Characterization of a Novel Bispecific Antibody With Improved Conformational and Chemical Stability

Journal of Pharmaceutical Sciences xxx (2019) 1-13

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Pharmaceutical Biotechnology

Characterization of a Novel Bispecific Antibody With Improved Conformational and Chemical Stability Prakash Manikwar 1, *, Sri Hari Raju Mulagapati 2, Srinath Kasturirangan 3, Khashayar Moez 1, Godfrey Jonah Rainey 3, Brian Lobo 1 1 2 3

Dosage Form Design & Development, AstraZeneca, Gaithersburg, Maryland 20878 Analytical Sciences, AstraZeneca, Gaithersburg, Maryland 20878 Antibody Discovery and Protein Engineering, AstraZeneca, Gaithersburg, Maryland 20878

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 March 2019 Revised 2 June 2019 Accepted 18 June 2019

Bispecific antibodies containing single-chain variable fragment (scFv) appended to immunoglobulins G offer unique development challenges. Here, we describe the stability of a novel bispecific format, BiS5, where the scFv is tethered to the CH3 domain. BiS5 showed an improved conformational and chemical stability compared with that of BiS4 in which the scFv is appended in the hinge region between the Fab and Fc. By switching the location of the scFv from hinge region to the CH3, there was an improved stabilization of CH2 and scFv domains. Interestingly, no noticeable impact was observed on the conformational stability of CH3 and Fab domains. BiS4 and BiS5 showed different aggregation and fragmentation rates under accelerated temperature stress conditions. BiS4 showed higher fragmentation rates compared with BiS5 likely owing to fragmentation in the linker region on either side of the scFv while BiS5 is more resistant toward fragmentation owing to tethering of scFv to the CH3 domain at its N and C terminus. In conclusion, the location of scFv affects both aggregation and fragmentation kinetics. These insights into the molecular structure and correlations with their physical and chemical stability will help formulation development of these novel bispecific antibodies. © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Keywords: bispecific antibody scFv antibody stability formulation fragmentation aggregation differential scanning calorimetry chromatography mass spectrometry

Introduction Bispecific antibodies (BiSAbs) belong to the next-generation antibody drugs which can bind simultaneously to 2 different epitopes either on a same antigen or 2 different antigens. Their dual target specificity provides distinct mechanisms of action compared with monoclonal antibodies (mAbs) or combination therapy alone,

Abbreviations used: BiSAb, bispecific antibody; scFv, single chain variable fragment; HMWF, high molecular weight fragment; LMWF, low molecular weight fragment; 2D-LC-UV/MS, two-dimensional liquid chromatography-ultraviolet/mass spectrometry. Current address for Srinath Kasturirangan: Biotherapeutics Discovery, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, Connecticut, 06877. Current address for Khashayar Moez: Waters Corporation, 34 Maple St, Milford, Massachusetts, 01757. Current address for Godfrey Jonah Rainey: Oriole Biotech, Inc., 8232 Hillandale Dr., San Diego, California, 92120. * Correspondence to: Prakash Manikwar (Telephone: þ1-301-398-1944). E-mail address: [email protected] (P. Manikwar).

thereby opening up new potential therapeutic applications.1,2 There has been a growing interest in developing BiSAbs in the industry, especially in the area of immunomodulating therapies including cancer.3 In addition to the 2 approved BiSAbs on the market, there are more than 30 BiSAbs in clinical development.4 There are multiple BiSAb scaffolds currently in clinical development including bispecific immunoglobulin G (IgG), single-chain variable fragment (scFv) appended to IgG (IgG-scFv fusion), bispecific fragments, bispecific fusion proteins, and bispecific conjugates.4,5 Each of these bispecific formats has its specific advantages and limitations. The biggest challenges during production and formulation of BiSAbs are purity and stability of the product, which is critical for the success of these therapeutics.6-8 To successfully apply BiSAbs in therapeutics practice, it is necessary to maintain the physical and chemical stability which helps ensure product safety and efficacy. To evaluate these critical factors, forced degradation studies9 play a critical role to provide comprehensive information for candidate selection,10 process development,11,12 formulation development,13 molecule characterization, assessment of critical quality attributes,14,15 comparability studies, and analytical method development.14,16-19

https://doi.org/10.1016/j.xphs.2019.06.025 0022-3549/© 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

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In this study, we compared the physical and chemical stability of 2 different scFv appended BiSAb formats, BiS4 where the scFv appended in the upper hinge region between the Fab and Fc20 and BiS5 where the scFv is inserted into a small surfaced exposed loop within the CH3 domain.21 These 2 BiSAbs were developed using the IgG1 structural frame work, which is considered one of the most widely used platforms for designing and developing novel therapeutic modalities.22,23 Formats generated through recombinantly fusing stability-engineered scFv to an IgG backbone enable us to generate BiSAbs with varying spacing, orientations, and geometries, which can impart unique function and biology to the molecule that cannot be achieved merely by combining the 2 parent antibodies. BiSAbs used in this study are large and complex therapeutic proteins corresponding to a molecular weight of 206.3 kDa. Like IgG, these BiSAbs consist of 4 different polypeptide chains (2 light chains [LCs] and 2 heavy chains [HC]) that are connected via disulfide bonds. As shown in Figure 1a, each of the polypeptide chains forms distinct globular-like domains. The LC forms VL and CL domains, and the HC forms VH, CH1, CH2, and CH3 domains. The LC and HC domains VL-CL and VH-CH1 form the 2 antigen binding fragments (Fab), and CH2-CH3 domains form 1 crystallizable fragment (Fc) that impart a Y-shaped structure to the molecule. The Fab domains are connected to the Fc domain by a proline-rich hinge region.24 In addition to the 2 Fab binding domains, these BiSAbs have a second binding moiety, scFv on each HC. In the BiS4 format, the scFv is appended in the upper hinge region between 231Cys-Asp232 residues, whereas in BiS5, the scFv is appended to a short surfaceeexposed loop within the CH3 domain between 395AsnGly396 residues.20,21 Furthermore, the scFv is linked to the antibody HC by a (Gly)4Ser linker (L1 or L3) on either side (Fig. 1a). As shown in the Figure 1b, the scFv has 2 domains, VL and VH, which are also connected by a (Gly)4Ser linker (L2) and a non-native disulfide bond between the position 44 (VH) and 100 (VL). Overall, in both the BiSAbs, there are 3 (Gly)4Ser linkers (L1, L2, and L3) per HC, and a total of 6 per antibody. Both the hinge region linker and the (Gly)4Ser linkers provide flexibility to the molecule. Although the physicochemical instabilities of these BiSAbs (scFv appended to IgG) have not been reported earlier, we can assume that the instabilities seen in the IgG1-based mAbs can generally be applied to BiSAbs as they share similar structural framework. The physicochemical instabilities of mAbs have previously been well documented,25 with aggregation and fragmentation as the 2 main routes of degradation pathways typically seen. In addition to the existing stability challenges of mAbs, the BiSAbs pose unique stability challenges that may be owing to the presence of an additional

