Harnessing Ceramic Hydroxyapatite as an Effective Polishing Strategy to Remove Product- and Process-related Impurities in Bispecific Antibody Purification

Harnessing Ceramic Hydroxyapatite as an Effective Polishing Strategy to Remove Product- and Process-related Impurities in Bispecific Antibody Purification | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Harnessing Ceramic Hydroxyapatite as an Effective Polishing Strategy to Remove Product- and Process-related Impurities in Bispecific Antibody Purification Wei Zhang, Nattha Ingavat, Wang Xinhui, Liew Jia Min, Farouq Bin Mahfut, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3402362/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Dec, 2023 Read the published version in Bioresources and Bioprocessing → Version 1 posted 5 You are reading this latest preprint version Abstract Bispecific antibody (bsAb), a novel therapeutic modality, provides excellent treatment efficacy, yet poses numerous challenges to downstream process development, which are mainly due to high diversity and complexity of bsAb structures and impurity profiles. The mixed-mode medium, ceramic hydroxyapatite (CHT), allows proteins to interact with its calcium sites (C-sites) through metal affinity and/or its phosphate sites (P-sites) through cation exchange interactions. This dual binding capability potentially offers unique bind and elute behaviours for different proteins of interest, resulting in optimal product purity when suitable elution conditions are employed. In this study, the effectiveness of CHT as a polishing step for bsAb purification was investigated across three model molecules and benchmarked against the traditional cation exchange chromatography (CEX). For both asymmetric and symmetric IgG-like bsAb post Protein A eluates, at least 97% product purity was achieved after CHT polishing. CHT delivered a superior aggregate clearance to CEX, resulting in low high molecular weight (HMW) impurities (0.5%) and low process-related impurities in the product pools. CHT also yielded significantly less “chromatography-induced aggregation” for all aggregation-prone bsAb molecules. Developability of CHT for more efficient low molecular weight (LMW) impurity removal was further demonstrated via post-load-wash (PLW) optimization with the selected bsAbs, resulting in up to 48% additional LMW reduction. Furthermore, possible mechanistic explanation about CHT performance on both process- and product-related impurity removal in bsAb polishing was proposed. Positive CHT C-site cooperation mediated effective impurity removal and mitigated “chromatography-induced aggregation”, and domain composition and size of bsAbs molecules may determine the effectiveness of such C-site cooperation. bispecific antibody mixed-mode chromatography product- and process-related impurity removal chromatography-induced aggregation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Bispecific antibodies (bsAbs) have become one of the most attractive therapeutic modalities over the past decade (Wei et al., 2022 ; Wu & Cheung, 2018 ). They are man-made proteins, deliberately engineered to possess two different antigen recognition sites instead of one that exists in traditional monoclonal antibodies (mAbs). A combination of the two antigen-binding sites in proximity provides dramatic improvement in therapeutic efficacy (Labrijn et al., 2019 ). Furthermore, bsAbs can potentially enable novel treatments, not offered by any combination of the natural mAbs. BsAbs are broadly categorized into three main formats (Chen & Zhang, 2021 ; Labrijn et al., 2019 ), including fragment-based bsAbs, asymmetric bsAbs and symmetric bsAbs. The fragment-based bsAbs are designed to incorporate at least two antigen-binding sites into one molecule without the Fc region to avoid any undesired chain-association issue (Labrijn et al., 2019 ). However, this format is likely to have a comparatively short plasma half-life (Labrijn et al., 2019 ). Both asymmetric bsAbs and symmetric bsAbs are Fc-containing molecules. Asymmetric bsAbs normally contain three to four polypeptides, which are derived from two different parental mAbs. Due to random chain pairing, it is possible for asymmetric bsAbs to generate up to ten different homodimers and heterodimers. Although a couple of bsAb engineering technologies, such as knobs-into-holes (Ridgway et al., 1996 ; Rouet & Christ, 2014 ), and CrossMAb™ technology (Surowka et al., 2021 ), have been developed to mitigate chain mispairings, none of these strategies can completely prevent the formation of mispaired byproducts. For symmetric bsAbs, both of their recognition sites are typically incorporated into a single polypeptide chain to allow co-expression of only one to two polypeptides. As a result, they are generally tetravalent (2 + 2) by design. Currently, among all bsAb molecules that have been under clinical development, asymmetric and symmetric IgG-like bsAbs are the most popular formats. BsAbs pose numerous challenges to downstream processing mainly due to high diversity and complexity of their structures and impurity profiles. The bsAb product-related impurities, including mispaired species, half antibodies, antibody fragments and aggregates, are difficult to remove because of their similarities in size and physiochemical properties to the desired end-product. Furthermore, engineered bsAbs are also reported to be highly aggregation-prone, making them less stable than their parental mAbs (Chen et al., 2022b ). Considering the differences between mAb and bsAb, traditional purification tools for mAbs are less likely to provide the same level of efficiency when it comes to bsAbs. In addition, “chromatography-induced aggregation” has been observed during both Protein A and cation exchange chromatography (CEX) for bsAb purification as reported by Serene Chen et al, and Lucus K. Kimerer et al. (Chen et al., 2022a , 2022b ; Kimerer et al., 2019 ). This “chromatography-induced aggregation” is more pronounced when bsAbs are subjected to high loading (Chen et al., 2022a , b ). This could be a crucial bottleneck for bsAb industrial production as lower loading leads to lower productivity. Given that ceramic hydroxyapatite (CHT) is one of the leading media for intermediate and polishing step development, it has been applied to the purification of various types of biological therapeutics, including antibodies, antibody fragments, recombinant proteins, viruses, and DNAs (Hilbrig & Freitag, 2012 ). CHT is a mixed-mode support that comprises two different binding sites as illustrated in Fig. 1 A. The first binding site which mainly retains solutes via calcium metal affinity is commonly known as the “C-site”. It primarily binds to carboxyl groups of proteins, phosphoryl residues of DNA, as well as phosphorylated solutes (Gagnon, 2009 ; Itoh et al., 2019 ). Elution of proteins bound to the C-sites can be achieved with solutions containing either phosphate or fluoride ions, which have higher affinity for calcium ions as compared to carboxyl groups (Gorbunoff & Timasheff, 1984 ). Retention of biomolecules via interactions with the C-sites (e.g. DNA and acidic proteins) can tolerate high concentration of sodium chloride (up to 4 M sodium chloride in the absence of phosphate), suggesting that the C-site interactions are significantly stronger than electrostatic forces (Gagnon, 2009 ; Gorbunoff & Timasheff, 1984 ; Itoh et al., 2019 ). The second binding site that acts as a cation exchanger is named as the “P-site”. P-sites primarily bind to ammonium groups on proteins and elution can be accomplished by raising conductivity using any types of salt (Gorbunoff, 1984 a; Gorbunoff & Timasheff, 1984 ). Sodium chloride gradient (CHT-NaCl) and phosphate gradients (CHT-P) have been used intensively for CHT elution. In addition, usage of borate and sulfate salts for elution has also been reported for certain applications (Gagnon, 2006 ). Buffer compositions and concentrations play important roles in binding strength, as well as protein binding capacity of CHT. Previous studies showed that sodium chloride can weaken repulsive forces between negatively charged biomolecules (e.g. phosphoryl DNA and carboxyl-proteins) and P-sites. Therefore, interactions of phosphoryl DNA and negatively charged protein with the C-sites are tremendously strengthened at high NaCl concentration (Fig. 1 B) (Gagnon, 2009 ; Gagnon et al., 2009 ; Hilbrig & Freitag, 2012 ). A similar concept applies to interactions between protein ammonium groups and P-sites. During a protein binding state, the presence of low phosphate concentration assists reducing repulsion between positively charged ammonium groups of proteins and the C-sites, as compared to when no phosphate is present. Therefore, strengthening cationic interactions between ammonium clusters of proteins and the P-sites (Gagnon et al., 2009 ; Gorbunoff, 1984 b). By tweaking the buffer compositions and concentrations, the binding of the biomolecules to CHT medium can be affected, leading to different binding strength and capacity. In addition, the presence of at least 5 mM phosphate in all buffers is required regardless elution mechanisms to maintain CHT stability (Gagnon, 2006 , 2009 ; Gagnon et al., 2009 ). With a combination of metal affinity and cation exchange interactions, CHT provides unique separation mechanisms with additional purification benefits, therefore improving the removal efficiency of both product- and process-related impurities (Gagnon, 2009 ). In this study, the effectiveness of CHT chromatography as a polishing step on bsAb purification was evaluated and compared to the traditional CEX, using three model bsAb molecules (Molecules A, B, and C) with different formats, molecular sizes and domain compositions as illustrated in Fig. 2 . The results showed that CHT chromatography can serve as an effective polishing step for both asymmetric and symmetric IgG-like bsAbs, interestingly, with different binding and elution mechanisms. The products of both bsAb formats were of 97% purity with acceptable levels of process-related impurities. Previously, Yiran Wang, et al. have conducted mechanistic studies on competitive binding kinetics and separation dynamics of monomeric and dimeric monoclonal antibodies using CHT (Wang & Carta, 2019 ; Wang & Carta, 2020a ). A hybrid model has also been developed to explain separation of monoclonal antibody monomer-dimer mixture using gradient elution strategies (Wang & Carta, 2020b ). On top of the previous published papers, herein, we extended CHT application to bsAb purification and elaborated the possible mechanistic insights. Our mechanistic explanation mainly focuses on how CHT medium mediates both product- and process-related impurity removal in bsAb purification. Materials and Methods Materials All buffers, salts, and reagents were purchased from Sigma-Aldrich except for sodium chloride and citric acid that were purchased from Merck Millipore. MabSelect PrismA, Capto SP Impress, and Tricon series columns were purchased from Cytiva. CHT Type II (40 µm) was provided by HOYA Technosurgical Corporation. BsAb culture production Stably transfected CHO K1 cell lines, producing bsAbs, were generated by site-specific integration of plasmid vectors, which carried genes encoding light chain (LC), heavy chain knob (HCK) and scFv-Fc hole (scFv-FcH) (for Molecule A), or VH-CH1-scFv-Fc hole (VH-CH1-scFv-FcH) (for Molecule B); LC and VH-CH1-scFv-Fc wild-type (VH-CH1-scFv-FcW) (for Molecule C) (Fig. 2 ). CH3 domains in the HCK and scFv-FcH/VH-CH1-scFv-FcH were engineered to form a knob (through mutations of S354C:T366W) and a hole (through mutations of Y349C:T366S:Y407V), respectively, to facilitate heterodimeric Fc pairing, based on a previous study (Merchant et al., 1998 ). For Molecule A, VH and VL in scFv were connected through a flexible linker (G4S)3, which was further linked to FcH through a G4 linker. The engineered Fc consisted of IgG1 positions 221 to 447 based on the Eu numbering system (Wang et al., 2018 ). For Molecule B and C, VH-CH1 was linked to the N-terminus of scFv through a G4S linker, which was further linked to either FcH (Molecule B) or FcW (Molecule C), respectively, through a G4 linker. The stably transfected cell lines were grown in a protein-free medium consisting of HyQ PF (Cytiva) and CD CHO (Thermo Fisher Scientific) at 1:1 ratio and supplemented with 1 g/L sodium carbonate (Sigma), 6 mM glutamine (Sigma), and 0.1% Pluronic F-68 (Thermo Fisher Scientific) in 50 mL tubespin (TPP) in a humidified Kuhner shaker (Adolf Kühner AG) with 8% CO 2 at 37°C. To produce these three molecules, 300 mL of cell culture at viable cell density of 3×10 5 cells/mL were inoculated into 600 mL tubespin (TPP) in the humidified Kuhner shaker (Adolf Kühner AG) with 8% CO 2 at 37°C. 30 mL of Ex-Cell Advanced CHO Feed 1 (with glucose) (SAFC, Sigma) was added at day 3, 5, 7, 9 and 11. Cell density and viability of each culture were monitored at day 3, 5, 7, 9, 11 and 14, using the Vi-Cell XR viability analyser (Beckman Coulter). d-Glucose concentration in the culture medium was quantified using Nova BioProfile 100plus analyzer (Nova Biomedical). When the glucose concentration