2 domains located in the Fc region (8). At the time, no struc—
`tural evidence was generated concerning the extent of CH2
`unfolding associated with this aggregation process. We are now
`filling this gap by gathering all of the necessary structural and
`stability data to implicate the CH2 domain. The scope of our
`study was limited to Fc fragments to allow for the use of high
`resolution two—dimensional NMR and to reduce the number of
`
`differential seaming calorimetry (DSC) transitions. In addi—
`tion, various forms of PC (Le. with respect to IgG subclass and
`degree of CH2 glycosylation) were analyzed under conditions
`promoting acid—induced aggregation. As a result, we revealed
`the aggregation rank order of the most typical lgG Fc domains
`currently used in biotechnology.
`
`EXPERIMENTAL PROCEDURES
`
`
`
`no:'9:KmuowensKqfilO'O‘iF/M‘Wfldnllwas
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`Page 2 of 17
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`S\l
`
`protein relative to an appropriate control stored refrigerated in
`10 mM sodium acetate, pH 5.2. Unstable proteins exhibited a
`faster decrease in the CEX monomer concentration compared
`with more stable proteins (8, 35).
`Cation—exchange Chromatography—Aggregation of mAb
`and PC mixtures was investigated by cation—exchange chroma—
`tography at pH 5.2 (35, 37). The method was run on an Agilent
`1100 series HPLC system. Chromatography was performed on
`a ProPac WCX—IO analytical column (weak cation exchange.
`4 X 250 mm; Dionex. Sunnyvale, CA) preceded by a ProPac
`WCX—IOG guard column (weak cation exchange, 4 X 50 mm;
`Dionex) at 25 "C. Protein samples were loaded onto the column
`and analyzed at a flow rate of 0.7 ml/min. The column was
`equilibrated with Buffer A (20 mM sodium acetate, pH 5.2), and
`protein was eluted with a linear gradient of Buffer B (20 mM
`sodium acetate, 300 mM sodium chloride, pH 5.2) from 0 to
`100% over 35 min. Following elution, the column was washed
`with Buffer C (20 mM sodium acetate, 1 M sodium chloride, pH
`5.2) for 5 min and then re—equilibrated with Buffer A for 16 min.
`Absorbance was measured at 215, 235, and 280 mm. Data were
`analyzed with Dionex Chromeleon® software, and the 280 nm
`signal was integrated to determine protein peak area.
`Difi‘erential Scanning Calorimetry—DSC measurements
`were taken using a VP-Capillary DSC system (MicroCal Inc.,
`Northampton, MA) equipped with tantalum 61 cells, each with
`an active volume of 135 p,l. Protein samples, typically at 0.5
`mg/ml, were scanned from 20 to 110 °C at a rate of 60 °C/h
`following an initial 15-min equilibration at 20 “C. A filtering
`period of 16 s was used, and the data were analyzed using Origin
`7.0 software (OriginLab® Corp., Northampton, MA). Resulting
`thermograms were corrected by subtraction of buffer control
`
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`Fc Aggregation in Acidic Conditions Is Determined by CH2 Stability
`
`tate at pH 4.7 (37). The sample was then cooled and maintained
`at 4 °C for immediate purification or frozen at —80 °C to arrest
`further enzyme activity.
`Glycosylated, partially deglycosylated, and fully deglycosy-
`lated Fc were purified using the CEX method described below
`(also see Ref 37). Digested IgGl-B material was loaded onto the
`column in multiple injections. An Agilent 1200 series HPLC
`system with a 12/ 13 SelValve external valve (Agilent Technol-
`ogies, Santa Clara, CA) was used to perform the fractionation.
`Resulting fractions containing the same species were pooled
`and concentrated and then reanalyzed by CEX. Purity greater
`than 90% was achieved for all
`three Fc species based on
`reversed-phase HPLC and mass spectrometry (see supplemen—
`tal Fig. S4).