domain, scFv. For example, a recent publication demonstrated lower bioactivity of BiS5 monomer variants and dimers owing to intermolecular disulfide bonds formed from the engineered disulfide bonds between VH and VL domains of the scFv.26 Aggregation in mAbs can be induced by structural changes (conformational) or solubility changes (colloidal) affecting domaindomain interactions.27,28 In general, aggregation is considered a multistep process that occurs as a result of changes in various conformational and colloidal properties.29,30 Subtle changes to the primary sequence of the CH2 and CH3 domains can dramatically alter the conformational stability and aggregation propensity of mAbs.31,32 Rose et al.32 reported that a point mutation in the CH3 domain (Y407E) of an IgG1 and IgG4 not only caused conformational changes in the CH3 domain but also led to remarkable structural changes in the CH2 domain and caused significant conformational changes via allosteric effects. Similarly, YTE mutation in the CH2 domain led to major conformational changes in the antibody structure, leading to pronounced aggregation of the mutant over its wild-type.31 Conformational and colloidal changes may also impact the antibody’s pharmacokinetic and pharmacodynamic properties.33 Protein concentration, hydrophobicity, ionic strength, surface charge, solution pH, excipients, agitation, freezethaw, and temperature also impact aggregation mechanisms. Even though the protein backbone is extremely stable under physiological conditions, it is susceptible to fragmentation in the presence of certain amino acid sequences (reactive side chains), enzymes, solvent conditions (such as pH and temperature), and impurities (such as metals and free radicals). The flexibility of the protein backbone, along with a reactive side chain, can have a detrimental effect on protein stability. The hinge region connecting Fab with Fc is considered as one of the most flexible regions of the mAb. The hinge region is comprised of upper (EPKSCDKTHT), middle (CPPCP), and lower (APELLGGP) hinge region.34 Two most common mechanisms of fragmentation seen in proteins are peptide bond hydrolysis and beta-elimination.35 The Ser-Cys and AspLys bonds in the upper hinge region are prone to fragmentation via beta-elimination and peptide bond hydrolysis.34 Similarly, the GlyGly bond in the lower hinge region is also prone to fragmentation under both acidic and basic conditions when present in the flexible and solvent exposed region.34-36 Liu et al.,36 reported extensive fragmentation in the hinge region of a mAb when incubated at different pH conditions at 40 C for 2.5 weeks. Previous studies have shown that the potential for fragmentation in mAbs increases when an Asp, Gly, Ser, Thr, Cys, or Asn residue is present in the primary sequence, as the side chains of all these residues (except Gly) can facilitate fragmentation via specific mechanisms.34 In addition to

Figure 1. (a) Schematic representation of BiS4 and BiS5 antibodies. (b) Schematic representation of scFv along with the (Gly)4Ser linkers (L1, L2, and L3).

P. Manikwar et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-13

hinge region fragmentation, BiS4 and BiS5 have (Gly)4Ser linkers which may also be prone to fragmentation. Despite structural similarities between BiS4 and BiS5dboth formats have single chain scFv tethered to different locations of the Fc with (Gly)4Ser linker, we suspect their physicochemical stabilities to be different owing to the difference in location of the scFv domain within the Fc. In this investigation, we performed a systematic study to compare the physical and chemical stability of BiS4 and BiS5.

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Storage Stability Study Both the BiSAb formulations were sterile filtered using a 0.22

mm filter (Millipore, Billerica, MA) in a presterilized laminar flow

Materials and Methods

hood. About 1.2 mL of each protein solution was dispensed into a 3mL type I borosilicate glass vials (West Pharmaceutical Services, Exton, PA) and sealed with 13 mm chlorobutyl Teflon-faced rubber stoppers (West Pharmaceutical Services) and aluminum TurEdge® Flip-Off® overseals (West Pharmaceutical Services). All samples were stored at 40 C and analyzed by high-performance sizeexclusion chromatography (HP-SEC) at time zero and after 1, 2, 4, 8, and 12 weeks.

Materials

High-Performance Size-Exclusion Chromatography

Both the BiSAbs (BiS4, and BiS5) and the mAb were produced within the Department of Antibody Discovery and Protein Engineering at Medimmune (Gaithersburg, MD) using standard molecular biology protocols as previously described.20 All reagents and chemicals purchased were of analytical grade from Sigma Aldrich (St Louis, MO), JT Baker (Center Valley, PA), or Pfanstiehl (Waukegan, IL).

The monomer purity of the stability samples was analyzed using HP-SEC. The analysis was performed using an Agilent 1100 HPLC system with UV detection at 280 nm absorbance spectra, a 7.8  30 cm Tosoh TSK-Gel BioAssist G3SWxL (TOSOH Biosciences, King of Prussia, PA), and a corresponding guard column. The column was precalibrated using molecular weight standards (Bio-Rad, Hercules, CA). All samples were injected neat (100 mL) and separated based on their size using a mobile phase containing 0.2 M sodium phosphate, pH 6.8, and a flow rate of 1.0 mL/min. The peak areas for each resolved species (aggregate, monomer, and fragments) were quantified using 280 nm wavelength quantification. The percentage of each of the species remaining was plotted against time.

Sample Preparation The stock solutions of the BiSAbs were provided between 5-10 mg/mL in phosphate-buffered saline. To conduct stability studies at 10 mg/mL in different pH conditions ranging from 5.0 to 7.5 (D0.5 pH units), the protein solution was first concentrated to 12 mg/mL using 30 kDa MWCO Millipore® Centriprep™ (Sigma-Aldrich, Austin, TX) filtration unit followed by dialysis at 5 C using Slide-ALyzer® Dialysis Cassettes (Thermo Fisher Scientific, Waltham, MA). Both BiSAbs were dialyzed into buffer containing either 20 mM succinate (pH5.0), 20 mM histidine (pH 5.5, 6.0, and 6.5), or 20 mM phosphate (pH 7.0 and 7.5) along with 240 mM sucrose. After dialysis, the protein solution was filtered using 0.22 mm filtration unit, protein concentration was adjusted to 10 mg/mL, and then a 0.02% (w/v) polysorbate-80 (PS-80) was spiked into solution. For differential scanning calorimetry (DSC) measurements, BiS4 and its corresponding mAb at pH 4.0 and 6.0 were dialyzed at 5 C into buffer containing 20 mM citrate-phosphate and 240 mM sucrose. The protein solution was then filtered using 0.22 mm filtration unit and followed by PS-80 spike to achieve a final concentration of 0.02% (w/v) PS80 into the solution. For the DSC runs, the protein solution was diluted to 1 mg/mL in its corresponding buffer. Protein concentration was determined with an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies, Palo Alto, CA) using an extinction coefficient 1.54 M1 cm1.

Differential Scanning Calorimetry To obtain DSC thermograms of each format under different pH conditions, a Microcal VP-Capillary DSC with autosampler (MicroCal, Northampton, MA) was used. A scan rate of 90 C/h was used over the temperature range of 20 C-100 C. After completion of each scan, the data were analyzed using the MicroCal LLC DSC plug-in for the Origin 7.0 software package. These results were fit to a multistate model with 2 transitions to calculate the midpoint of thermal unfolding (Tm) values. The heat capacity (Cp) of 500 cal/mol/ C was considered to calculate the onset temperature (Tonset) as described previously.37 The processed thermograms of the BiSAbs in different pH conditions were compared with each other.