in the media dropped below 2 g/L, d-glucose (Sigma) was added to the culture to adjust glucose concentration above 6 g/L. Culture supernatant was harvested at day 14, then centrifuged and filtrated to remove cells and cell debris before proceeding to purification. ÄKTA ™ chromatography BsAb harvest cell culture fluids (HCCFs) were purified using MabSelect PrismA resin to obtain post Protein A products, which were adjusted to pH 6.5, using 1 M Tris-base. The neutralized bsAb samples were then aliquoted and frozen at -20 o C. These samples were used for the polishing step study. Prior to CEX/CHT purification, the frozen bsAb samples were thawed at room temperature, then adjusted to pH 5.5 for CEX load and pH 6.8 for CHT load, all conductivities were kept ≤ 5 mS/cm. 1 mL of CHT Type II resin (Bio-Rad) and Capto SP ImpRes resin (Cytiva) were packed in Tricorn™ series columns (Cytiva) respectively with a bed height of 5.1 cm. Purifications were conducted using AKTA™ Avant 25 (Cytiva) with a flow rate of 150 cm/hr unless otherwise stated. Eluates were collected as fractions (1 mL/fraction) into a 96-well deep well plate. Fractions with higher purity than loading material were mock pooled with equal volume and re-analysed by HPLC-SEC to determine yield and purity. For CHT evaluation runs, the packed column was pre-equilibrated with 500 mM sodium phosphate pH 6.8 (3 CV), then equilibrated with equilibration buffer (10 mM sodium phosphate pH 6.8) (10 CV) prior to loading (10 ± 1 mg protein/mL resin; monomer concentration of 1 ± 0.1 mg/mL). After loading, a post-load-wash (PLW) was conducted using the equilibration buffer (5 CV). Linear gradient elution was performed using either 10–400 mM sodium phosphate pH 6.8 (for phosphate gradient elution) or 10 mM sodium phosphate pH 6.8 with 0–400 mM sodium chloride (for sodium chloride gradient elution), both over 40 CV with additional 10 CV hold at 400 mM phosphate or sodium chloride concentration. For optimized CHT sodium chloride purification, PLW was modified by using 45 mM or 60 mM sodium phosphate pH 6.8 (10 CV, flowrate = 75 cm/hr) for Molecule A and B, respectively, followed by 10 mM sodium phosphate pH 6.8 (3 CV, flowrate = 75 cm/hr) before proceeding to the same linear sodium chloride gradient elution profile as performed in the scouting runs. For CEX evaluation runs, the column was pre-equilibrated with 50 mM sodium acetate pH 5.5, 1 M sodium chloride (5 CV) then equilibrated with an equilibration buffer (50 mM sodium acetate pH 5.5) (5 CV) prior to loading (10 ± 1 mg protein/mL resin; monomer concentration of 1 ± 0.1 mg/mL), being followed by a PLW using the equilibration buffer (5 CV). Linear gradient elution was performed using 50 mM sodium acetate pH 5.5 with 0–400 mM sodium chloride over 40 CV with 10 CV additional hold at 400 mM sodium chloride concentration. BsAb concentration and purity determination BsAb concentration and purity analysis were performed by size exclusion chromatography-HPLC (HPLC-SEC), using a TSKgel G3000SWXL column (7.8 mm i.d. x 30 cm; Tosoh Bioscience); flow rate: 0.6 mL/min; mobile phase: 200 mM L-arginine, 50 mM MES, 5 mM EDTA, 0.05% sodium azide (w/w), pH 6.5, UV 280 nm. BsAb concentrations were determined using the main peak area, compared to a calibration curve generated using antibody standards with known concentrations and rectified using respective extinction coefficient (Chen et al., 2022b ). To ensure that other proteins in HCCF were excluded from bsAb concentration calculation, the bsAb titer determination was calculated by subtracting monomeric peak integration of HCCF with that of the flowthrough from PrismA chromatography. Amounts of HMW and LMW were determined from area of peaks pre- and post-main peak, respectively. SDS-PAGE gels (BioRad, non-reducing Gel: 4–15% Criterion TGX Stain-Free Protein Gel, reducing Gel: Any KD Criterion TGX Stain-Free Protein Gel) were used to visualize target proteins and impurities. 0.2 µg of monomeric bsAb was loaded per lane. The gels were stained using eLuminol™ (GeneCopoeia). Residual host cell protein (HCP) and host cell DNA (HCDNA) determination CHO HCP contents were determined by CHO HCP ELISA Kit, 3G (Cygnus Technologies). The protocol was executed, following manufacturer’s instructions. Data acquisition was performed on the Synergy™ 2 plate reader (BioTek). CHO HCDNA contents were determined using a qPCR assay. Briefly, all bsAb samples were digested with proteinase K (0.2 mg/mL in 0.5% SDS, 1 h, 50°C), followed by heat inactivation (10 min, 95°C) and DNA extraction using QIAamp® viral RNA mini kit (Qiagen). HCDNA contents were then determined from the extracted samples, using resDNASEQ Quantitative CHO DNA Kit (Thermo Fisher Scientific), following manufacturer’s instructions. Data acquisition was performed on LightCycler 96 (Roche). Intact mass analysis by LC-MS Purified bsAb samples were diluted with 0.3% formic acid, and 200 ng of each sample was separated on nanoACQUITY UPLC (Waters) with Waters BioResolve RP mAb Polyphenyl column (450Å, 2.7 µm, 1 mm X 150 mm) at a flow rate of 50 µL/min, with 2 min desalting, 6 min gradient from 20–90% mobile phase (0.1% formic acid in acetonitrile), followed by 4 min equilibration of 20% mobile phase. Protein intact mass data were acquired using TripleTOF 6600 mass spectrometry (SCIEX) in a positive ion mode at 600-3,500 m/z, spray voltage of 5000 V, source temperature of 400 o C, and operated by Analyst 1.8. MS data were processed and deconvoluted using Byos 4.3 Intact Mass™ software (Protein Metrics Inc). Results Resin evaluation for polishing asymmetric and symmetric IgG-Like bsAbs The performance of CHT for bsAb polishing was evaluated in comparison to CEX resin. We explored CHT Type II, which is widely used in biopharmaceutical manufacturing and is expected to offer even higher separation resolution as compared to that of the CHT type I (BioRad; Wang & Carta, 2020a ). Capto SP ImpRes was chosen as the CEX resin for comparison with CHT because it is a popular resin, established for polishing mAbs industrially (Luo et al., 2014 ; Sharkey et al., 2017 ). In addition, Capto SP ImpRes possesses a comparable particle size to that of the CHT Type II (40 µm) (BioRad; Cytiva, 2020 ). As CHT is a mixed-mode medium, runs with CHT-NaCl and CHT-P were performed to explore both elution mechanisms. As a result, a total of nine purification runs were conducted on the model bsAbs to evaluate the performance of CHT and CEX as a polishing step. Gradient elution profiles of the nine purification runs are illustrated in Fig. 3 . The results revealed that CHT chromatography resulted in higher product purity for all three bsAbs, compared to CEX chromatography. The highest monomer purity for both asymmetric bsAbs (Molecule A and B), were obtained from CHT-NaCl runs (%Main = 97.5% (Molecule A); and 96.5% (Molecule B)). Instead, a symmetric bsAb (Molecule C) achieved the best monomer purity of 97.7% from the CHT-P run (Fig. 4 and Table 1 ). As such, CHT-NaCl and CHT-P were chosen for asymmetric and symmetric bsAbs purification, respectively. Table 1 Product purity (%), HCP and HCDNA contents in loads and product pools of Molecule A, B and C after Protein A, CEX, CHT-P, and CHT-NaCl chromatography. Indicated as N.D. means the amount was not detectable. Molecule Protein samples Purity (%) HCP (ppm) HCDNA (ppm) HMW Main LMW A HCCF 21.32 35.39 43.29 1012636 100223 Post Protein A 4.73 93.55 1.72 5839 39 Post CEX 3.23 95.74 1.03 301 0.008 Post CHT-P 1.82 96.49 1.68 378 N.D. Post CHT-NaCl 0.53 97.49 1.98 73 N.D. Post CHT-NaCl (optimized-PLW) 0.47 98.49 1.04 62 0.009 B HCCF 30.98 34.18 34.84 391286 51832 Post Protein A 4.10 91.69 4.22 4578 17 Post CEX 2.85 94.81 2.34 43 0.006 Post CHT-P 0.88 96.08 3.04 155 N.D. Post CHT-NaCl 0.28 96.46 3.26 34 N.D. Post CHT-NaCl (optimized-PLW) 0.15 97.36 2.50 12 N.D. C HCCF 25.16 30.97 43.87 380062 108858 Post Protein A 2.28 91.47 6.25 1304 7 Post CEX 2.04 92.90 5.05 243 0.026 Post CHT-P 0.39 97.70 1.91 70 N.D. Post CHT-NaCl 0.33 95.36 4.31 35 0.012 More efficient aggregate removal for all three bsAbs was observed through the employment of CHT chromatography, compared to CEX. For asymmetric bsAbs, post CEX product pools still contained up to 3.2% HMW species, while as low as 0.3% HMW was present in post CHT products. CHT-NaCl showed higher efficiency in removing HMW species than CHT-P, with approximately 3-fold less HMW contents, detected in post CHT-NaCl products for both Molecule A and B. Interestingly, the HMW removal efficiency by CHT-NaCl and CHT-P became more convergent for the symmetric bsAb Molecule C (%HMW = 0.39% (CHT-P); and 0.33% (CHT-NaCl), suggesting merely equivalent performance for aggregate removal by both CHT-P and CHT-NaCl. Regarding LMW removal, small LMW species, such as light chain monomers (23.4 kDa) and dimers (46.8 kDa), could be fully removed by all tested chromatography runs as evidenced by SDS-PAGE, showing no light chain co-eluted with the products (Fig. 5 , data not shown for CEX). However, larger LMW species, including hole-hole homodimer (103.6 kDa) and monomer without one light chain (101.1 kDa) in Molecule A; knob-knob homodimer (145.6 kDa) in Molecule B; monomers, lacking one or two light chains (153.0 and 176.4 kDa, respectively) in Molecule C, were still co-eluted with the targets in high purity elution fractions. As evidenced by SDS-PAGE that the light chain monomers and dimers were completely removed from the products, the LMW species, being detected by SEC-HPLC, were primarily larger LMW species. The SEC-HPLC results indicated that CEX offered the best large LMW removal for asymmetric bsAbs Molecule A and B, followed by CHT-P runs that did slightly better than the CHT-NaCl runs (Molecule A: %LMW = 1.0% (CEX); 1.7% (CHT-P); 2.0% (CHT-NaCl); and Molecule B: %LMW = 2.34% (CEX); 3.04% (CHT-P); 3.26% (CHT-NaCl)) (Fig. 3 , Fig. 4 , and Table 1 ). Nevertheless, the best large LMW removal performance was achieved by CHT-P in symmetric bsAb Molecule C (%LMW = 1.9% (CHT-P); 4.3% (CHT-NaCl); 5.1% (CEX)). Product yields across different medium/elution mechanisms were also evaluated and compared within the same molecule (Fig. 6 (left)). %Yield of asymmetric bsAbs Molecule A and B across all purification runs are generally comparable (Molecule A: %yield = 76.4% (CHT-NaCl); 82.2% (CHT-P); 80.3% (CEX); Molecule B: %yield = 73.9% (CHT-NaCl); 80.4% (CHT-P); 69.4% (CEX)) (Fig. 6 (left)). However, %yield for Molecule C was relatively lower than that of Molecule A and B. This was potentially because Molecule C is the most aggregation-prone molecule among all tested bsAbs. The monomers could have been associated to form aggregates on the medium during purification, thus leading to lower monomer yield. %Yield of the symmetric bsAb Molecule C was significantly improved when using CHT chromatography as CHT provided higher separation resolution than that of the CEX (%yield = 67.8% (CHT-NaCl); 53.6% (CHT-P); 31.4% (CEX)) (Fig. 6 (left)). In addition, CHT caused much less “chromatography induced aggregation” (Fig. 6 (right)). One of the most challenging issues, reported for bsAb purification is so called “chromatography-induced aggregation” (Chen et al., 2022a , b ). This phenomenon is where monomeric bsAbs are self-associated during chromatographic purification, resulting in more HMWs in the product pools, together with reduction in monomer yield. Herein, we defined ratios of [HMW contents in all elution fractions] over [HMW contents in loads] to represent degrees of “chromatography-induced aggregation”. The higher ratios indicate that more HMWs are generated during purification. Our study showed that less “chromatography-induced aggregation” was detected in CHT runs as compared to CEX runs (Fig. 6 (right)). For both asymmetric bsAbs, up to ~ 5-fold more HMWs were generated during CEX chromatography than those observed during CHT runs. The effect was more exaggerated for the symmetric bsAb, Molecule C, where almost 8-fold more aggregates were formed during CEX purification, while the “chromatography-induced aggregates” were barely detected in post CHT products. These results implied the symmetric Molecule C expressed much higher aggregation propensity than both asymmetric bsAbs, but the CHT medium could impressively mitigate aggregation formation during the process, while CEX was unable to. Developability of CHT purification methods to enhance LMW removal Since post CHT-NaCl eluates of asymmetric bsAbs Molecule A and B still contained higher levels of LMWs, comparing to those from CEX-NaCl runs, optimization of CHT-NaCl runs were conducted to evaluate the developability of CHT purification methods for further LMW removal. As shown in Fig. 3 (indicated by red boxes), LMW species could be removed from Molecule A and B at elevated phosphate concentrations (> 10 mM, used during the resin evaluation study) with minimal loss of monomers. Therefore, a modified post-load-wash (PLW) step with slightly higher phosphate concentration was implemented for both asymmetric bsAbs. Phosphate concentrations were increased from 10 mM to 45 mM for Molecule A, and to 60 mM for Molecule B, respectively. The CVs of PLW was also extended from 5 CV to 10 