`Aggregation Experiments—Previously, we demonstrated that
`CEX can be used to measure the aggregation propensity of both
`intact and fragmented mAbs (8, 35). This same method was
`applied in the current study, where mAb and Fc mixtures were
`exposed to different solution conditions. In our experiments,
`each protein was at 0.5 mg/ml final concentration unless noted
`otherwise. mAb and Fc mixtures were prepared in a native
`buffer composed of 10 mM sodium acetate at pH 5.2. They were
`subsequently diluted into various solutions of interest and incu—
`bated quiescently at 30 °C for up to several days. Sample ali—
`quots were taken at predetermined intervals and analyzed
`immediately or stored on ice to reduce further aggregation. The
`loss of soluble monomer was determined for each individual
`
`scans. The corrected thermograms were normalized for protein
`concentration.
`
`NuclearMagnetic Resonance—PC NMR measurements were
`performed at 25 °C using a Varian [NOVA 800—MHz NMR
`spectrometer (Varian Inc., Palo Alto, CA) equipped with a
`5—mm triple resonance probe. The uniformly 2H,15N—labeled
`E. coli-derived IgGl PC was tested at 5 mg/ml in 10 mM sodium
`acetate adjusted by HCl to pH 2.5, 3.1, 3.5, and 4.7.
`lH-ISN HSQC spectra were acquired with 64 experiments
`run in the 15N dimension (t1) consisting of 16 scans and 1024
`data points in the 1H dimension (£2). The total experimental
`time for each spectral acquisition was 37 min. Spectra were
`processed using NMRPipe (38) and analyzed using NMRView
`(39). The lH-‘sN cross—peak assignments from Liu et al. (40)
`were used. The weighted average chemical shift difference was
`calculated as described previously (41).
`Reversed-phase Chromatography and Mass Spectrometry—
`Reversed—phase analysis of the E. coli—derived IgGl Fc, CHO-
`derived IgGl PC, and CHO-derived IgG2 PC was carried out on
`a Waters (Milford, MA) Acquity system, equipped with a
`Diphenyl 3 pm, 1 X 50—mm column (Varian Inc.) as described
`previously (42). Typically, 5 pg of protein was injected on the
`column. The column was held at 95% solvent A (0.1% TFA in
`water) and 5% solvent B (90% acetonitrile and 0.085% TFA in
`water) for 5 min followed by a gradient from 5% B to 38% B over
`13 min. Fc elution was achieved with a linear gradient from 38%
`B to 46% B in 40 min at a flow rate of 0.05 ml/min. The column
`
`temperature was maintained at 75 °C throughout the run, and
`detection was at 214 nm.
`
`Reversed—phase analysis of the IgGl—B—derived PC was car—
`ried out on a Waters Acquity UPLC system as previously
`described (42). Typically, 5 ug of sample was injected onto an
`Acquity BEH 1.7 pm 1 X 50 mm phenyl column. The column
`was held at 72% solvent A (0.1% TFA in water) and 28% solvent
`B (90% acetonitrile and 0.085% TFA in water) for 0.7 min. $01—
`vent B was increased to 31.4% at 0.9 min, to 49.4% at 3.4 min,
`and to 90% at 3.5 min. At 4.10 min, solvent B returned to the
`
`starting level (28%) and remained constant until the end of the
`assay at 5 min. The column temperature was maintained at
`80 r‘C throughout the run, the flow rate was kept constant at
`0.35 ml/ min, and the detection was at 214 nm.
`
`The mass spectrometric analysis was carried out in positive
`ion mode on a Waters Q—TOF Premier or LCT Premier mass
`spectrometer equipped with an electrospray ionization source.
`The capillary and cone voltages were set at 3200 and 60 V,
`respectively. The desolvation and source temperatures were set
`at 350 and 80 "C, respectively. All other voltages were optimized
`to provide maximal signal intensity. The instrument was cali—
`brated in the m/z range of 1500—4000 using multiply charged
`ions of a standard antibody with a calculated molecular mass
`value of 148.2512 Da or commercial trypsinogen with a mass of
`32,300 Da. All raw data were processed using Waters MassLynx
`MaxEnt 1 software to obtain the deconvoluted mass.