Reduced-GXII Fragment quantification was carried out using a size-based separation method under reduced conditions. Samples were analyzed using Perkin Elmer LabChip® GXII. GXII is a high throughput microfluidic-based capillary electrophoresis instrument consisting of microfabricated channels designed to incorporate simultaneous protein separation, staining, de-staining, and detection. All samples were mixed with a lithium dodecyl sulfate sample buffer, denatured for 2 min at 100 C, then diluted to achieve the final working concentration. The percent purity of the main band and the fragments were quantified using the LabChip GX software. Two-Dimensional Liquid Chromatography-Ultraviolet/Mass Spectrometry Automated two-dimensional LC (2D-LC) was performed on ACQUITY UPLC H-Class Bio system with 2D technology featuring a heart-cut technique. The configuration was constructed with ACQUITY UPLC Quaternary Solvent Manager (first dimension), ACQUITY UPLC Binary Solvent Manager (second dimension), ACQUITY UPLC Column manager (2 six-port switching valve and 2 column holders), ACQUITY H Class UPLC biosample manager, ACQUITY UPLC Photodiode Array (PDA) Detector, and Xevo G2 QTof mass spectrometry (MS). The first dimensional separation was achieved on HP-SEC column (ACQUITY UPLC Protein BEH SEC column with 4.6 mm  150 mm, 1.7 mm particle size), and the column was maintained at 25 C with a flow rate of 0.25 mL/min. SEC mobile phase contains 0.1 M sodium phosphate dibasic anhydrous, 0.1 M sodium sulfate, pH 6.8. UV detection for the first-dimension separation was monitored at 280 nm. The second-dimensional separation was carried out on reversed-phase (RP) column (ACQUITY UPLC Protein BEH C4 Column with 300Å, 2.1 mm  50 mm, 1.7 mm particle size) with mobile phase A water/formic acid (100:0.1, v/v) and mobile phase B acetonitrile/formic acid (100:0.1, v/v). The column was maintained

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Figure 2. (a) DSC thermograms of BiS4 in formulations containing 20 mM succinate, histidine, or phosphate, 240 mM sucrose, 0.02% (w/v) PS-80 at indicated pH conditions. (b) DSC thermograms of BiS5 in formulations containing 20 mM succinate, histidine, or phosphate, 240 mM sucrose, 0.02% (w/v) PS-80 at indicated pH conditions. (c) Representative deconvoluted DSC thermogram of BiS4 at pH 6.0 with Tonset, Tm1, Tm2, and Tm3. (d) Representative deconvoluted DSC thermogram of BiS5 at pH 6.0 with Tonset, Tm1, Tm2, and Tm3.

at 65 C with a flow rate of 0.15 mL/min. A 30-min gradient (5%-45% B in 11-20 min; 45%B-80% in 20-24 min) was used to separate the components in the second dimension. All samples were diluted with water to 1 mg/mL before injection (injection volume of 5 mL). The heart cutting intervals were set based on the elution profile on the first-dimension column, the process is carried by temporarily switching right valve position 1 (off) to position 2 (on) resulting in combining the flow paths where effluent from column 1 (SEC) is redirected to column 2 (RP). Heart cuts were made at 5 different time ranges 4.40-4.60 min (peak 1/peak a), 4.65-4.85 min (peak 2/peak b), 4.85-5.10 min (peak 3), 5.40-5.70 min (peak 4), and 6.10-6.40 min (peak 5/peak c). Because the 2D-LC configuration is equipped with single loop to capture first dimension fraction, 1 heart cut per injection was made to carry out second-dimension separation followed by MS (Xevo G2 Q-Tof) identification. MassLynx™ software (Waters) was used to control the instrument for data acquisition and data processing. Deconvolution of multiply charged ions was performed with the MaxEnt 1 software provided with MassLynx™ software.

240 mM sucrose, and 0.02% (w/v) PS80 at several different pH conditions starting from pH 5.0 to 7.5 with 0.5 pH unit increments (Figs. 2a and 2b). Sucrose and polysorbatedthe most commonly used excipients for immunoglobulinsdwere selected to stabilize both the BiSAbs. At all pH conditions, both BiSAbs displayed twopeak transitions, indicating 2 thermal unfolding events. Figures 2c and 2d show a representative DSC thermogram of BiS4 and BiS5 at pH 6.0. Using the curve-fitting tool, we could assign the

Results Improved Conformational Stability of BiS5 Over BiS4 The conformational stability of BiS4 and BiS5 were examined in a formulation containing 20 mM succinate, histidine or phosphate,

Figure 3. DSC thermograms of mAb at pH 4 and BiS4 at pH 4.0 and 6.0 formulated in 20 mM citrate-phosphate buffer.

P. Manikwar et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-13

Tm2 ¼ 65.1 C) corresponding to the unfolding of CH2 and scFv domains, showed a single peak at pH 6.0 (Tm1 ¼ 68.0 C) likely owing to simultaneous unfolding of CH2 and scFv. Additionally, the unfolding of CH2 and scFv domains shifts to higher temperature, suggesting stabilization of both the domains at higher pH. Furthermore, at pH 6.0, an increase in the height (enthalpy) of the first peak as compared with the enthalpy of first (Tm1 ¼ 57.9 C) and second peak (Tm2 ¼ 65.1 C) at pH 4.0 further supports the possibility of simultaneous unfolding of CH2 and scFv domains. Similarly, at pH 4.0, the third left shoulder peak (Tm3 ¼ 78.7 C) corresponding to the unfolding of the CH3 domain showed a shift to higher temperature at pH 6.0 (Tm3 ¼ 85.8 C), suggesting stabilization of the CH3 domain at higher pH. Moreover, the left shoulder peak associated at pH 6.0 becomes less prominent as it coincides along with the unfolding of the Fab domain. BiS4 also showed a shift in the fourth peak to higher temperature, relating to the unfolding of Fab at pH 6.0 (Tm4 ¼ 89.5 C) in comparison to pH 4.0 (Tm4 ¼ 84.4 C) indicating thermal stabilization at pH 6.0. Because the thermal profiles of BiS5 are similar to BiS4 at all pH (5.0-7.5) conditions, we assumed that the first and the second peaks observed in BiS5 were also owing to the simultaneous unfolding of CH2/scFv and CH3/Fab (Figs. 2a and 2b). The Tm values associated with these thermograms are summarized in Table 2. At all the pH (5.0-7.5) conditions tested, there was an increase in Tm1 (D of 1 C-2 C) associated with the unfolding of peak 1 for BiS5 as compared with BiS4, indicating thermal stabilization of either CH2, scFv, or both the domains in BiS5. Interestingly, there was no noticeable change in the Tonset of BiS4 and BiS5 at the varying pH conditions. Additionally, the Tm2 and Tm3 associated with unfolding of peak 2 did not show any major changes between BiS4 and BiS5, indicating minimal impact via modification of the scFv location on the thermal stability of CH3 and Fab domains (Table 2).