CV, and the wash flow rate was reduced by half to potentially improve LMW removal efficiency. Modifications on the PLW step led to further reduction of LMW contents in product pools by 48% for Molecule A, and 23% for Molecule B, as compared to the CHT-NaCl evaluation run (Fig. 7 (left)). Consequently, product purity was improved from 97.5–98.5% for Molecule A, and from 96.5–97.4% for Molecule B (Fig. 7 (middle)). Although some larger LMW species still co-eluted with the target proteins ( Supplementary Fig. 1 ), over 97% purity is typically considered as acceptable for bsAb products (Li et al., 2020 ; Tustian et al., 2016 ). Expectedly, improvement upon separation resolution led to slight increase in %yield for both asymmetric bsAb Molecule A and B (%yield from 76.4–78.9% for Molecule A, and from 73.9–74.4% for Molecule B, respectively) (Fig. 7 (right)). Determination of HCP and HCDNA contents Residual CHO HCP and HCDNA contents were evaluated on the load and product from each purification step (Table 1 ). As expected, reduction of these process-related impurities after each purification step was observed with depletion of HCDNA contents to undetectable levels across all tested polishing media and elution mechanisms (Table 1 ). However, the amount of HCP present in product pools after each chromatography run differed. Interestingly, high purity products from CHT-NaCl consistently expressed minimal HCP contents (HCP = 12–73 ppm), whereas those from both CEX and CHT-P showed higher levels of HCP contaminants (HCP = 43–378 ppm) (Table 1 ). The results suggested that the CHT-NaCl has unique mechanisms to efficiently modulate HCP removal from the product pools. Characterization of purified bsAbs Intact mass analysis confirmed that identities of all three bsAb molecules were conserved after each chromatography step (Protein A, CEX and CHT) (Supplementary Fig. 2–4) . For Molecule C, CEX purification showed two dominant elution peaks (Fig. 3 ), both of which were confirmed to be the target molecule (Supplementary Fig. 4) . Discussion Higher conductivity was required during CHT-NaCl than CHT-P gradient elution for bsAb to be eluted out Conductivity, required for bsAb to be eluted out from CHT medium, was higher with CHT-NaCl gradient elution than with CHT-P gradient elution (Fig. 3 ), suggesting that bsAb binding to CHT medium was stronger during NaCl gradient elution in comparison with that of the phosphate gradient. This phenomenon could be the outcome of C-site cooperation, which behaved differently during CHT-NaCl vs CHT-P elution. Figure 8 illustrates the possible differences in binding and elution mechanisms for CHT-NaCl and CHT-P. Even though Molecule A, B, and C binding to CHT medium was P-site dominant as indicated by pI their values (pI = 8.52 (Molecule A); 8.59 (Molecule B); 8.70 (Molecule C), parts of the protein surfaces could still interact with C-sites via carboxyl clusters (Gorbunoff, 1984 b). Prior to elution, bsAb binding to CHT medium was at the same state with the same strength in both CHT-NaCl and CHT-P runs, as the same loading and washing conditions were applied. When the CHT-NaCl gradient started, higher conductivity weakened the electrostatic interactions between P-sites and ammonium clusters on proteins, which served as the main driving force for bsAb elution from CHT medium. However, higher NaCl concentration also enhanced C-site interactions to carboxyl groups on protein surfaces, which concurrently leading to further protein retention on CHT. Consequently, higher elution conductivity was required for CHT-NaCl elution due to C-site cooperation. When it came to CHT-P, the increase of phosphate concentration gradually saturated C-sites and led to the termination of C-site cooperation, which facilitated bsAb elution from CHT. This explains why lower conductivity for elution was required for CHT-P, as compared to CHT-NaCl. This evidence demonstrated that buffer composition and concentration have direct impacts on protein-CHT binding, either strengthening or weakening the binding strength. Positive cooperation from C-sites enhanced CHT performance for product-related impurity removal Efficient HMW removal by CHT CHT chromatography proved to offer better aggregate removal than CEX for all three tested bsAbs (Molecule A, B, and C), and CHT-NaCl showed the best performance Fig. 3 , Table 1 ). For Molecule A and B, CHT-NaCl significantly outperformed CHT-P, as evidenced by approximately 3-fold less HMWs detected in post CHT-NaCl product. However, CHT-P showed almost equivalent HMW removal efficiency to CHT-NaCl for Molecule C, as closer HMW contents were detected in both post CHT-P and CHT-NaCl products (%HMW = 0.39% (CHT-P); and 0.33% (CHT-NaCl). The results implied CHT-NaCl has a distinctive mechanism to improve separation resolution, and molecular size might be a key factor to promote HMW removal efficiency by CHT-P for Molecule C (MW = 124.6 kDa (Molecule A); = 172.5 kDa (Molecule B); = 199.8 kDa (Molecule C)). For Molecule A and B, better HMW removal by CHT-NaCl could be simply explained by the different C-site cooperation during CHT-NaCl and CHT-P elution as well. Aggregates are significantly larger than monomers in size and have higher surface charges. They had stronger interactions to both P-sites and C-sites on CHT thus requiring higher conductivity for elution. During CHT-NaCl gradient elution, higher conductivity triggered protein elution but at the same time high NaCl concentration also strengthened affinity between C-sites and carboxyl groups on protein surfaces (Fig. 8 , top right). Since aggregates were larger than monomers, this C-site retention effect was stronger towards aggregates than monomers, thus leading to even higher conductivity required for aggregates elution. The difference in C-site cooperation between aggregates and monomers during elution enhanced their separation resolution. However, such mechanism was not applicable when performing CHT-P elution. Our observation is also supported by the previous work, demonstrating that a monomeric IgG and tetra-aggregates were eluted at significantly different NaCl concentrations when using CHT-NaCl (Gagnon et al., 2009 ). Interestingly, HMW removal performance by CHT-P was on par with that of CHT-NaCl for Molecule C, of which the molecular size is larger than both Molecule A and B due to the presence of two flexible scFv domains (Fig. 2 ). This was potentially because Molecule C had stronger initial binding strength to both P-sites and C-sites due to its larger protein surface, especially for aggregates. Therefore, the difference in binding strength between aggregates and monomers were significant enough to provide good separation resolution during elution, no matter CHT-NaCl or CHT-P was applied. This evidence implied the “molecular size” could be a notable factor, accounting for different binding and separation behaviors. As proven by our study, CHT-P could also yield great HMW removal efficiency to the same level as provided by CHT-NaCl when it came to larger bsAbs Molecule C. A similar observation was reported in previous studies, where CHT-P was successfully applied to purify large monoclonal antibody types, like IgA (385 kDa) and IgM (900 kDa) (Aoyama & Chiba, 1993 ; Gagnon, 2009 ; Luellau et al., 1998 ; Lüllau et al., 1997 ). Obviously, CEX separation was solely achieved by the difference in electrostatic interactions between aggregates and monomers. As such, HMW removal by CEX was lower than that of both CHT-P and CHT-NaCl. Efficient LMW removal by CHT Various LMW populations existed in post Protein A for all three bsAbs (Molecule A, B and C), which could be broadly categorized into small and large LMW species (Table 2 ). Light chain monomers and dimers could be efficiently removed by both CHT and CEX chromatography as they were small, hence containing lesser binding surfaces and net charges, as indicated by low pI values (Table 2 ). This left mainly larger LMWs to co-elute with the target monomers. Interestingly, different media and elution mechanisms demonstrated different performance on large LMW removal. By comparing two CHT elution mechanisms, CHT-P always provided better large LMW removal than CHT-NaCl for all tested bsAbs, yet to different extents (Table 1 ). Better separation resolution by CHT-P suggested that monomers and large LMW impurities expressed divergent binding preference to CHT binding sites, where ones could be more favored to C-sites than the others. Herein, we defined “C-site binding ratios” (Eq. 1) to evaluate on CHT binding site preference for three domain compositions of bsAb Molecule A, B and C (scFv, Fab, and Fc), then identified which domain binding was the most favoured to C-sites. To simplify the calculations, only amino acids, giving major contributions to C-site (Asp and Glu) and P-site (Arg and Lys) binding were considered. A protein domain with a higher C-site binding ratio was supposed to be more desirable to bind to C-sites. As illustrated in Table 1 , Fc domains (Fc (knob), Fc(hole), and Fc (wild-type)) owned higher C-site binding ratios than both scFv and Fab domains. Hence, Fc domains would favourably bind to C-sites, while binding of both scFv and Fab had higher tendency towards P-sites. This was also in-line with the pI values (Table 3 ). In addition, our rationale was supported by the previous empirical study, explaining different domain compositions of IgGs demonstrated different preference to CHT binding sites, where binding of Fab and (Fab)2 regions is P-site dominant but binding of a Fc domain is more favoured to the C-sites (Gagnon et al., 2009 ). Table 3 C-site binding ratios, pI values, and C-site binding preference for scFv, Fab, Fc domain compositions for bsAbs Molecule A, B, and C. Fc (knob) and Fc (hole) are Fc domains of Molecule A, and B, while Fc (wild-type) is the Fc domain for Molecule C. bsAb Domain C-site binding ratio pI Value C-site binding preference scFv 0.70 9.09 Less favoured Fab 0.85 8.64 Fc (knob) 1.0 6.94 More favoured Fc (hole) 1.0 6.94 Fc (wild-type) 1.0 7.18 We then further investigated on domain compositions of larger LMWs and monomers for bsAb Molecule A, B and C, and defined Fc(%) (Eq. 2) and ∆Fc(%) (Eq. 3) (See Appendices) to explain our results in a mechanistic point of view. Table 2 illustrated that large LMW impurities for all three bsAbs (Molecule A, B, and C) contained higher percentage of the Fc region (Fc (%)) than their monomers (positive ∆Fc(%)). Therefore, these large LMWs were likely to have more preferential binding towards the C-sites, as compared to the monomers. Greater large LMW removal efficiency by CHT-P than CHT-NaCl seemed to also be driven by the positive cooperation from C-sites. As showed in Fig. 9 , during CHT-P gradient elution, gradual increase in amounts of phosphate could trigger elution of large LMWs prior to monomer elution (Fig. 5 , Supplementary Fig. 5 ) as those impurities expressed more C-site cooperation. While those impurities were eluting, monomers, which had stronger electrostatic interactions, were still hold mainly on P-sites, leading to higher separation resolution by CHT-P. Instead, during CHT-NaCl gradient, hiking in NaCl concentration strengthened protein affinity to C-sites, leading to further retention of both impurities and the target monomers. Once proper conductivity was achieved, large LMWs were simply co-eluted with the target monomers, resulting in lower separation efficiency by CHT-NaCl as compared to CHT-P. Interestingly, CHT-P provided just slightly better large LMW removal than CHT-NaCl for Molecule A and B (Molecule A: %LMW = 1.7% (CHT-P); 2.0% (CHT-NaCl); Molecule B: %LMW = 3.0% (CHT-P); 3.3% (CHT-NaCl)), while the difference was more pronounced for Molecule C (%LMW = 1.9% (CHT-P); 4.3% (CHT-NaCl), and this can be potentially attributed to the difference in their molecular sizes. As bsAb Molecule A and B were smaller than Molecule C, their concurrent binding with both C- and P-sites on CHT was potentially less pronounced than Molecule C. Thus, C-site cooperation was more beneficial for larger protein like Molecule C, enabling higher separation resolution between large LMWs and the target monomers. The C-site cooperation also well explained better large LMW separation by CEX than CHT-NaCl for Molecule A and B. As CEX did not have C-sites to retard elution of large LMWs at high NaCl concentration, LMW species were likely to elute earlier by CEX than CHT-NaCl, resulting in better separation resolution. Impressively, CHT-P could even outperform CEX for Molecule C to remove large LMW species. This evidence was also driven by larger molecular size of Molecule C, allowing the protein to make the most use of C-site cooperation for the best performance on LMW removal efficiency among all tested chromatographic runs. CHT-NaCl showed great performance on process-related impurity removal Consistently low HCP contents were observed in post CHT-NaCl products for all three bsAbs (Table 1 ), indicating that the sodium chloride elution mechanism can efficiently remove HCPs from the product pools. Other previous studies also demonstrated similar observations (Gagnon, 2009 ). This could be explained by pI values of