`
`RESULTS
`
`Effect of Acidic Conditions on IgGJ Fc Structure via NMR
`Analysis—Although PC is a relatively large protein (~50 kDa),
`recent studies demonstrated that it is amenable to high resolu-
`
`JANUARY 6, 2012-VOLUME 287-NUMBER 2
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`m
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`JOURNAL OF BIOLOGICAL CHEMISTRY 1 383
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`S\l
`
`tion two—dimensional NMR analysis (7, 43). Furthermore, res—
`onance assignments from Liu et al. (40) made investigation of
`pH effects on IgGl Fc structure straightforward. In the current
`study, the Fc conformation was assessed between pH 2.5 and
`4.7 by acquiring a series of ‘H—lsN HSQC spectra of the uni—
`formly 2H,15N—labeled E. coli—derived IgGl Fc. Due to the low
`ionic strength of the protein solutions (see “Experimental Pro—
`cedures"), no evidence of aggregation was seen in any of the Fc
`samples throughout the experiment. At pH 4.7, the amide
`peaks of Fc were highly dispersed, which was consistent with a
`folded conformation (see Fig. 2A). A similar degree of disper—
`sion was seen at pH 3.5, although a number of peaks were
`reduced in intensity (Fig. 28). In addition, some new, low inten—
`sity peaks emerged that were not present at higher pH. At pH
`3.1, a subset of native resonances disappeared, whereas a differ—
`ent set of peaks appeared (Fig. 2C). Spectral properties of these
`new peaks were characteristic of a disordered, largely unfolded
`protein conformation. They resembled the minor resonances
`that were barely visible at pH 3.5. The remaining native reso-
`nances disappeared at pH 2.5, where NMR showed limited peak
`dispersion, consistent with an unfolded state (Fig. 2D).
`At pH 3.5 and 4.7, the number of assigned amide resonances
`available for analysis was 116 and 117, respectively. This repre-
`sented 51% of the 227 PC amino acid residues. Many of the
`missing peaks originated from the vicinity ofthe hinge region or
`
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`Fc Aggregation in Acidic Conditions Is Determined by CH2 Stability
`
`
`
`
`
`15NChemicalShift(ppm)
`
`1H Chemical Shift (ppm)
`FIGURE 2. 1H-“N HSQC spectra of the uniformly 2H.15N-labeled E. coli-derived lgGl F: at pH 4.7 (A), 3.5 (B), 3.1 (C), and 2.5 (D). The spectra were
`recorded at 25 °C.
`
`were due to peak overlap between the different pH spectra. The
`number of native resonances dropped to 46 at pH 3.1, and none
`were present at pH 2.5.
`shift
`residue—specific, chemical
`The weighted average,
`changes between pH 3.5 and 4.7 and between 3.1 and 3.5 are
`shown in Fig. 3. The most prominent changes between pH 3.5
`and 4.7 were clustered around residue positions 250—255 and
`310 —315 (Fig. 3A). These regions overlap with two short CH2
`a—helices that interface with the CH3 domains (see “Discus—
`sion”). In addition, notable chemical shift changes (20.05 ppm)
`were associated with positions corresponding to Asp—280, Gln—
`295, Leu—306, and Thr—335 of CH2 and Gly—385 and Lys—447 of
`CH3. Similar regions produced peaks with reduced intensity at
`pH 3.5, which probably reflected changes in the CH2 conforma—
`tional dynamics. Moreover, some of the native resonances that
`were present at pH 4.7 apparently disappeared at pH 3.5, among
`them resonances from Lys—290 and possibly Trp—277 and Val—
`412 (see Table 1).