Table 1 Effect of pH on Thermal Melting Temperatures (Tm1, Tm2, Tm3, and Tm4) for BiS4 and Its Corresponding mAb as Measured by DSC Construct/pH

Tm1 ( C)

Tm2 ( C)

Tm3 ( C)

Tm4 ( C)

mAb/pH 4.0 BiS4/pH 4.0 BiS4/pH 6.0

56.6 57.9 68.0

76.2 65.1

83.6 78.7 85.8

-ND84.4 89.5

5

Formulations contained 1 mg mL1 protein in citrate-phosphate buffer at the indicated pH condition. The values are based on single DSC measurements. ND, not detected.

first peak to a single transition (Tm1), whereas the second peak was fit to 2 transitions (Tm2 and Tm3). Because both these BiSAbs included mAb structural framework, we assumed that the first peak (Tm1) may be associated with the unfolding of the CH2 domain, and the second peak (Tm2 and Tm3) may be associated with the simultaneous unfolding of the CH3 and Fab domains.38-42 However, we were unable to distinguish whether the unfolding of scFv was associated with the first or the second peak within the pH range (5.0-7.5) studied. To confirm association of the unfolding of scFv with either the first or second peak, we compared the DSC thermograms of BiS4 with its corresponding mAb at pH 4.0 along with BiS4 at pH 6.0 in a separate experiment (Fig. 3). The Tm values associated with these 3 thermograms are shown in Table 1. These experiments were performed in 20 mM citrate-phosphate buffer to minimize variability owing to the buffer component. As shown in Figure 3 and Table 2, the DSC thermogram of the mAb (black line) showed 3 peaks. Based on previously reported mAb thermal transitions,38-42 the first peak (Tm1 ¼ 56.6 C), second shoulder peak (Tm2 ¼ 76.2 C), and third peak (Tm3 ¼ 83.6 C) may correspond to the unfolding of CH2, CH3, and Fab domains, respectively. Under similar pH conditions, BiS4 (red line) showed 4 distinct peaks. The first peak (Tm1 ¼ 56.6 C) of the mAb coincides with the first transition (Tm1 ¼ 57.9 C) of BiS4, indicating that this first transition in BiS4 may correspond to the unfolding of the CH2 domain. The unfolding temperature associated with the second (Tm2 ¼ 76.2 C) and third (Tm3 ¼ 83.6 C) peaks of the mAb coincide with the third (Tm3 ¼ 78.7 C) and fourth (Tm4 ¼ 84.4 C) peaks of BiS4 at pH 4.0. This indicates that the third transition in BiS4 may correspond to the unfolding of CH3 domain, and the fourth transition may correspond to the unfolding of the Fab domain. Interestingly, the second peak (Tm2 ¼ 65.1 C) in BiS4 does not coincide with any of the 3 transitions seen in the mAb, suggesting that this new transition seen in BiS4 may correspond to the unfolding of the scFv. As shown in the Figure 3 and Table 1, at pH 6.0, the DSC thermograms of BiS4 (green line) showed 2 distinct peaks compared with 4 distinct transitions at pH 4.0 (red line), indicating possibility of simultaneous unfolding of multiple domains at higher pH. At pH 4.0, the first and second peak observed in BiS4 (Tm1 ¼ 57.9 C and

Improved Accelerated Storage Stability of BiS5 Compared With BiS4 To better understand the effects of changing the location of scFv from the hinge region to the CH3 domain and its impact on longterm storage of BiS4 and BiS5, protein samples in different pH (5.0-7.5) conditions were stored in stoppered sterile glass vials under accelerated temperature conditions at 40 C for up to 3 months. The HP-SEC chromatograms at pH 6.0 on day 0 for BiS4 (Fig. 4a; blue lines) and BiS5 (Fig. 4a; red lines) were compared with each other. Both formats primarily contain monomer with low levels of aggregate speciesdlikely the dimer. In addition to the aggregate species, BiS4 also showed low levels of fragment species, whereas BiS5 showed trace amount of fragmentation (<0.1%). Figure 4b shows an overlay of the HP-SEC chromatograms of BiS4 (blue lines) and BiS5 (red lines) at pH 6.0 after storage for 3 months at 40 C. Under accelerated temperature, both these bispecifics showed an additional, earlier eluting peak (multimeric species) along with a decrease in monomer, and elevated levels of fragment

Table 2 Effect of pH on Thermal Onset Temperature (Tonset) and Thermal Melting Temperatures (Tm1, Tm2, and Tm3) for BiS4 and BiS5 as Measured by DSC pH

BiS4

BiS5 

5.0 5.5 6.0 6.5 7.0 7.5







Tonset ( C)

Tm1 ( C)

Tm2 ( C)

Tm3 ( C)

Tonset ( C)

Tm1 ( C)

Tm2 ( C)

Tm3 ( C)

56.3 56.8 59.3 60.6 60.6 60.3

67.8 67.8 69.3 70.3 70.2 70.0

81.9 81.6 83.1 83.8 82.2 81.5

84.1 83.8 85.3 86.0 84.7 84.2

55.9 57.0 59.4 60.6 59.9 59.6

69.5 69.4 70.7 71.6 71.2 71.0

81.8 81.7 83.1 83.8 81.5 81.0

83.9 83.7 85.2 85.9 84.3 83.9

Formulations contained 1 mg mL1 protein in 20 mM Histidine, 240 mM Sucrose, and 0.02% PS80 at the indicated pH condition. The values are based on single DSC measurements.

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Figure 4. Representative HP-SEC analysis of BiS4 and BiS5 samples at pH 6.0 before and after storage at 40 C for 3 months. (a) Overlay of HP-SEC chromatograms of BiS4 (blue trace) and BiS5 (red trace) samples at time zero. Inset shows the zoomed in chromatogram. (b) Overlay of HP-SEC chromatograms of BiS4 (blue trace) and BiS5 (red trace) samples incubated at 40 C for 3 months. Inset shows the zoomed in chromatogram. The bar chart represents the effect of thermal stress on different forms (monomer, fragment, and soluble aggregates) of BiS4 and BiS5 as measured on (c) time zero and (d) after 3 months at 40 C.

species. Figures 4a and 4b inset shows the zoomed HP-SEC chromatograms of BiS4 and BiS5. BiS5 showed different aggregation and fragmentation profiles compared to BiS4. BiS5 showed only 1 fragment species (fragment 1), whereas 3 fragment species (shoulder fragment, fragment 2, and fragment 1) were seen for BiS4 under similar accelerated temperature stress. To quantify the changes in the formation of aggregate and fragment species in both the bispecific antibodies, HP-SEC results were plotted in a bar graph at time zero and for samples incubated for 3 months at 40 C (Figs. 4c and 4d). On day zero, there was no difference observed in the aggregate levels between BiS4 (0.5%) and BiS5 (0.4%). Similarly, no differences were observed in the fragment levels between BiS4 (0.2%) and BiS5 (0%). After 3 months of storage at 40 C and in the same formulation conditions, samples of both bispecifics showed drastic differences in the level of aggregate and fragment species. The extent of aggregation in BiS5 (4.7%) was found to be slightly higher than BiS4 (3.7%). However,

fragmentation was much lower in BiS5 compared with BiS4. For BiS5, fragmentation comprised 1.3% whereas BiS4 showed 12.2% of fragmentation (i.e., 6.4% shoulder fragment, 3.6% fragment 2%, and 2.2% fragment 1). Interestingly, the retention time (RT) for the smallest fragment (fragment 1) for BiS5 (10.8 min) matched with that of BiS4 (10.8 min). To understand the extent of degradation in BiS4 and BiS5 across several different pH conditions, the rate of monomer loss along with aggregation and fragmentation at 40 C were plotted against pH (Fig. 5). As shown in Figure 5a, the rate of aggregation between BiS4 and BiS5 showed similar trend up to pH 7.0. At lower pH conditions (pH 5.0, 5.5, and 6.0), the rate of aggregation in BiS5 (i.e., 2.7%/m, 1.4%/m, and 1.5%/m) is slightly higher than in BiS4 (i.e., 1.6%/ m, 1.0%/m, and 1.2%/m). However, at pH 6.5 and 7.0, the rate of aggregation between BiS5 (i.e., 1.6%/m and 2.4%/m) and BiS4 (i.e., 1.6%/m and 2.4%/m) are similar. At pH 7.5, BiS5 showed a higher aggregation rate (i.e., 4.2% per month) as compared with BiS4 (i.e.,

Figure 5. pH-rate profile of BiS4 (blue) and BiS5 (red) for samples incubated at 40 C. (a) Aggregation rate plotted as a function of pH. (b) Fragmentation plotted as a function of pH. (c) Monomer loss rate plotted as a function of pH.