HCPs, along with purification process conditions. The CHO HCPs have a wide range of pI values (pI = 2–11), majority of which are between pI of 4.5–7.0 (Chollangi et al., 2015 ). After a neutralization step (post Protein A, at pH of 6.5), some HCPs with neutral pI values could potentially be eliminated via precipitation, leaving majority of the acidic HCPs in CHT and CEX loads. As acidic proteins were well-coordinated with the C-sites on the CHT medium, products eluted via sodium chloride gradient contained less HCPs. This was because those acidic HCPs still bound to the C-sites during elution with sodium chloride, whereas application of phosphate solutes totally abolished this acidic HCP holding mechanism. Obviously, the CEX chromatography had no such mechanism to hold these acidic HCPs, resulting in co-elution of the acidic HCPs with the target proteins. Although HCDNA contents in both post CHT and post CEX products were undetectable in our study (Table 1 ), other previous work demonstrated that CHT-NaCl provided not only lower HCP contents than CHT-P performed, but it also yielded lower contents of other process-related impurities, including HCDNA and endotoxins (Gagnon, 2009 ). CHT-NaCl and CHT-P significantly mitigated “chromatography-induced aggregation” against highly aggregation-prone bsAbs In general, bsAbs are highly aggregation-prone proteins, especially for the scFv-containing molecules (Chen et al., 2022a , 2022b ; Li et al., 2016 ). Chromatography-induced aggregation for both Molecule A and B have been previously reported, when performing a bind-elute mode for both protein A and CEX chromatography (Chen et al., 2022a , b ). Our study further confirmed the phenomenon. In addition, we observed that symmetric bsAb Molecule C is more prone to aggregation than the other two asymmetric bsAbs Molecule A and B. This is potentially because Molecule C contains two scFv domains, which are known to be less folded, more hydrophobic and with higher aggregation propensity (Fig. 2 ). One of the most impactful advantages of CHT is that it mitigated aggregate formation during purification, while significantly higher aggregate contents were generated during CEX chromatography (Fig. 6 (right)). A possible mechanistic explanation for minimal aggregate formation by CHT is likely due to the presence of calcium ions at C-sites. During column chromatography, proteins are bound on the medium at high concentrations, making intermolecular interactions more favorable than intramolecular interactions (Baek & Zydney, 2018 ). Thus, more aggregate formation could be expected during the purification process given hydrophobic interactions as key driving forces. Chaotropic salts, such as calcium chloride and arginine hydrochloride, have been commonly used as additives to suppress “chromatography-induced aggregation” in protein purification (Chen et al., 2020 ; Luo et al., 2014 ; Song et al., 2023 ). Based on this evidence, we hypothesized that calcium structures, installed on CHT as the C-sites, could potentially mitigate the issue as well. Chaotropic calcium salts can disrupt hydrogen bonding among water molecules, leading to increase in solution hydrophobicity (Jacob, 2000 ). This eventually reduces intermolecular hydrophobic interactions among protein molecules, leading to lesser protein aggregation (Pham & Meng, 2020 ). This phenomenon explained why CHT could mitigate “on-column aggregation” whereas CEX lacked this capability. The potential of CHT to minimize process-induced aggregates accentuates its suitability for bsAb purification in a commercial scale to achieve high productivity. Conclusion Our study demonstrated the effectiveness of using CHT Type II medium for bsAb purification, covering both asymmetric and symmetric IgG-like bispecific formats. Utilizing CHT as a polishing step yielded bsAb products with at least 97% purity. Dual binding mechanisms by CHT offered such great advantages to remove both product- and process-related impurities. Excellent HMW removal capability of CHT makes it a suitable medium for bsAb purification, as bsAbs are commonly associated with high levels of aggregates due to their high aggregation propensity. HMWs are also able to trigger severe immunogenicity, which poses more concerns to drug regulatory authorities (Lundahl et al., 2021 ; Ratanji et al., 2014 ). Great aggregate removal efficiency by CHT was observed across all tested bsAb molecules. In general, CHT-NaCl showed the best aggregate removal performance than both CEX and CHT-P, but CHT-P could express similar HMW removal efficiency when applied for large bsAbs, where C-site cooperation was more beneficial for better separation. For large bsAbs, CHT-P may also render good large LMWs removal provided that there is divergent binding preference between monomers and large LMW species towards C-sites. Therefore, for bsAbs with similar size to IgG (e.g. Molecule A and B), CHT-NaCl may be selected as the elution strategy to achieve the best HMW removal, whereas CHT-P elution strategy can be explored for larger bsAbs (e.g. Molecule C) to potentially remove both HMW and large LMW impurities. More impressively, “chromatography-induced aggregation”, which was previously reported on the same bsAb molecules during Protein A and CEX (Chen et al., 2022b ), was barely detected in post CHT products, especially, those from CHT-NaCl runs. Finally, CHT-NaCl offered a unique mechanism to yield bsAb products with the lowest HCP contents among all tested runs. Among all benefits, which CHT provided to serve efficient purification, C-site cooperation was a key player behind the success. Lastly, there are other possible solutions to resolve certain imperfections of CHT chromatography. CHT-NaCl might not be able to efficiently remove large LMW species for certain bsAb molecules (e.g. Molecule A and B). However, other resin types could be used in conjunction with CHT to assist in separating those impurities. The previous study showed that great LMW removal (including half antibodies and homodimers) from asymmetric bsAbs could be achieved during Protein A affinity purification with an implementation of low pH intermediate washes (Chen et al., 2022b ). Therefore, CHT-NaCl can potentially be coupled with Protein A chromatography and the low pH wash strategy to achieve even higher product purity with minimal LMWs and HMWs. Although CHT-P was less effective to remove process-related contaminants (e.g. HCPs and HCDNA), this elution mechanism can still be utilized, mainly for the challenging large LMW and aggregate removal applications. An orthogonal polishing strategy (e.g. anion exchange chromatography (Li, 2017 )) can always be coupled with CHT-P to further remove these process-related impurities in order to meet drug safety compliance. Abbreviations bsAbs: bispecific antibodies CEX: cation exchange chromatography CHT: ceramic hydroxyapatite CHT-NaCl: ceramic hydroxyapatite with sodium chloride linear gradient elution CHT-P: ceramic hydroxyapatite with sodium phosphate linear gradient elution HCCF: harvest cell culture fluid HCP: host cell proteins HMW: high molecular weight LMW: low molecular weight mAbs: monoclonal antibodies PLW: post-load-wash Declarations Acknowledgements This research was supported by HOYA Technosurgical Corporation, Singapore Branch and the Agency for Science, Technology and Research of Singapore. Author contributions NI designed experiments, analysed results, and wrote the manuscript draft; WX did experiments; LJM did experiments and assisted reviewing the manuscript draft; FBM did cell culture; BKP did MS experiments; KYJ did MS analysis; XB oversaw the MS analysis; YY oversaw the cell culture; KS and TM designed experiments and reviewed the manuscript draft; ZW conceived, oversaw the project, and revised the manuscript. Funding This research was supported by HOYA Technosurgical Corporation, Singapore Branch and the Agency for Science, Technology and Research, Singapore. Availability of data and materials All data generated or analysed during this study are included in this published article [and its supplementary information files]. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Aoyama, K., & Chiba, J. (1993). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3402362","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":237954680,"identity":"c7351769-3624-4cd4-b23a-ee09f660bc71","order_by":0,"name":"Wei Zhang","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-8597-5357","institution":"Bioprocessing Technology Institute","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Zhang","suffix":""},{"id":237954681,"identity":"10b18ba8-5378-4879-bce5-b4cdbf600a32","order_by":1,"name":"Nattha Ingavat","email":"","orcid":"","institution":"Bioprocessing Technology Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nattha","middleName":"","lastName":"Ingavat","suffix":""},{"id":237954682,"identity":"54dee025-b060-45ee-806d-bb77f21ac7b6","order_by":2,"name":"Wang Xinhui","email":"","orcid":"","institution":"Bioprocessing Technology Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wang","middleName":"","lastName":"Xinhui","suffix":""},{"id":237954683,"identity":"10cdfa6b-e629-4404-8b08-2e113bb53477","order_by":3,"name":"Liew Jia Min","email":"","orcid":"","institution":"Bioprocessing Technology Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Liew","middleName":"Jia","lastName":"Min","suffix":""},{"id":237954684,"identity":"1a19bd5d-8716-4701-8a76-aadaa5d439f1","order_by":4,"name":"Farouq Bin Mahfut","email":"","orcid":"","institution":"Bioprocessing Technology Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Farouq","middleName":"Bin","lastName":"Mahfut","suffix":""},{"id":237954685,"identity":"052f8ffd-78ac-4a49-a7fe-c18ba2d57f35","order_by":5,"name":"But Ka Pui","email":"","orcid":"","institution":"Bioprocessing Technology Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"But","middleName":"Ka","lastName":"Pui","suffix":""},{"id":237954686,"identity":"d7fa6a7c-c3a4-42b0-9c61-3e4161106d5b","order_by":6,"name":"Kok Yee Jiun","email":"","orcid":"","institution":"Bioprocessing Technology Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kok","middleName":"Yee","lastName":"Jiun","suffix":""},{"id":237954687,"identity":"4f4d6cd3-f8f5-4165-8703-824ead6883f2","order_by":7,"name":"Xuezhi Bi","email":"","orcid":"","institution":"Bioprocessing Technology Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xuezhi","middleName":"","lastName":"Bi","suffix":""},{"id":237954688,"identity":"0224926d-09a0-4714-b9ae-043bbc6ddd98","order_by":8,"name":"Yuansheng Yang","email":"","orcid":"","institution":"Bioprocessing Technology Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuansheng","middleName":"","lastName":"Yang","suffix":""},{"id":237954689,"identity":"2b4dfbaf-389b-46da-9287-6de45c650808","order_by":9,"name":"Kobayashi Shintaro","email":"","orcid":"","institution":"HOYA Technosurgical Corporation","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kobayashi","middleName":"","lastName":"Shintaro","suffix":""},{"id":237954690,"identity":"ba16a27a-4f7c-4db3-8769-96d62c077391","order_by":10,"name":"Tsoumpra Maria","email":"","orcid":"","institution":"HOYA Technosurgical Corporation","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tsoumpra","middleName":"","lastName":"Maria","suffix":""}],"badges":[],"createdAt":"2023-10-01 10:04:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3402362/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3402362/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40643-023-00713-9","type":"published","date":"2023-12-13T15:01:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":44385099,"identity":"36900682-f0e9-4640-bcd4-c90244392c28","added_by":"auto","created_at":"2023-10-10 18:58:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":184270,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Ceramic hydroxyapatite (CHT) binding (left) and elution (right) mechanisms. There are two types of binding sites on CHT medium, a calcium-site (C-site) (top) and a phosphate-site (P-site) (bottom). (B) The addition of suitable salts (high concentration of NaCl (top); low concentration of phosphate (bottom)) enables stronger binding between biomolecules and the CHT binding sites.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3402362/v1/ff817f1cc3df27f1ab12c005.png"},{"id":44386124,"identity":"bdddabad-90a0-4da1-a565-981520c74fe6","added_by":"auto","created_at":"2023-10-10 19:06:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":54576,"visible":true,"origin":"","legend":"\u003cp\u003eThree bispecific antibodies (bsAbs), used as model molecules in this study, including two asymmetric molecules with heterodimeric knob-in-to-hole Fc and one symmetric molecule with homodimeric wild-type Fc. Molecule A, “1+1” valency; Molecule B, “2+1” valency; Molecule C, “2+2” valency. Abbreviations: LC = Light chain; HCK = Heavy chain knob; scFv-FcH = scFv-Fc hole; VH-CH1-scFv-FcH = VH-CH1-scFv-Fc hole; and VH-CH1-scFv-FcW = VH-CH1-scFv-Fc wild-type.