`Peaks indicating the presence of a folded CH2 domain were
`virtually non—existent at pH 3.1, suggesting that major unfold-
`ing had occurred (Fig. 2C). Therefore, estimates for the chem—
`ical shift changes between pH 3.1 and 3.5 were only available for
`CH3 domains (Fig. 3B). At least three residue positions showed
`significant chemical shift changes at pH 3.1: Arg-34-4, Trp-381,
`and Lys-447. Of interest is the Arg-34-4 residue, the residue near
`
`1384 JOURNAL OF BIOLOGICAL CHEM/577W
`
`m VOLUME 287-NUMBER 2-JANUARY 6, 2012
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`Page 4 of 17
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`

`conditions to induce Fc aggregation within a short period of
`time at moderately elevated temperatures. First, we performed
`a pH screening experiment using protein solutions buffered
`with 10 mM sodium acetate to mimic the conditions that were
`
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`340
`Residue Number
`
`
`300
`
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` WeightedAverageChemicalsnmcnange(ppm)
`
`used for NMR. The samples contained a mixture of four differ—
`ent molecules: three full—length mAbs (IgGl—A, IgGZ—B, and
`IgG2—C) and E. colt—derived IgG] Fc. The choice to use the
`three mAbs was dictated by our previous experience with these
`molecules (8, 35). They served as internal controls to optimize
`solvent composition and incubation time to assess Fc aggrega—
`tion. In agreement with the NMR results, aggregation was not
`observed in these low ionic strength solutions at pH 3—5 even
`after 2 days of storage at 30 °C (data not shown). This was con—
`sistent with the important role of the ionic strength and acid
`concentration in low pH mAb aggregation (8). Subsequently,
`the pH screening was repeated in the presence of high (100 mM)
`sodium acetate with and without 50 mM NaCl (the correspond—
`ing solutions are abbreviated as 100Ax and 100AxN, where A
`represents sodium acetate, x is the pH, and N is NaCl). All four
`molecules were premixed in 10 mM sodium acetate at pH 5.2
`prior to being exposed to the low pH conditions. Fig. 4, A and B,
`shows CEX chromatographic traces for samples incubated in
`100A37N and 100A34, respectively. Fig. 5 summarizes results
`from various conditions in terms of percentage of monomer
`recovery based on CEX. It is evident that IgGl—A aggregated
`only at pH 3.4 (Fig. 5, A and E), whereas aggregation of Fc and
`the two lgGZs occurred at pH 3.7— 4.1 (Fig. 5, B and F, C and G,
`andD and H. respectively). Previously, we observed that low pH
`aggregation of mAbs was dependent on CH2 glycosylation
`and the IgG subclass (8). In particular, glycosylated IgGl
`mAbs were more resistant to aggregation compared with
`their glycosylated IgG2 counterparts, whereas an aglyco—
`IgGl (an IgGl mAb devoid of CH2 glycosylation) was the
`least stable molecule tested (8). Consistent with these find—
`ings, Fig. 5 reveals the following aggregation rank order of
`the four molecules (listed from the highest aggregation pro—
`pensity to the lowest): E. colt—derived IgGl Fc (Le. aglyco—
`IgG] PC) > IgGZ-C > IgG2—B > IgGl—A. Aggregation pro—
`pensity of Fc in 100A34 and 100A34N was particularly high
`and resulted in the loss of 30 — 40% of monomer at t = 0 (Fig.
`5, D and H, respectively). Thus, the CEX data demonstrated
`an increased instability of aglyco—IgGl Fc compared with
`glycosylated mAbs. Furthermore, the aggregation rank order
`for these molecules was the same in either the 100Ax or
`
`meow/ma
`
`Fc Aggregation in Acidic Conditions Is Determined by CH2 Stability
`
`pH 3.5—4.7
`
`
`
`
`
`
`
`
`
`220
`
`260
`
`300
`
`340
`Residue Number
`
`380
`
`420
`
`FIGURE 3. The weighted average chemical shift changes of the uniformly
`2H,1 sN-labeled E. coIi-derived IgG1 Fe. The graphs show residue-specific
`perturbations induced by a pH reduction from 4.7 to 3.5 (A) and from 3.5 to 3.1
`(B). The location of the hinge region as well as the CH2/CH3 domains is indi-
`cated in B. Residue numbering is listed in Eu format (44).
`
`the loop connecting the CH2 and CH3 domains. Changes at this
`position are probably the result of structural changes in the
`adjacent CH2 domain. This is supported by the simultaneous
`disappearance of many of the CH3 resonances originating from
`positions 366 —380 and 428 —438 (Table 1). Both of these seg—
`ments contain residues forming the CHZ—CH3 domain inter—
`face. which probably gets disrupted because of CH2 unfolding
`(see "Discussion").