P. Manikwar et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-13

2.3% per month). Curiously, the rate of fragmentation between BiS4 and BiS5 showed very different trends at all the pH conditions studied. BiS5 showed resistance to fragmentation with minimal pH impact on fragmentation rate across all the pH conditions, whereas BiS4 showed a varying degree of fragmentation rates with

7

increasing pH (Fig. 5b). The fragmentation rates for BiS5 after storage at 40 C for 3 months were found to be 0.7%/m, 0.4%/m, 0.4%/m, 0.5%/m, 0.6%/m, and 1.2%/m at pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5, respectively. Under similar storage conditions, the total fragmentation rate (i.e., combined rates of fragmentation for the

Figure 6. Reduced GXII electropherogram analysis of BiS4 and BiS5 samples before and after storage at 40 C for up to 4 weeks. (a) Representative overlay of electropherograms of BiS4 formulated in pH 6.0 at time zero (blue trace), 1 week (red), 2 weeks (brown), and 4 weeks (green). (b) Overlay of electropherograms of BiS5 formulated in pH 6.0 at time zero (blue trace), 1 week (red), 2 weeks (brown), and 4 weeks (green). (c) pH-rate profile of BiS4 (red) and BiS5 (purple) for samples incubated at 40 C. Solid lines correspond to change in light chain (LC) content and dotted lines correspond to change in heavy chain (HC) content.

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shoulder fragment, fragment 2 and fragment 1) for BiS4 was found to be 5.2%/m, 4.1%/m, 4.0%/m, 5.4%/m, 10.4%/m, and 19.4%/m at pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5, respectively. Overall, BiS5 showed lower monomer loss as compared with BiS4 throughout all the pH conditions tested (Fig. 5c). Monomer loss in BiS5 was found to be 3.4%/m, 2.8%/m, 1.9%/m, 2.1%/m, 3.0%/m, and 5.4%/m at pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5, respectively. Under similar pH conditions, monomer loss in BiS4 was found to be 6.8%/m, 5.1%/m, 5.2%/m, 7.0%/m, 12.8%/m, and 21.7%/m. Lower HC Fragmentation in BiS5 Compared With BiS4 Because fragment quantification using HP-SEC is not a reliable method, owing to poor resolution between monomer and fragment species, we tested all the stability samples of BiS4 and BiS5 using an orthogonal technique, reduced GXII. Samples from both the BiSAbs at pH 5.0-7.5 that were stored at 40 C for up to a month were analyzed using reduced GXII. Representative overlays of reduced GXII electropherograms of BiS4 and BiS5 at pH 6.0 at time zero, 1 week, 2 weeks, and 4 weeks are shown in Figures 6a and 6b. Upon reduction, both the BiSAbs show 2 major peaksdone peak at ~95 kDa corresponding to HC and the other peak ~27 kDa corresponding LC. An increase in fragment species between the molecular weight of 27-95 kDa was observed with time. To understand the extent of the changes in the HC and LC in BiS4 and BiS5 across the pH range, the rate of change of HC and LC content per month at 40 C was plotted against pH (Fig. 6c). As shown in Figure 6c, the rate of change in LC per month between BiS4 and BiS5 showed no change up to pH 7.0. However, at pH 7.5, BiS5 showed an increase in LC content (5.5%) compared with BiS4 (0.8%). Under similar pH conditions, the rate of HC loss between BiS4 and BiS5 demonstrated similar trends; however, the loss of HC was much more pronounced in BiS4 compared with BiS5 up to pH 6.5. At pH 5.0, 5.5, 6.0, and 6.5, the HC loss for BiS4 was found to be 9.8%/m, 6.9%/m, 9.1%/m, and 10.7%/m; and for BiS5, the HC loss was 5.8%/m, 3.6%/m, 4.3%/m, and 5.4%/m, respectively. However, at pH 7.0 and 7.5, there was no difference in the HC loss rate per month between BiS4 (25.0%/m and 36.8%/m) and BiS5 (24.0%/m and 35.3%/m) was observed.

with molecular mass of 206340 Da. Chromatographic Peak 2 corresponds to truncated BiS4, lacking 1 of the Fab domains, with a molecular mass of 158057 Da. Peak 3 corresponds to truncated BiS4, lacking 1 Fab and 1 scFv fragment, with a molecular mass of 130156 Da. Interestingly, Peak 4 showed 2 speciesda minor and a major species with a molecular mass of 75884 Da and 76200 Da. It is possible the major species corresponds to Fab and scFv fragments along with the linker (503GGGGSGGGG511). The minor species corresponds to truncated Fab and scFv fragment with a smaller halflinker (503GGGG506). The difference in the molecular masses between the major and minor species is ~316 Da, corresponding to the truncated linker portion (507SGGGG511). Surprisingly, Peak 5 showed 2 separate peaks on 2D RP-HPLC (data not shown)deach peak displaying two different m/z ranges (Fig. 8, Peak 5a and 5b). Peak 5a showed presence of 2 species, a minor species with a molecular mass of 47985 Da and a major species with a molecular mass of 48300 Da. Similar to Peak 4, the major species corresponds to the Fab fragment along with the linker (232GGGGSGGGG241), and the minor species corresponds to the truncated Fab fragment with the half-linker (232GGGG235) present. The difference in the molecular masses between the major and minor species is ~315 Da, corresponding to the rest of the truncated linker portion (236SGGGG240). Peak 5b showed the presence of 2 species as well, a

Fragmentation in (Gly)4Ser Linkers To identify the regions of fragmentation, multi heartcutting 2DLC-MS analysis was performed using unstressed (T0) and stressed (3 months at 40 C) BiS4 and BiS5 samples. The first-dimension chromatography was achieved using HP-SEC. In both BiS4 and BiS5, unstressed samples (Fig. 7; black traces) showed predominantly monomeric species, whereas stressed samples (Fig. 7; red traces) showed both aggregate and fragment species along with the monomer. Because the main purpose of this study was to identify the fragmentation site(s) in these BiSAbs, we omitted the aggregate peak from further analysis. As shown in Figure 7a (highlighted regions), 5 cuts (Peak 1-5) from unstressed and stressed BiS4 samples were transferred from 1D (HP-SEC) to 2D reversed-phase highperformance liquid chromatography, (RP-HPLC) followed by online MS identification. The results from 2D-LC MS analysis identified cut-1 as monomer species, and cuts 2-5 were identified as the fragment species. Similarly, for BiS5 (Fig. 7b; lower panel), 3 cuts (Peaks a-c) were transferred from 1D (HP-SEC) to 2D (RP-HPLC) and followed by online MS identification. As highlighted in the Figure 7b, cut a was identified as monomer, and cuts b and c were identified as the fragments. Figure 8 shows electrospray ionization mass spectra of BiS4 stressed sample from peaks 1-5. The corresponding deconvoluted masses of peaks 1-5 were summarized in Table 3. The peak assignment is as follows: Peak 1 was identified as BiS4 monomer

Figure 7. Represents UV-280 nm chromatographic overlay of unstressed (black) and 3 months, 40 C stressed (red) samples. The samples were separated by HP-SEC (first dimension). (a) BiS4 samples with different heart cut time ranges (peak 1, peak 2, peak 2, peak 4, and peak 5*). (b) BiS5 samples with different heart cutting time ranges (peak a, peak b and peak c*). *Represents 1 peak in HP-SEC (first dimension); 2 peaks in RPHPLC (second dimension).