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3402362/v1/1293c0a3cdf53eece1f05e6b.png"},{"id":44385104,"identity":"32a6cd46-0a56-4459-a540-e8b65c50e657","added_by":"auto","created_at":"2023-10-10 18:58:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":245973,"visible":true,"origin":"","legend":"\u003cp\u003eElution profiles of Molecule A (top); Molecule B (middle); and Molecule C (bottom) from CHT phosphate gradient elution (CHT-P, left column); CHT NaCl gradient elution (CHT-NaCl, middle column); and CEX (right column). Each plot demonstrates an overlay of chromatogram (A280) with protein mass in each elution fraction (bar graph), comprising Main (white); HMW (red); and LMW (black). The x-axis demonstrates volume during an elution phase (40 CV). Dotted lines demonstrate conductivity (mS/cm), required for corresponding elution fractions. Red boxes indicate elution fractions, containing mainly LMW species.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3402362/v1/7e20bc0e3afbe16fd51332d5.png"},{"id":44386125,"identity":"82ea2168-bb6a-438d-824b-7c9608913e75","added_by":"auto","created_at":"2023-10-10 19:06:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":76731,"visible":true,"origin":"","legend":"\u003cp\u003eBar graphs, demonstrating product purity (%Main (left); %HMW (middle); %LMW (right)) of Molecule A, B and C after each chromatography step (post Protein A, post CEX, post CHT phosphate gradient elution (CHT-P), and post CHT sodium chloride gradient elution (CHT-NaCl)).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3402362/v1/bafffbb6ddbb22b1960afdbb.png"},{"id":44385107,"identity":"fc9e827c-585b-4770-91c3-722a8d8e495a","added_by":"auto","created_at":"2023-10-10 18:58:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":396918,"visible":true,"origin":"","legend":"\u003cp\u003eNon-reducing SDS-PAGEs, illustrating protein populations in HCCF, Post Protein A, Post CHT-P (top), Post CHT-NaCl (bottom), and early elution fractions prior to main product peaks (Fx) of Molecule A (left column); Molecule B (middle column); and Molecule C (right column), respectively. Marker units are in kDa. Protein populations, indicated as (*), were proposed as suggested by molecular weights, which were calculated from amino acid sequences. Abbreviations used are listed as followed; M = monomer; HH = hole-hole homodimer; KK = knob-knob homodimer; LC = light chain; w/o = without. Existence of large LMWs in early elution fractions indicated early elution of large LMWs, as compared to monomers.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3402362/v1/4da1e8c915e9ed454cb0d7f0.png"},{"id":44386127,"identity":"491d7593-172c-4ab0-b190-5dc535ff27b7","added_by":"auto","created_at":"2023-10-10 19:06:14","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":185076,"visible":true,"origin":"","legend":"\u003cp\u003eBar graphs, illustrating %yield (left); and “chromatography-induced aggregation (right) of Molecule A, B, and C after CHT sodium chloride gradient elution (CHT-NaCl), CHT phosphate gradient elution (CHT-P), and CEX. Levels of “chromatography-induced aggregation”, was defined by the ratios of [HMW contents in all elution fractions] over [HMW contents in loads]. Higher ratios indicated more aggregates were generated during chromatography.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3402362/v1/334f84328a1f2748936c8ec2.jpg"},{"id":44385105,"identity":"a2ec9cec-b2b8-4d86-bfb8-6eb8e2ef6bfe","added_by":"auto","created_at":"2023-10-10 18:58:14","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":160201,"visible":true,"origin":"","legend":"\u003cp\u003eBar graphs, showing %LMW (left); %Main (middle); and %Yield (right)) of Molecule A, and B post CHT-NaCl vs post CHT-NaCl (optimized-PLW).\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3402362/v1/13eaf719dad815739cb2db7c.jpg"},{"id":44385106,"identity":"783eda98-3fcb-4658-84c2-650153bcd323","added_by":"auto","created_at":"2023-10-10 18:58:14","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":97232,"visible":true,"origin":"","legend":"\u003cp\u003eBinding (left) and elution mechanisms for CHT-NaCl (top right) and CHT-P (bottom right).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-3402362/v1/2e5f4f72e2604ab15d474839.png"},{"id":44386126,"identity":"1a67474e-0501-4b83-853f-39e0d62b6239","added_by":"auto","created_at":"2023-10-10 19:06:14","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":143395,"visible":true,"origin":"","legend":"\u003cp\u003eMechanistic illustrations during CHT-P (top) and CHT-NaCl (bottom) gradient elution. Differences in interactions between large LMWs vs monomers to C- and P-sites on CHT resulted in better separation resolution by CHT-P over CHT-NaCl. Numbers of dotted lines, NaCl and phosphate molecules are used for an illustration purpose to compare protein-CHT binding behavior (from left to right) during CHT-NaCl and CHT-P gradient elution, but not quantitatively.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-3402362/v1/a7115aa66190a50047a2970b.png"},{"id":48401333,"identity":"1343088d-30e1-493b-b7c6-3c4899e59a66","added_by":"auto","created_at":"2023-12-18 15:08:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1986768,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3402362/v1/817fe3c9-b563-4912-a939-b64c77e34cc0.pdf"},{"id":44385102,"identity":"bb1d6668-8332-4715-b8dd-058b92eea84a","added_by":"auto","created_at":"2023-10-10 18:58:14","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":102303,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-3402362/v1/e5b66359f6deb0751769d3b3.docx"},{"id":44386415,"identity":"9563bab7-ea7a-449a-a470-eec7016ba86e","added_by":"auto","created_at":"2023-10-10 19:14:14","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":13094,"visible":true,"origin":"","legend":"","description":"","filename":"Appendices.docx","url":"https://assets-eu.researchsquare.com/files/rs-3402362/v1/dd36c7643a389863992c793f.docx"},{"id":44385112,"identity":"7fa86a49-9dba-4e53-89d3-903a5af3db8e","added_by":"auto","created_at":"2023-10-10 18:58:15","extension":"pptx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":487027,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstractfinal.pptx","url":"https://assets-eu.researchsquare.com/files/rs-3402362/v1/b7b709de3e5341dd0431712d.pptx"},{"id":44385110,"identity":"03cd93d4-7383-49a4-ad85-f2994233c2bd","added_by":"auto","created_at":"2023-10-10 18:58:14","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":793673,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-3402362/v1/41ad91bcd99380986720e67a.docx"}],"financialInterests":"","formattedTitle":"Harnessing Ceramic Hydroxyapatite as an Effective Polishing Strategy to Remove Product- and Process-related Impurities in Bispecific Antibody Purification","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBispecific antibodies (bsAbs) have become one of the most attractive therapeutic modalities over the past decade (Wei et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wu \u0026amp; Cheung, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). They are man-made proteins, deliberately engineered to possess two different antigen recognition sites instead of one that exists in traditional monoclonal antibodies (mAbs). A combination of the two antigen-binding sites in proximity provides dramatic improvement in therapeutic efficacy (Labrijn et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Furthermore, bsAbs can potentially enable novel treatments, not offered by any combination of the natural mAbs.\u003c/p\u003e \u003cp\u003eBsAbs are broadly categorized into three main formats (Chen \u0026amp; Zhang, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Labrijn et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), including fragment-based bsAbs, asymmetric bsAbs and symmetric bsAbs. The fragment-based bsAbs are designed to incorporate at least two antigen-binding sites into one molecule without the Fc region to avoid any undesired chain-association issue (Labrijn et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, this format is likely to have a comparatively short plasma half-life (Labrijn et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Both asymmetric bsAbs and symmetric bsAbs are Fc-containing molecules. Asymmetric bsAbs normally contain three to four polypeptides, which are derived from two different parental mAbs. Due to random chain pairing, it is possible for asymmetric bsAbs to generate up to ten different homodimers and heterodimers. Although a couple of bsAb engineering technologies, such as knobs-into-holes (Ridgway et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Rouet \u0026amp; Christ, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), and CrossMAb\u0026trade; technology (Surowka et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), have been developed to mitigate chain mispairings, none of these strategies can completely prevent the formation of mispaired byproducts. For symmetric bsAbs, both of their recognition sites are typically incorporated into a single polypeptide chain to allow co-expression of only one to two polypeptides. As a result, they are generally tetravalent (2\u0026thinsp;+\u0026thinsp;2) by design. Currently, among all bsAb molecules that have been under clinical development, asymmetric and symmetric IgG-like bsAbs are the most popular formats.\u003c/p\u003e \u003cp\u003eBsAbs pose numerous challenges to downstream processing mainly due to high diversity and complexity of their structures and impurity profiles. The bsAb product-related impurities, including mispaired species, half antibodies, antibody fragments and aggregates, are difficult to remove because of their similarities in size and physiochemical properties to the desired end-product. Furthermore, engineered bsAbs are also reported to be highly aggregation-prone, making them less stable than their parental mAbs (Chen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). Considering the differences between mAb and bsAb, traditional purification tools for mAbs are less likely to provide the same level of efficiency when it comes to bsAbs. In addition, \u0026ldquo;chromatography-induced aggregation\u0026rdquo; has been observed during both Protein A and cation exchange chromatography (CEX) for bsAb purification as reported by Serene Chen et al, and Lucus K. Kimerer et al. (Chen et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e; Kimerer et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This \u0026ldquo;chromatography-induced aggregation\u0026rdquo; is more pronounced when bsAbs are subjected to high loading (Chen et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003eb\u003c/span\u003e). This could be a crucial bottleneck for bsAb industrial production as lower loading leads to lower productivity.\u003c/p\u003e \u003cp\u003eGiven that ceramic hydroxyapatite (CHT) is one of the leading media for intermediate and polishing step development, it has been applied to the purification of various types of biological therapeutics, including antibodies, antibody fragments, recombinant proteins, viruses, and DNAs (Hilbrig \u0026amp; Freitag, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). CHT is a mixed-mode support that comprises two different binding sites as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. The first binding site which mainly retains solutes via calcium metal affinity is commonly known as the \u0026ldquo;C-site\u0026rdquo;. It primarily binds to carboxyl groups of proteins, phosphoryl residues of DNA, as well as phosphorylated solutes (Gagnon, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Itoh et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Elution of proteins bound to the C-sites can be achieved with solutions containing either phosphate or fluoride ions, which have higher affinity for calcium ions as compared to carboxyl groups (Gorbunoff \u0026amp; Timasheff, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Retention of biomolecules via interactions with the C-sites (e.g. DNA and acidic proteins) can tolerate high concentration of sodium chloride (up to 4 M sodium chloride in the absence of phosphate), suggesting that the C-site interactions are significantly stronger than electrostatic forces (Gagnon, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Gorbunoff \u0026amp; Timasheff, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Itoh et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The second binding site that acts as a cation exchanger is named as the \u0026ldquo;P-site\u0026rdquo;. P-sites primarily bind to ammonium groups on proteins and elution can be accomplished by raising conductivity using any types of salt (Gorbunoff, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1984\u003c/span\u003ea; Gorbunoff \u0026amp; Timasheff, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1984\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSodium chloride gradient (CHT-NaCl) and phosphate gradients (CHT-P) have been used intensively for CHT elution. In addition, usage of borate and sulfate salts for elution has also been reported for certain applications (Gagnon, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Buffer compositions and concentrations play important roles in binding strength, as well as protein binding capacity of CHT. Previous