`A further reduction in pH from 3.1 to 2.5 resulted in the
`disappearance of all remaining folded resonances (Fig. 2D). The
`NMR spectrum of PC at pH 2.5 was now consistent with an
`unfolded protein conformation devoid of stable tertiary or sec—
`ondary structure. The high affinity CH3—CH3 interaction was
`probably disrupted also, as demonstrated by the lack of native
`resonances originating from the domain contact area. Specifi—
`cally, this was reflected by the absence of native amide peaks
`from the following residues: Leu-351, Glu—357, Ser—364. Leu—
`368, Lys—370, Thr—394, Asp—399, Phe—405, and Lys—409. Because
`all of these positions were in a native-like environment at higher
`pH, the data were consistent with a scenario where dissociation
`and unfolding of the CH3—CH3 interchain complex occurred
`simultaneously (see "Discussion”).
`CEX Analysis of PC Aggregation—Previously, we demon—
`strated the utility of CEX in measuring the aggregation propen—
`sity of both intact and fragmented mAbs (8, 35). Similar to size
`exclusion HPLC, CEX is a nondenaturing chromatographic
`technique that can effectively separate aggregates from mono—
`mers. However, in contrast to size exclusion HPLC, CEX can
`
`resolve complex mixtures composed of similarly sized proteins.
`Working with protein mixtures allows us to monitor the aggre—
`gation of different molecules simultaneously and under identi—
`cal conditions. Hence, CEX was selected to establish the rank
`order of Fc aggregation as a function of CH2 glycosylation and
`subclass (IgGl versus IgG2).
`In addition to separating aggregates from monomers, CEX is
`useful in detecting degraded or chemically modified proteins
`(31, 37). This was an added benefit because our goal was to
`measure Fc aggregation with minimal interference from chem—
`ical degradations. Our initial studies were focused on finding
`
`IOOAxN conditions, which indicated the following: 1) the
`underlying aggregation mechanism was largely unaffected
`by NaCl, and 2) the rate and extent of Fc aggregation could
`be appropriately modulated by varying the ionic strength.
`Because covalent modification and fragmentation were not
`evident in these experiments (see Fig. 4), protein aggregation
`was the major degradation process. In summary, sufficient
`evidence was gathered to support the low pH approach for
`generating Fc aggregation data.
`Our next experiment was performed on a mixture composed
`of three different Fc moieties: E. colt—derived IgGl Fc, CHO—
`derived IgGl Fc, and CHO—derived IgGZ PC. This mixture was
`subjected to aggregation in the 100A31N and 100A35N condi—
`tions as outlined above. The CEX overlays corresponding to
`
`JANUARY 6, 2012-VOLUME 287- NUMBER 2
`
`me
`
`JOURNAL OF BIOLOGICM CHEM/SEW 1 385
`
`Page 5 of 17
`
`

`

`Fc Aggregation in Acidic Conditions ls Determined by CH2 Stability
`
`TABLE 1
`
`Fc residues that lost their natlve amlde resonances upon pH reductlon (relatlve to the 1H-‘SN HSQC spectrum acqulred at pH 4.7 (Fig. 2A))
`pH
`Hinge
`CH2
`CH3“
`3.5
`3.1
`
`Tilt-223
`
`Trp—277", Lys—290
`Val’240, Phe-243, Lys—248, Asp-249, Thr-ZSO, Leu’
`251, Met—252, SET—254. Arg—255, Glu—258, Tin—260,
`Cys—26l, Val—263, Lys—274. Asn—276, Trp—277", Tyr—
`278, Val-279, Asp-280, Gly-281, Val-282, Glu-283,
`Lys-290, Glu-294, Ser—298, Arg-301.Val-303. Ser—
`304. Leu—306, Thr—307, Val—308, His—310, Gin—311,
`Asp-312, Trp—313, Asn»315, Gly»316, Lys»317, Tyr-
`319, Lys—320, Val—323, Ala-330, Thr-335, Ile-336,
`Ser—337, Ala—339
`All
`All
`2.5
`An"
`“ Disappearance of native CH3 resonances at pH 3.5 and 3.1 may not necessarily reflect structural changes in CH3. it may also result from peak overlap with resonances origi-
`nating from denatured CH2.