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9

Figure 8. The ESI-mass spectra obtained after the chromatographic separation of BiS4 stress sample (3 months, 40 C stress) with online 2D-LC coupled with MS (HP-SEC/RP-HPLC/ MS). The peaks (1-5) represents different heart cutting time ranges on HP-SEC. (Note: peak 5 was represented as peak-5a and peak-5b, because it was resolved into 2 separate peaks on second dimension). ESI, electrospray ionization.

minor species with a molecular mass of 22820 Da corresponding to the fragments associated with LC (1-209) and a major species with a molecular mass of 23040 Da corresponding to the fragments associated with HC (1-215). Figure 9 shows electrospray ionizationemass spectra of BiS5 stressed sample from Peak a-c. The corresponding deconvoluted masses of Peak a-c are summarized in Table 4. The peak assignment is as follows: chromatographic Peak “a” was identified as BiS5 monomer with molecular mass of 206315 Da. Peak “b” corresponds to truncated BiS5 lacking 1 Fab domain with a molecular mass of 158077 Da. Similar to Peak 5 seen in BiS4, Peak c showed 2 separate peaks on 2D RP-HPLC (data not shown)deach peak with 2 different m/z ranges, as shown in the Figure 9, Peak c1 and c2. Peak “c1” showed presence of 2 species, a minor and a major species, with a molecular mass of 48000 Da and 48240 Da, respectively. The major species was identified as Fab fragment along with a small portion of the upper hinge region (232DKTH235), whereas the minor species corresponds to the Fab fragment with additional 232DK233 present. The difference in the molecular masses between the major and minor species is ~240 Da, likely corresponding to the molecular weight of the dipeptide, 234TH235. Peak c2 showed a minor and a major species with a molecular mass of 22820 Da and 23040 Da, corresponding to fragments associated with LC (1-209) and HC (1-

215), respectively. To confirm the identity of fragments observed in 2D-LC-MS, the samples were also analyzed on direct LC-MS (RPHPLC-MS) under reduced and nonreduced conditions (data not shown). Similar masses were observed from traditional LC-MS and 2D LC-MS. Discussion Physical Stability of BiS4 and BiS5 DSC is routinely used in formulation development to probe conformational stability of mAbs.39 For mAbs, 3 thermal transitions are typically seen on a DSC thermogram, corresponding to the unfolding of CH2, CH3, and Fab domains.38-40,42 Depending on the thermal stability of the individual domains and solution conditions, it is not uncommon to observe a 2-peak transition in mAbs.38-40,42 Often, a 2-peak transition is a result of simultaneous unfolding of CH2 and Fab domains followed by the unfolding of CH3 domain or the unfolding of CH2 domain followed by the simultaneous unfolding of Fab and CH3 domains. In either case, the unfolding of CH2 domain takes place at lower temperature than the CH3 and Fab domains and is the least thermally stable domain in mAbs. Like mAbs, the DSC thermograms of BiS4 and BiS5 showed a two-peak

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Table 3 2D-LC-MS Identification of BiS4 Fragments Generated for 3 Mo, 40 C Stress Condition (Elution Profile Shown in Fig. 1a) SEC Peaks

Description

Predominant Masses (Da)

Position

1

BiS4 monomer (intact)

206340

LC intact: 1-210 HC: Intact: 1-738

2

One cleavage site in linker 1 (Fab-GGGGGSGGGG240….S241)

158057

Intact minus (1LC þ1HC (1-240))

3

One cleavage site in linker 3 (Fab-GGGGSGGGG511….S512)

130156

Intact minus (1LC þ1HC (1-511))

4

Two cleavage sites in linker 3 (Fab-GGGG….S507GGGG511….S512)

75884 (Minor species) 76200 (Major species)

1LC þ1HC (1-507) 1LCþ1HC (1-511)

5a

Two cleavage sites in linker 1 (Fab-GGGG….S235GGGG240….S241)

47985 (Minor species) 48300 (Major species)

1LC þ1HC (1-235) 1LCþ1HC (1-240)

Nonlinker cleavage sites

22820 (Minor species) 23040 (Major species)

LC (1-209) HC (1-215)

5b

Protein Variants

Fragments

Mass values are within 30 ppm accuracy. Linker 1: 232-241 (G4S)2, linker 2: 349-368 (G4S)4, and linker 3: 503-512 (G4S)2.

transition for the pH (5.0-7.5) conditions, indicating a possibility of simultaneous unfolding of multiple domains (Figs. 2a and 2b). Upon deconvolution, both BiS4 and BiS5 showed 3 peaks at all pH conditions. Only pH 6.0 data are being shown in Figures 2c and 2d, and the rest of the pH conditions (5.0, 5.5, 6.5, 7.0, and 7.5) showed similar 3 peak transitions (deconvoluted peaks not shown). As described in the results section, the 2 peaks observed for BiS4 and BiS5 (Figs. 2a and 2b) can be assigned to the simultaneously unfolding of CH2/scFv followed by the unfolding of CH3/Fab domains. Moreover, the deconvoluted thermogram of first peak showed one transition (with a midpoint Tm1) corresponding to simultaneous unfolding of CH2/scFv, whereas the second peak showed 2 partially overlapping peaks (with their midpoints corresponding to Tm2 and Tm3), which denotes the unfolding of CH3 and Fab (Figs. 2c and 2d). Together, these results suggest that the unfolding of CH2 and scFv takes place at similar temperature, whereas the unfolding of CH3 and Fab domains happens at different temperatures. For both BiS4 and BiS5, an increase in Tonset, Tm1, Tm2, and Tm3 were observed with increasing pH, especially between pH 5.5 and 6.5 (Table 2), suggesting an increase in thermal stability of CH2/scFv, CH3, and Fab. Interestingly, at all the pH conditions tested, BiS5 showed an increase in Tm1 but no impact on Tonset, Tm2, and Tm3 as compared with BiS4. These data suggest an improvement in the conformational stability of either in CH2 or scFv domain when switching the location of scFv from the hinge region (in BiS4) to the CH3 domain (in BiS5). Numerous studies, including ours,37 have shown that the CH2 domain is least conformationally stable domain compared with CH3 and Fab.43,44 The stability of the CH2 domain can be influenced by several factors, including point mutations,31 solution pH conditions,45 excipients,37 and deglycosylation.43 By changing the location of scFv from the hinge region (in BiS4) to the CH3 domain (in BiS5), we may have improved the conformational stability of the CH2 domain when compared with BiS4dhence an increase in Tm1 was observed for BiS5 (Table 2). To further examine the effects of changing the location of scFv on the physical stability of BiS5, the extent of aggregation occurring over time was measured in different solution pH conditions (pH 5.0-7.5) under accelerated temperature of 40 C for up to 3 months and compared with that of BiS4. BiS5 showed slightly higher amount of soluble aggregates when compared with BiS5 (Figs. 4d and 5a). An increase in aggregation as measured by HP-SEC does not correlate with an increase in Tm1 as measured by