studies showed that sodium chloride can weaken repulsive forces between negatively charged biomolecules (e.g. phosphoryl DNA and carboxyl-proteins) and P-sites. Therefore, interactions of phosphoryl DNA and negatively charged protein with the C-sites are tremendously strengthened at high NaCl concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) (Gagnon, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Gagnon et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Hilbrig \u0026amp; Freitag, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). A similar concept applies to interactions between protein ammonium groups and P-sites. During a protein binding state, the presence of low phosphate concentration assists reducing repulsion between positively charged ammonium groups of proteins and the C-sites, as compared to when no phosphate is present. Therefore, strengthening cationic interactions between ammonium clusters of proteins and the P-sites (Gagnon et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Gorbunoff, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1984\u003c/span\u003eb). By tweaking the buffer compositions and concentrations, the binding of the biomolecules to CHT medium can be affected, leading to different binding strength and capacity. In addition, the presence of at least 5 mM phosphate in all buffers is required regardless elution mechanisms to maintain CHT stability (Gagnon, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Gagnon et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). With a combination of metal affinity and cation exchange interactions, CHT provides unique separation mechanisms with additional purification benefits, therefore improving the removal efficiency of both product- and process-related impurities (Gagnon, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, the effectiveness of CHT chromatography as a polishing step on bsAb purification was evaluated and compared to the traditional CEX, using three model bsAb molecules (Molecules A, B, and C) with different formats, molecular sizes and domain compositions as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The results showed that CHT chromatography can serve as an effective polishing step for both asymmetric and symmetric IgG-like bsAbs, interestingly, with different binding and elution mechanisms. The products of both bsAb formats were of 97% purity with acceptable levels of process-related impurities. Previously, Yiran Wang, et al. have conducted mechanistic studies on competitive binding kinetics and separation dynamics of monomeric and dimeric monoclonal antibodies using CHT (Wang \u0026amp; Carta, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang \u0026amp; Carta, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e). A hybrid model has also been developed to explain separation of monoclonal antibody monomer-dimer mixture using gradient elution strategies (Wang \u0026amp; Carta, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e). On top of the previous published papers, herein, we extended CHT application to bsAb purification and elaborated the possible mechanistic insights. Our mechanistic explanation mainly focuses on how CHT medium mediates both product- and process-related impurity removal in bsAb purification.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eAll buffers, salts, and reagents were purchased from Sigma-Aldrich except for sodium chloride and citric acid that were purchased from Merck Millipore. MabSelect PrismA, Capto SP Impress, and Tricon series columns were purchased from Cytiva. CHT Type II (40 \u0026micro;m) was provided by HOYA Technosurgical Corporation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBsAb culture production\u003c/h2\u003e \u003cp\u003eStably transfected CHO K1 cell lines, producing bsAbs, were generated by site-specific integration of plasmid vectors, which carried genes encoding light chain (LC), heavy chain knob (HCK) and scFv-Fc hole (scFv-FcH) (for Molecule A), or VH-CH1-scFv-Fc hole (VH-CH1-scFv-FcH) (for Molecule B); LC and VH-CH1-scFv-Fc wild-type (VH-CH1-scFv-FcW) (for Molecule C) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). CH3 domains in the HCK and scFv-FcH/VH-CH1-scFv-FcH were engineered to form a knob (through mutations of S354C:T366W) and a hole (through mutations of Y349C:T366S:Y407V), respectively, to facilitate heterodimeric Fc pairing, based on a previous study (Merchant et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). For Molecule A, VH and VL in scFv were connected through a flexible linker (G4S)3, which was further linked to FcH through a G4 linker. The engineered Fc consisted of IgG1 positions 221 to 447 based on the Eu numbering system (Wang et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). For Molecule B and C, VH-CH1 was linked to the N-terminus of scFv through a G4S linker, which was further linked to either FcH (Molecule B) or FcW (Molecule C), respectively, through a G4 linker. The stably transfected cell lines were grown in a protein-free medium consisting of HyQ PF (Cytiva) and CD CHO (Thermo Fisher Scientific) at 1:1 ratio and supplemented with 1 g/L sodium carbonate (Sigma), 6 mM glutamine (Sigma), and 0.1% Pluronic F-68 (Thermo Fisher Scientific) in 50 mL tubespin (TPP) in a humidified Kuhner shaker (Adolf K\u0026uuml;hner AG) with 8% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. To produce these three molecules, 300 mL of cell culture at viable cell density of 3\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL were inoculated into 600 mL tubespin (TPP) in the humidified Kuhner shaker (Adolf K\u0026uuml;hner AG) with 8% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. 30 mL of Ex-Cell Advanced CHO Feed 1 (with glucose) (SAFC, Sigma) was added at day 3, 5, 7, 9 and 11. Cell density and viability of each culture were monitored at day 3, 5, 7, 9, 11 and 14, using the Vi-Cell XR viability analyser (Beckman Coulter). d-Glucose concentration in the culture medium was quantified using Nova BioProfile 100plus analyzer (Nova Biomedical). When the glucose concentration in the media dropped below 2 g/L, d-glucose (Sigma) was added to the culture to adjust glucose concentration above 6 g/L. Culture supernatant was harvested at day 14, then centrifuged and filtrated to remove cells and cell debris before proceeding to purification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e\u0026Auml;KTA\u003csup\u003e\u0026trade;\u003c/sup\u003e chromatography\u003c/h2\u003e \u003cp\u003eBsAb harvest cell culture fluids (HCCFs) were purified using MabSelect PrismA resin to obtain post Protein A products, which were adjusted to pH 6.5, using 1 M Tris-base. The neutralized bsAb samples were then aliquoted and frozen at -20\u003csup\u003eo\u003c/sup\u003e C. These samples were used for the polishing step study. Prior to CEX/CHT purification, the frozen bsAb samples were thawed at room temperature, then adjusted to pH 5.5 for CEX load and pH 6.8 for CHT load, all conductivities were kept\u0026thinsp;\u0026le;\u0026thinsp;5 mS/cm.\u003c/p\u003e \u003cp\u003e1 mL of CHT Type II resin (Bio-Rad) and Capto SP ImpRes resin (Cytiva) were packed in Tricorn\u0026trade; series columns (Cytiva) respectively with a bed height of 5.1 cm. Purifications were conducted using AKTA\u0026trade; Avant 25 (Cytiva) with a flow rate of 150 cm/hr unless otherwise stated. Eluates were collected as fractions (1 mL/fraction) into a 96-well deep well plate. Fractions with higher purity than loading material were mock pooled with equal volume and re-analysed by HPLC-SEC to determine yield and purity.\u003c/p\u003e \u003cp\u003eFor CHT evaluation runs, the packed column was pre-equilibrated with 500 mM sodium phosphate pH 6.8 (3 CV), then equilibrated with equilibration buffer (10 mM sodium phosphate pH 6.8) (10 CV) prior to loading (10\u0026thinsp;\u0026plusmn;\u0026thinsp;1 mg protein/mL resin; monomer concentration of 1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 mg/mL). After loading, a post-load-wash (PLW) was conducted using the equilibration buffer (5 CV). Linear gradient elution was performed using either 10\u0026ndash;400 mM sodium phosphate pH 6.8 (for phosphate gradient elution) or 10 mM sodium phosphate pH 6.8 with 0\u0026ndash;400 mM sodium chloride (for sodium chloride gradient elution), both over 40 CV with additional 10 CV hold at 400 mM phosphate or sodium chloride concentration. For optimized CHT sodium chloride purification, PLW was modified by using 45 mM or 60 mM sodium phosphate pH 6.8 (10 CV, flowrate\u0026thinsp;=\u0026thinsp;75 cm/hr) for Molecule A and B, respectively, followed by 10 mM sodium phosphate pH 6.8 (3 CV, flowrate\u0026thinsp;=\u0026thinsp;75 cm/hr) before proceeding to the same linear sodium chloride gradient elution profile as performed in the scouting runs.\u003c/p\u003e \u003cp\u003eFor CEX evaluation runs, the column was pre-equilibrated with 50 mM sodium acetate pH 5.5, 1 M sodium chloride (5 CV) then equilibrated with an equilibration buffer (50 mM sodium acetate pH 5.5) (5 CV) prior to loading (10\u0026thinsp;\u0026plusmn;\u0026thinsp;1 mg protein/mL resin; monomer concentration of 1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 mg/mL), being followed by a PLW using the equilibration buffer (5 CV). Linear gradient elution was performed using 50 mM sodium acetate pH 5.5 with 0\u0026ndash;400 mM sodium chloride over 40 CV with 10 CV additional hold at 400 mM sodium chloride concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBsAb concentration and purity determination\u003c/h2\u003e \u003cp\u003eBsAb concentration and purity analysis were performed by size exclusion chromatography-HPLC (HPLC-SEC), using a TSKgel G3000SWXL column (7.8 mm i.d. x 30 cm; Tosoh Bioscience); flow rate: 0.6 mL/min; mobile phase: 200 mM L-arginine, 50 mM MES, 5 mM EDTA, 0.05% sodium azide (w/w), pH 6.5, UV 280 nm. BsAb concentrations were determined using the main peak area, compared to a calibration curve generated using antibody standards with known concentrations and rectified using respective extinction coefficient (Chen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). To ensure that other proteins in HCCF were excluded from bsAb concentration calculation, the bsAb titer determination was calculated by subtracting monomeric peak integration of HCCF with that of the flowthrough from PrismA chromatography. Amounts of HMW and LMW were determined from area of peaks pre- and post-main peak, respectively.\u003c/p\u003e \u003cp\u003eSDS-PAGE gels (BioRad, non-reducing Gel: 4\u0026ndash;15% Criterion TGX Stain-Free Protein Gel, reducing Gel: Any KD Criterion TGX Stain-Free Protein Gel) were used to visualize target proteins and impurities. 0.2 \u0026micro;g of monomeric bsAb was loaded per lane. The gels were stained using eLuminol\u0026trade; (GeneCopoeia).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eResidual host cell protein (HCP) and host cell DNA (HCDNA) determination\u003c/h2\u003e \u003cp\u003eCHO HCP contents were determined by CHO HCP ELISA Kit, 3G (Cygnus Technologies). The protocol was executed, following manufacturer\u0026rsquo;s instructions. Data acquisition was performed on the Synergy\u0026trade; 2 plate reader (BioTek).\u003c/p\u003e \u003cp\u003eCHO HCDNA contents were determined using a qPCR assay. Briefly, all bsAb samples were digested with proteinase K (0.2 mg/mL in 0.5% SDS, 1 h, 50\u0026deg;C), followed by heat inactivation (10 min, 95\u0026deg;C) and DNA extraction using QIAamp\u0026reg; viral RNA mini kit (Qiagen). HCDNA contents were then determined from the extracted samples, using resDNASEQ Quantitative CHO DNA Kit (Thermo Fisher Scientific), following manufacturer\u0026rsquo;s instructions. Data acquisition was performed on LightCycler 96 (Roche).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIntact mass analysis by LC-MS\u003c/h2\u003e \u003cp\u003ePurified bsAb samples were diluted with 0.3% formic acid, and 200 ng of each sample was separated on nanoACQUITY UPLC (Waters) with Waters BioResolve RP mAb Polyphenyl column (450\u0026Aring;, 2.7 \u0026micro;m, 1 mm X 150 mm) at a flow rate of 50 \u0026micro;L/min, with 2 min desalting, 6 min gradient from 20\u0026ndash;90% mobile phase (0.1% formic acid in acetonitrile), followed by 4 min equilibration of 20% mobile phase. Protein intact mass data were acquired using TripleTOF 6600 mass spectrometry (SCIEX) in a positive ion mode at 600-3,500 m/z, spray voltage of 5000 V, source temperature of 400 \u003csup\u003eo\u003c/sup\u003eC, and operated by Analyst 1.8. MS data were processed and deconvoluted using Byos 4.3 Intact Mass\u0026trade; software (Protein Metrics Inc).