`1’ Disappearance of the native resonance from this residue is uncertain because of peak overlap.
`‘ This residue resides between CH2 and CH3 domains.
`“ Because native resonance assignments for the hinge residues were incomplete (see “Experimental Procedures"), implications of CH2/CH3 denaturation on the hinge confor—
`mation are not fully understood.
`
`meow/ma
`
`Val»412"
`Gly-Mlc, Glu-345, Gln-34-7, Thr-366, Val-
`369, Gly—37l, Set—375, Asp—376, Ile—377,
`Ala—378, Glu—380, Gln—386, Tyr—391. Val—
`397, Tyr-407, Val—412b, Gln-418, Met-428,
`HisA29, Ala431, LeuAB2, His»433, Tyr-
`436, Gln—438
`
`no:‘szKewuoIssnflKqfilo'oqi'MMW/iduuwoe
`
`nature of the Fc samples, the CHO—derived IgGl Fc (including
`all minor forms) was evidently more resistant to aggregation
`compared with its aglycosylated (E. coli) variant or the CH0—
`derived IgG2 Fc. Aggregation of the latter two molecules
`appeared similar in 100A35N but differed in 100A31N. In par—
`ticular, aglyco—IgGl Fc lost ~55% of monomer at t = 0 but
`aggregated more slowly afterward (gray symbols in Fig. 6C). The
`initial monomer loss of the CHO—derived IgG2 PC was less than
`40%, but the remaining monomer disappeared rapidly (open
`symbols in Fig. 6C). Because all three molecules were premixed
`at pH 5.2 prior to the low pH exposure, this result indicated a
`lack of stability of the aglyco—IgGl and glyco—IgG2 Fc. Conse—
`quently, the rank order of PC aggregation was found to be as
`follows: aglyco—IgGl Fe 2 IgG2 Fc (CHO) > IgGl Fc (Cl-[0).
`This was consistent with our earlier findings (8) as well as the
`aggregation rank order that was drawn from Fig. 5. Therefore, a
`conclusion was made that the 100A31N and 100A35N condi—
`
`tions primarily promoted a CH2—dependent aggregation
`mechanism.
`
`Our last aggregation experiment utilized differentially glyco—
`sylated Fc fractions generated from another IgGl mAb, IgGl—B.
`The success of this experiment depended on 1) the ability of
`CEX to resolve Fc fragments with different levels of CH2 glyco—
`sylation and 2) the optimization of PNGase F treatment to
`achieve an optimal ratio of glycosylated, partially deglycosy—
`lated, and fully deglycosylated PC for purification. The ability of
`CEX to separate differentially glycosylated PC was verified by
`analyzing PNGase F—treated and -untreated IgGl-B following
`Lys—C limited proteolysis (data not shown). Subsequently,
`IgGl—B and PNGase P concentrations were varied along with
`incubation temperature and duration to achieve an optimal
`rate of digestion and desired ratio of glycosylated, partially
`deglycosylated, and fully deglycosylated Fc. Storage temper—
`ature and duration after digestion were also assessed to
`ensure that this ratio was sufficiently maintained over the
`course of purification (see "Experimental Procedures"). The
`resulting Fc fractions were verified by reversed—phase HPLC
`and mass spectrometry (see supplemental Fig. S4), mixed
`together, and subjected to an aggregation process in
`100A31N. The corresponding CEX results are shown in Fig.
`7, A and B. In agreement with the data in Fig. 6C, glycosy—
`lated PC was more resistant to aggregation compared with its
`
`A 7°
`60
`
`50
`
`4o
`
`30
`20
`
`
`
`UVAbs(280nm)
`
`lgG2-B
`
`IgGZ—C
`
`E. coli lgG1 Fc
`
`Aggregates
`
`18 2124 27
`
`30
`
`33
`
`36
`
`Elution time, min
`
`E. coli lgG1 Fc
`
`Aggregates
`
`CD
`
`
`
`uvAbs(280nm)
`
`
`
`18
`
`21
`
`24
`
`27
`
`30
`
`33
`
`36
`
`l—I—I—I
`41