DSCdindicating the possibility of colloidal stability playing a role in BiS5 aggregation. Chemical Stability of BiS4 and BiS5 To assess the impact of changing the location of scFv on the chemical stability of BiS4 and BiS5, the aggregation and fragmentation rates were measured using HP-SEC. BiS5 and BiS4 solutions prepared at different pH (5.0-7.5) were subjected to accelerated temperature conditions of 40 C for up to 3 months. HP-SEC data showed a lower amount of fragment species in BiS5 as compared with BiS4 at pH 6.0 (Fig. 4d). Furthermore, the fragmentation rate measured by HP-SEC remained unchanged for BiS5 over a broader pH range (5.0-7.5) as compared with BiS4. This indicates a significant improvement in BiS5 chemical stability by changing the location of scFv from hinge region to CH3 domain (Fig. 5b). Using HP-SEC under native conditions is not a reliable method of fragment quantification for the following reasons: first, there is poor resolution between monomer and high molecular-weight fragment (HMWF) and second, the fragments held together by disulfide bonds cannot be resolved under native conditions. Owing to these limitations of native HP-SEC, fragmentation in BiS4 and BiS5 was compared using an orthogonal technique (GXII) under reduced conditions. Reduced GXII data revealed 2 major peaks (HC and LC), along with multiple small peaks. The molecular weight of the small peaks corresponded to an intermediate molecular weightethat which is in between the LC and HC for both BiSAbs (Figs. 6a and 6b). Because the molecular weight of these small peaks is between the molecular weight of LC and HC, we assumed that these small peaks are formed as a result of fragmentation in the HC, but not in the LC. The fluorescence intensity associated with these small peaks increases with time for both BiS4 and BiS5, suggesting an increase in HC fragmentation during heat stress. However, we did not observe an appreciable increase in fragments smaller than the molecular weight of LC. To further quantify the fragmentation, a plot of LC and HC loss per month under accelerated temperature is shown in Figure 6c. A positive value on the Y-axis indicates an increase in either LC or HC content over time whereas a negative value denotes a decrease in either LC or HC content over time. As shown by the solid lines in Figure 6c, there was no change in LC content throughout all the pH conditions, with the exception at pH 7.5 for both BiS4 and BiS5,

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Figure 9. The ESI-mass spectra obtained after the chromatographic separation of BiS5 stress sample (3 months, 40 C stress) with online 2D-LC coupled with MS (HP-SEC/RP-HPLC/ MS). The peaks (a-c) represents different heart cutting time ranges on HP-SEC. (Note: peak c was represented as peak-c1 and peak-c2, because it was resolved into 2 separate peaks on second dimension).

indicating no degradation in the LC under temperature stressed conditions with time. However, at pH 7.5, an increase in LC content was observed for BiS5 compared with BiS4. This is only possible if the HC degrades into smaller fragment species, with molecular weight equivalent to the molecular weight of LC. As a result of this mechanism, an increase in the LC content would occur. Interestingly, a negative value for HC content was observed at all pH conditions, indicating a decrease in HC content over time at accelerated temperature conditions (Fig. 6c; dotted lines). However, these negative values were much higher at pH 7.0 and 7.5 when compared with pH 5.0-6.5, indicating faster rates of fragmentation at higher pH. This is likely a result of high base catalyzed hydrolysis. In general, HC content in BiS5 was less negative as compared to BiS4 in the pH range 5.0-6.5, indicating decreased fragmentation in BiS5. This could be attributed to a significant improvement in the chemical stability by switching the location of scFv from hinge

region to the CH3 domain. However, at pH 7.0 and 7.5, HC loss between BiS4 and BiS5 showed similar negative values, indicating similar HC fragmentation occurring over time in both these molecules. Both native HP-SEC and reduced GXII data suggest a decrease in fragmentation in BiS5 compared with BiS4. However, the sensitivity in detecting fragment species under reduced conditions is much higher than in native conditions, because the fragments held together by disulfide bonds can be separated under reduced conditions. Location of Fragmentation in BiS4 and BiS5 Under thermal stress conditions, both the BiSAbs showed multiple peaks. As shown in Figures 7a and 7b, the main peak decreased, and both aggregate and fragment species increased over the duration of exposure. Higher number of distinct peaks in BiS4

Table 4 2D-LC-MS Identification of BiS5 Fragments Generated for 3 Mo, 40 C Stress Condition (Elution Profile Shown in Fig. 1b). SEC Peaks

Description

Predominant Masses (Da)

Position

a

BiS5 monomer (intact)

206315

LC intact: 1-210 HC: Intact: 1-738

b

One cleavage site in the upper hinge region (Fab-EPKSCDKTH…236T)

158077

Intact minus (1LC þ1HC (1-235))

c1

Two cleavage sites in the upper hinge region between Fab-EPKSCD233K…234T and 235 …236T

48000 (Minor species) 48240 (Major species)

1LC (1-211) þ1HC (1-233) 1LC (1-211) þ1HC (1-235)

Nonlinker cleavage sites

22820 (Minor species) 23040 (Major species)

LC (1-209) HC (1-215)

c2

Protein Variants

Fragments

Mass values are within 30 ppm accuracy. Upper hinge region: 227-236 (EPKSCDKTHT), middle hinge region: 237-241 (CPPCP), lower hinge region: 242-249 (APELLGGP), disulfide bond between LC (Cys 210), and HC (Cys 231).