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eResin evaluation for polishing asymmetric and symmetric IgG-Like bsAbs\u003c/h2\u003e \u003cp\u003eThe performance of CHT for bsAb polishing was evaluated in comparison to CEX resin. We explored CHT Type II, which is widely used in biopharmaceutical manufacturing and is expected to offer even higher separation resolution as compared to that of the CHT type I (BioRad; Wang \u0026amp; Carta, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e). Capto SP ImpRes was chosen as the CEX resin for comparison with CHT because it is a popular resin, established for polishing mAbs industrially (Luo et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Sharkey et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In addition, Capto SP ImpRes possesses a comparable particle size to that of the CHT Type II (40 \u0026micro;m) (BioRad; Cytiva, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As CHT is a mixed-mode medium, runs with CHT-NaCl and CHT-P were performed to explore both elution mechanisms. As a result, a total of nine purification runs were conducted on the model bsAbs to evaluate the performance of CHT and CEX as a polishing step.\u003c/p\u003e \u003cp\u003eGradient elution profiles of the nine purification runs are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The results revealed that CHT chromatography resulted in higher product purity for all three bsAbs, compared to CEX chromatography. The highest monomer purity for both asymmetric bsAbs (Molecule A and B), were obtained from CHT-NaCl runs (%Main\u0026thinsp;=\u0026thinsp;97.5% (Molecule A); and 96.5% (Molecule B)). Instead, a symmetric bsAb (Molecule C) achieved the best monomer purity of 97.7% from the CHT-P run (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As such, CHT-NaCl and CHT-P were chosen for asymmetric and symmetric bsAbs purification, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProduct purity (%), HCP and HCDNA contents in loads and product pools of Molecule A, B and C after Protein A, CEX, CHT-P, and CHT-NaCl chromatography. Indicated as N.D. means the amount was not detectable.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMolecule\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eProtein samples\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003ePurity (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHCP\u003c/p\u003e \u003cp\u003e(ppm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHCDNA\u003c/p\u003e \u003cp\u003e(ppm)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHMW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLMW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHCCF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e43.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1012636\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100223\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePost Protein A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e93.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5839\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePost CEX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e301\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePost CHT-P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e378\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN.D.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePost CHT-NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e97.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN.D.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePost CHT-NaCl (optimized-PLW)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHCCF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e391286\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e51832\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePost Protein A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e91.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4578\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePost CEX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e94.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePost CHT-P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e155\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN.D.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePost CHT-NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN.D.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePost CHT-NaCl (optimized-PLW)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e97.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN.D.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHCCF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e43.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e380062\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e108858\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePost Protein A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e91.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1304\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePost CEX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e243\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.026\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePost CHT-P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e97.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN.D.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePost CHT-NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.012\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMore efficient aggregate removal for all three bsAbs was observed through the employment of CHT chromatography, compared to CEX. For asymmetric bsAbs, post CEX product pools still contained up to 3.2% HMW species, while as low as 0.3% HMW was present in post CHT products. CHT-NaCl showed higher efficiency in removing HMW species than CHT-P, with approximately 3-fold less HMW contents, detected in post CHT-NaCl products for both Molecule A and B. Interestingly, the HMW removal efficiency by CHT-NaCl and CHT-P became more convergent for the symmetric bsAb Molecule C (%HMW\u0026thinsp;=\u0026thinsp;0.39% (CHT-P); and 0.33% (CHT-NaCl), suggesting merely equivalent performance for aggregate removal by both CHT-P and CHT-NaCl.\u003c/p\u003e \u003cp\u003eRegarding LMW removal, small LMW species, such as light chain monomers (23.4 kDa) and dimers (46.8 kDa), could be fully removed by all tested chromatography runs as evidenced by SDS-PAGE, showing no light chain co-eluted with the products (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, data not shown for CEX). However, larger LMW species, including hole-hole homodimer (103.6 kDa) and monomer without one light chain (101.1 kDa) in Molecule A; knob-knob homodimer (145.6 kDa) in Molecule B; monomers, lacking one or two light chains (153.0 and 176.4 kDa, respectively) in Molecule C, were still co-eluted with the targets in high purity elution fractions. As evidenced by SDS-PAGE that the light chain monomers and dimers were completely removed from the products, the LMW species, being detected by SEC-HPLC, were primarily larger LMW species. The SEC-HPLC results indicated that CEX offered the best large LMW removal for asymmetric bsAbs Molecule A and B, followed by CHT-P runs that did slightly better than the CHT-NaCl runs (Molecule A: %LMW\u0026thinsp;=\u0026thinsp;1.0% (CEX); 1.7% (CHT-P); 2.0% (CHT-NaCl); and Molecule B: %LMW\u0026thinsp;=\u0026thinsp;2.34% (CEX); 3.04% (CHT-P); 3.26% (CHT-NaCl)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Nevertheless, the best large LMW removal performance was achieved by CHT-P in symmetric bsAb Molecule C (%LMW\u0026thinsp;=\u0026thinsp;1.9% (CHT-P); 4.3% (CHT-NaCl); 5.1% (CEX)).\u003c/p\u003e \u003cp\u003eProduct yields across different medium/elution mechanisms were also evaluated and compared within the same molecule (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (left)). %Yield of asymmetric bsAbs Molecule A and B across all purification runs are generally comparable (Molecule A: %yield\u0026thinsp;=\u0026thinsp;76.4% (CHT-NaCl); 82.2% (CHT-P); 80.3% (CEX); Molecule B: %yield\u0026thinsp;=\u0026thinsp;73.9% (CHT-NaCl); 80.4% (CHT-P); 69.4% (CEX)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (left)). However, %yield for Molecule C was relatively lower than that of Molecule A and B. This was potentially because Molecule C is the most aggregation-prone molecule among all tested bsAbs. The monomers could have been associated to form aggregates on the medium during purification, thus leading to lower monomer yield. %Yield of the symmetric bsAb Molecule C was significantly improved when using CHT chromatography as CHT provided higher separation resolution than that of the CEX (%yield\u0026thinsp;=\u0026thinsp;67.8% (CHT-NaCl); 53.6% (CHT-P); 31.4% (CEX)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (left)). In addition, CHT caused much less \u0026ldquo;chromatography induced aggregation\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (right)).\u003c/p\u003e \u003cp\u003eOne of the most challenging issues, reported for bsAb purification is so called \u0026ldquo;chromatography-induced aggregation\u0026rdquo; (Chen et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003eb\u003c/span\u003e). This phenomenon is where monomeric bsAbs are self-associated during chromatographic purification, resulting in more HMWs in the product pools, together with reduction in monomer yield. Herein, we defined ratios of [HMW contents in all elution fractions] over [HMW contents in loads] to represent degrees of \u0026ldquo;chromatography-induced aggregation\u0026rdquo;. The higher ratios indicate that more HMWs are generated during purification. Our study showed that less \u0026ldquo;chromatography-induced aggregation\u0026rdquo; was detected in CHT runs as compared to CEX runs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (right)). For both asymmetric bsAbs, up to ~\u0026thinsp;5-fold more HMWs were generated during CEX chromatography than those observed during CHT runs. The effect was more exaggerated for the symmetric bsAb, Molecule C, where almost 8-fold more aggregates were formed during CEX purification, while the \u0026ldquo;chromatography-induced aggregates\u0026rdquo; were barely detected in post CHT products. These results implied the symmetric Molecule C expressed much higher aggregation propensity than both asymmetric bsAbs, but the CHT medium could impressively mitigate aggregation formation during the process, while CEX was unable to.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDevelopability of CHT purification methods to enhance LMW removal\u003c/h2\u003e \u003cp\u003eSince post CHT-NaCl eluates of asymmetric bsAbs Molecule A and B still contained higher levels of LMWs, comparing to those from CEX-NaCl runs, optimization of CHT-NaCl runs were conducted to evaluate the developability of CHT purification methods for further LMW removal. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (indicated by red boxes), LMW species could be removed from Molecule A and B at elevated phosphate concentrations (\u0026gt;\u0026thinsp;10 mM, used during the resin evaluation study) with minimal loss of monomers. Therefore, a modified post-load-wash (PLW) step with slightly higher phosphate concentration was implemented for both asymmetric bsAbs. Phosphate concentrations were increased from 10 mM to 45 mM for Molecule A, and to 60 mM for Molecule B, respectively. The CVs of PLW was also extended from 5 CV to 10 CV, and the wash flow rate was reduced by half to potentially improve LMW removal efficiency. Modifications on the PLW step led to further reduction of LMW contents in product pools by 48% for Molecule A, and 23% for Molecule B, as compared to the CHT-NaCl evaluation run (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e (left)). Consequently, product purity was improved from 97.5\u0026ndash;98.5% for Molecule A, and from 96.5\u0026ndash;97.4% for Molecule B (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e (middle)). Although some larger LMW species still co-eluted with the target proteins (\u003cem\u003eSupplementary Fig.\u0026nbsp;1\u003c/em\u003e), over 97% purity is typically considered as acceptable for bsAb products (Li et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tustian et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Expectedly, improvement upon separation resolution led to slight increase in %yield for both asymmetric bsAb Molecule A and B (%yield from 76.4\u0026ndash;78.9% for Molecule A, and from 73.9\u0026ndash;74.4% for Molecule B, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e (right)).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of HCP and HCDNA contents\u003c/h2\u003e \u003cp\u003eResidual CHO HCP and HCDNA contents were evaluated on the load and product from each purification step (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As expected, reduction of these process-related impurities after each purification step was observed with depletion of HCDNA contents to undetectable levels across all tested polishing media and elution mechanisms (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, the amount of HCP present in product pools after each chromatography run differed. Interestingly, high purity products from CHT-NaCl consistently expressed minimal HCP contents (HCP\u0026thinsp;=\u0026thinsp;12\u0026ndash;73 ppm), whereas those from both CEX and CHT-P showed higher levels of HCP contaminants (HCP\u0026thinsp;=\u0026thinsp;43\u0026ndash;378 ppm) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The results suggested that the CHT-NaCl has unique mechanisms to efficiently modulate HCP removal from the product pools.