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(2-5) compared with BiS5 (b and c), suggests an increase in the number of different fragment species that are formed when BiS4 is subjected to heat stress. The RT associated with these fragments species is also different, suggesting that these fragments are of different molecular weight sizes. Interestingly, the fragment species associated with Peak 5* and c* for BiS4 and BiS5, respectively, elute at same RT (6.1-6.4 min) on the 1D HP-SEC column (Figs. 7a and 7b). These species formed 2 peaks on 2D RP-HPLC column (data not shown) suggesting a possibility of these fragments to be of similar molecular weight sizes and hydrophobicity. As shown in Figure 7, Tables 3 and 4, the molecular mass of peak 1 and peak “a” are ~206300 Da, which coincide with the theoretical molecular mass of BiS4 (206337 Da) and BiS5 (206311 Da) monomer. Peak 2 is associated with a molecular mass of 158057 Da, corresponding to the molecular mass of HMWF species without Fab-GGGGSGGGG240, suggesting truncation between FabGGGGSGGGG240 and S241 in the first G4S linker (L1). The corresponding LMWF species, Fab-GGGGSGGGG240 (48300 Da) elutes in Peak 5 along with a small population of slightly lower molecular weight species, Fab-GGGG235 (47,985 Da). This suggests possible truncation between Fab-GGGG235 and S236 in the linker L1. In addition, Peak 5 also showed presence of other LC1-209 (22820 Da) and HC1-215 (23040 Da) fragment species generated during heat stress, resulting in truncation within non-linker cleavage sites present in the antibody. Interestingly, all these LMWF fragments (Fab-GGGGSGGGG240, Fab-GGGG235, LC1-209 and HC1-215) co-elute on 1D HP-SEC (Peak 5). However, when transferred to 2D RP-HPLC, these fragment species are separated into 2 peaks, Peak 5a and 5b, corresponding to Fab-GGGGSGGGG240/Fab-GGGG235 and LC1-209/ HC1-215, respectively, owing to differences in their hydrophobicity. Peak 3 is associated with a molecular mass of 130156 Da, corresponding to the HMWF species without Fab-scFv-GGGGSGGGG511, suggesting truncation between Fab-scFv-GGGGSGGGG511 and S512 in the third G4S linker (L3). The corresponding LMWF species, Fab-scFv-GGGGSGGGG511 (76200 Da), elutes in Peak 4 along with a small population of Fab-scFv-GGGG506 (75884 Da) that is formed owing to truncation between Fab-GGGG506 and S507 in the linker L3. As shown in Table 4, Peak “b” is associated with a molecular mass of 158077 Da, corresponding to the molecular mass of HMWF species without Fab-EPKSCDKTH235. This peak is likely generated as a result of truncation between Fab-EPKSCDKTH235 and 236T in the upper hinge region. The corresponding LMWF species, FabEPKSCDKTH235 (48240 Da) elutes in Peak “c” along with a small population of Fab-EPKSCDK233 (48000 Da), suggesting truncation likely between Fab-EPKSCDK233 and T234. Fragmentation within the hinge region of the mAb is considered as most common fragmentation site, which has been previously reported in several mAbs.34 In addition to these fragments, peak “c” contained other nonlinker LC1-209 (22820 Da) and HC1-215 (23040 Da) fragment species that were generated during the heat stress. Like BiS4, all LMWF (Fab-EPKSCDKTH235, Fab-EPKSCDK233, LC1-209, and HC1-215) of BiS5 also co-eluted as a single peak on 1D HP-SEC and as 2 peaks (Peak c1 and c2) on 2D RP-HPLC. Hence, they show similar RT on the chromatograms (Peak 5* and c* in Figs. 7a and 7b). The molecular masses of LC1-209 and HC1-215 fragments generated during heat stress have similar molecular weight in both BiS4 and BiS5, because they share identical Fab region. These fragmentation sites are most commonly observed in mAbs and other recombinant proteins during processing and storage; owing to commonality of these fragmentation patterns, they have been well-characterized.34,46-48 However, the low molecular weight Fab fragments observed in this study for BiS4 (Fab-GGGGSGGGG240/Fab-GGGG235) and BiS5 (Fab-EPKSCDKTH235/Fab-EPKSCDK233) co-eluting in Peak 5* and c*, respectively, are of different molecular masses, mainly owing to

their C-terminal sequence differences. In BiS4, the Fab is connected to the rest of the antibody via G4S linker (L1), while in BiS5, the Fab is connected to the rest of the antibody via the hinge region (Fig. 1). Observed Fragmentation in BiS5 Is Lower than in BiS4 Overall, BiS4 showed more fragment peaks with different molecular masses as compared with BiS5 under similar stress conditionsdresulting from temperature induced truncation in the linkers (L1 and L3)dmeasured via native HP-SEC (Figs. 4a and 4b). Furthermore, the fragmentation rates were found to be much lower for BiS5 compared with BiS4 across the pH range (Fig. 5b). We measured fragmentation rates under reduced conditions using GXII to assess their true fragmentation rates. Interestingly, the fragmentation rates for BiS5 were found to be lower than BiS4, even under reduced conditions and across the majority of the pH range (Fig. 6c). The possible explanation for this lies in the unique design of BiS5 construct. As shown in Figure 1, in BiS5, the scFv is tethered to CH3 domain on both sides via G4S linkers (L1 and L3). If a truncation takes place in either one of these G4S linkers, the scFv is still attached to the mAb. For the scFv to be completely separated from the rest of its antibody, truncation needs to happen on both the linkers (L1 and L3). The possibility of truncation on both of the linkers (L1 and L3) of BiS5 is less likely to occur, which is seen by the scFv molecular mass not being detected during 2D-LC-MS analysis. In addition, if a truncation takes place in the linker L2 region, connecting between VL and VH domains of scFv, the scFv is still held together to its parent mAb via a non-native disulfide bond connecting between VL and VH domains. However, under reduced conditions, in the absence of the disulfide bond connection between the VL and VH domains of scFv, these fragments can be easily detected. This explains why the absolute fragmentation rates measured by GXII under reduced conditions (Fig. 6c) are higher than the fragmentation rates measured by HP-SEC under native conditions (Fig. 5b). These results further confirm that these fragments may be held together by either native or non-native disulfide bonds. In BiS4, scFv is in the upper hinge region, and it is connected on either side with G4S linkers (L1 and L3) to the rest of the antibody (Fig. 1). If a truncation takes place in either one of these G4S linkers, a HMWF (BiS4 either without Fab or Fab-scFv) and a LMWF (Fab or Fab-scFv) is readily formed (which is separate from rest of the antibody). However, if a truncation takes place in the linker L2 region, the corresponding HMWF and LMWF may not be detected under native conditions owing to the presence of a non-native disulfide bond connecting between VL and VH. Only under reduced conditions can these fragment species be detected. Overall, in BiS4, higher fragmentation rates were observed across all pH conditions when analyzed under native conditions because truncation in one of the G4S linkers can readily release the fragment species from its parent antibody. Whereas, in BiS5, lower fragmentation is observed despite truncation in one of the G4S linkers, as the scFv is still attached to its parent mAb via the other G4S linker. Based on the results from this work, despite having same number of G4S-linkers, BiS5 showed higher monomer stability as compared with BiS4. Fragmentation is the major route of degradation across the pH range as compared with aggregation. The location of scFv affects both aggregation and fragmentation kinetics; however, its impact is much higher on fragmentation compared with aggregation. BiS5 showed higher resistance toward fragmentation mainly owing to unique design of the construct, which was achieved by tethering scFv on both sides of the CH3 domain via the G4S linkers. In the event of truncation in one of the G4S linkers, the scFv can still maintain its connectivity to the parent antibody. This study also demonstrates that the susceptibility of

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G4S linkers toward fragmentation may be high, causing instability during storage, which can potentially hinder development of a liquid stable formulation of these novel molecules. There is certainly a need for developing new linkers with higher resistance toward chemical degradation to prevent fragmentation in these types of molecules. Even though, BiS5 showed higher conformational stability compared with BiS4 (Tm1; Table 2), the aggregation rates seemed to be higher in BiS5, suggesting that there may be some contribution from colloidal instability. It will be interesting to investigate the potential cause for higher aggregation in BiS5 despite a significant improvement in its conformational stability.

Acknowledgments The authors would like to thank MedImmune for supporting this work. The authors would like to thank Cuihua Gao from the Medimmune Antibody Discovery and Protein Engineering and Yanhong Yang, Samuel Aaron Korman, Arun Parupudi, Dengfeng Liu, and Xiangyang Wang from the Analytical Sciences Department for their contributions to this work. The authors also thank Marissa Minor for proofreading the manuscript. Finally, the authors would also like to thank Steve Bishop and Christopher Van Der Walle for critical review and suggestions.

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