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of purified bsAbs\u003c/h2\u003e \u003cp\u003eIntact mass analysis confirmed that identities of all three bsAb molecules were conserved after each chromatography step (Protein A, CEX and CHT) \u003cem\u003e(Supplementary Fig.\u0026nbsp;2\u0026ndash;4)\u003c/em\u003e. For Molecule C, CEX purification showed two dominant elution peaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), both of which were confirmed to be the target molecule \u003cem\u003e(Supplementary Fig.\u0026nbsp;4)\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003eHigher conductivity was required during CHT-NaCl than CHT-P gradient elution for bsAb to be eluted out\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConductivity, required for bsAb to be eluted out from CHT medium, was higher with CHT-NaCl gradient elution than with CHT-P gradient elution (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e), suggesting that bsAb binding to CHT medium was stronger during NaCl gradient elution in comparison with that of the phosphate gradient. This phenomenon could be the outcome of C-site cooperation, which behaved differently during CHT-NaCl vs CHT-P elution.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e illustrates the possible differences in binding and elution mechanisms for CHT-NaCl and CHT-P. Even though Molecule A, B, and C binding to CHT medium was P-site dominant as indicated by pI their values (pI\u0026thinsp;=\u0026thinsp;8.52 (Molecule A); 8.59 (Molecule B); 8.70 (Molecule C), parts of the protein surfaces could still interact with C-sites via carboxyl clusters (Gorbunoff, \u003cspan class=\"CitationRef\"\u003e1984\u003c/span\u003eb). Prior to elution, bsAb binding to CHT medium was at the same state with the same strength in both CHT-NaCl and CHT-P runs, as the same loading and washing conditions were applied. When the CHT-NaCl gradient started, higher conductivity weakened the electrostatic interactions between P-sites and ammonium clusters on proteins, which served as the main driving force for bsAb elution from CHT medium. However, higher NaCl concentration also enhanced C-site interactions to carboxyl groups on protein surfaces, which concurrently leading to further protein retention on CHT. Consequently, higher elution conductivity was required for CHT-NaCl elution due to C-site cooperation. When it came to CHT-P, the increase of phosphate concentration gradually saturated C-sites and led to the termination of C-site cooperation, which facilitated bsAb elution from CHT. This explains why lower conductivity for elution was required for CHT-P, as compared to CHT-NaCl. This evidence demonstrated that buffer composition and concentration have direct impacts on protein-CHT binding, either strengthening or weakening the binding strength.\u003c/p\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003ePositive cooperation from C-sites enhanced CHT performance for product-related impurity removal\u003c/h2\u003e\n\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\n\u003ch2\u003eEfficient HMW removal by CHT\u003c/h2\u003e\n\u003cp\u003eCHT chromatography proved to offer better aggregate removal than CEX for all three tested bsAbs (Molecule A, B, and C), and CHT-NaCl showed the best performance Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). For Molecule A and B, CHT-NaCl significantly outperformed CHT-P, as evidenced by approximately 3-fold less HMWs detected in post CHT-NaCl product. However, CHT-P showed almost equivalent HMW removal efficiency to CHT-NaCl for Molecule C, as closer HMW contents were detected in both post CHT-P and CHT-NaCl products (%HMW\u0026thinsp;=\u0026thinsp;0.39% (CHT-P); and 0.33% (CHT-NaCl). The results implied CHT-NaCl has a distinctive mechanism to improve separation resolution, and molecular size might be a key factor to promote HMW removal efficiency by CHT-P for Molecule C (MW\u0026thinsp;=\u0026thinsp;124.6 kDa (Molecule A); = 172.5 kDa (Molecule B); = 199.8 kDa (Molecule C)).\u003c/p\u003e\n\u003cp\u003eFor Molecule A and B, better HMW removal by CHT-NaCl could be simply explained by the different C-site cooperation during CHT-NaCl and CHT-P elution as well. Aggregates are significantly larger than monomers in size and have higher surface charges. They had stronger interactions to both P-sites and C-sites on CHT thus requiring higher conductivity for elution. During CHT-NaCl gradient elution, higher conductivity triggered protein elution but at the same time high NaCl concentration also strengthened affinity between C-sites and carboxyl groups on protein surfaces (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e, top right). Since aggregates were larger than monomers, this C-site retention effect was stronger towards aggregates than monomers, thus leading to even higher conductivity required for aggregates elution. The difference in C-site cooperation between aggregates and monomers during elution enhanced their separation resolution. However, such mechanism was not applicable when performing CHT-P elution. Our observation is also supported by the previous work, demonstrating that a monomeric IgG and tetra-aggregates were eluted at significantly different NaCl concentrations when using CHT-NaCl (Gagnon et al., \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e). Interestingly, HMW removal performance by CHT-P was on par with that of CHT-NaCl for Molecule C, of which the molecular size is larger than both Molecule A and B due to the presence of two flexible scFv domains (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). This was potentially because Molecule C had stronger initial binding strength to both P-sites and C-sites due to its larger protein surface, especially for aggregates. Therefore, the difference in binding strength between aggregates and monomers were significant enough to provide good separation resolution during elution, no matter CHT-NaCl or CHT-P was applied. This evidence implied the \u0026ldquo;molecular size\u0026rdquo; could be a notable factor, accounting for different binding and separation behaviors. As proven by our study, CHT-P could also yield great HMW removal efficiency to the same level as provided by CHT-NaCl when it came to larger bsAbs Molecule C. A similar observation was reported in previous studies, where CHT-P was successfully applied to purify large monoclonal antibody types, like IgA (385 kDa) and IgM (900 kDa) (Aoyama \u0026amp; Chiba, \u003cspan class=\"CitationRef\"\u003e1993\u003c/span\u003e; Gagnon, \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e; Luellau et al., \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e; L\u0026uuml;llau et al., \u003cspan class=\"CitationRef\"\u003e1997\u003c/span\u003e). Obviously, CEX separation was solely achieved by the difference in electrostatic interactions between aggregates and monomers. As such, HMW removal by CEX was lower than that of both CHT-P and CHT-NaCl.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eEfficient LMW removal by CHT\u003c/h2\u003e\n\u003cp\u003eVarious LMW populations existed in post Protein A for all three bsAbs (Molecule A, B and C), which could be broadly categorized into small and large LMW species (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Light chain monomers and dimers could be efficiently removed by both CHT and CEX chromatography as they were small, hence containing lesser binding surfaces and net charges, as indicated by low pI values (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). This left mainly larger LMWs to co-elute with the target monomers. Interestingly, different media and elution mechanisms demonstrated different performance on large LMW removal.\u003c/p\u003e\n\u003cp\u003eBy comparing two CHT elution mechanisms, CHT-P always provided better large LMW removal than CHT-NaCl for all tested bsAbs, yet to different extents (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Better separation resolution by CHT-P suggested that monomers and large LMW impurities expressed divergent binding preference to CHT binding sites, where ones could be more favored to C-sites than the others. Herein, we defined \u0026ldquo;C-site binding ratios\u0026rdquo; (Eq.\u0026nbsp;1) to evaluate on CHT binding site preference for three domain compositions of bsAb Molecule A, B and C (scFv, Fab, and Fc), then identified which domain binding was the most favoured to C-sites. To simplify the calculations, only amino acids, giving major contributions to C-site (Asp and Glu) and P-site (Arg and Lys) binding were considered. A protein domain with a higher C-site binding ratio was supposed to be more desirable to bind to C-sites. As illustrated in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, Fc domains (Fc (knob), Fc(hole), and Fc (wild-type)) owned higher C-site binding ratios than both scFv and Fab domains. Hence, Fc domains would favourably bind to C-sites, while binding of both scFv and Fab had higher tendency towards P-sites. This was also in-line with the pI values (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In addition, our rationale was supported by the previous empirical study, explaining different domain compositions of IgGs demonstrated different preference to CHT binding sites, where binding of Fab and (Fab)2 regions is P-site dominant but binding of a Fc domain is more favoured to the C-sites (Gagnon et al., \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eC-site binding ratios, pI values, and C-site binding preference for scFv, Fab, Fc domain compositions for bsAbs Molecule A, B, and C. Fc (knob) and Fc (hole) are Fc domains of Molecule A, and B, while Fc (wild-type) is the Fc domain for Molecule C.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ebsAb Domain\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC-site binding ratio\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epI Value\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC-site binding preference\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003escFv\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.70\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.09\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eLess favoured\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.85\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.64\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFc (knob)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.94\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eMore favoured\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFc (hole)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.94\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFc (wild-type)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.18\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eWe then further investigated on domain compositions of larger LMWs and monomers for bsAb Molecule A, B and C, and defined Fc(%) (Eq.\u0026nbsp;2) and ∆Fc(%) (Eq.\u0026nbsp;3) (See Appendices) to explain our results in a mechanistic point of view. Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e illustrated that large LMW impurities for all three bsAbs (Molecule A, B, and C) contained higher percentage of the Fc region (Fc (%)) than their monomers (positive ∆Fc(%)). Therefore, these large LMWs were likely to have more preferential binding towards the C-sites, as compared to the monomers.\u003c/p\u003e\n\u003cp\u003eGreater large LMW removal efficiency by CHT-P than CHT-NaCl seemed to also be driven by the positive cooperation from C-sites. As showed in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e, during CHT-P gradient elution, gradual increase in amounts of phosphate could trigger elution of large LMWs prior to monomer elution (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cem\u003eSupplementary Fig.\u0026nbsp;5\u003c/em\u003e) as those impurities expressed more C-site cooperation. While those impurities were eluting, monomers, which had stronger electrostatic interactions, were still hold mainly on P-sites, leading to higher separation resolution by CHT-P. Instead, during CHT-NaCl gradient, hiking in NaCl concentration strengthened protein affinity to C-sites, leading to further retention of both impurities and the target monomers. Once proper conductivity was achieved, large LMWs were simply co-eluted with the target monomers, resulting in lower separation efficiency by CHT-NaCl as compared to CHT-P.\u003c/p\u003e\n\u003cp\u003eInterestingly, CHT-P provided just slightly better large LMW removal than CHT-NaCl for Molecule A and B (Molecule A: %LMW\u0026thinsp;=\u0026thinsp;1.7% (CHT-P); 2.0% (CHT-NaCl); Molecule B: %LMW\u0026thinsp;=\u0026thinsp;3.0% (CHT-P); 3.3% (CHT-NaCl)), while the difference was more pronounced for Molecule C (%LMW\u0026thinsp;=\u0026thinsp;1.9% (CHT-P); 4.3% (CHT-NaCl), and this can be potentially attributed to the difference in their molecular sizes. As bsAb Molecule A and B were smaller than Molecule C, their concurrent binding with both C- and P-sites on CHT was potentially less pronounced than Molecule C. Thus, C-site cooperation was more beneficial for larger protein like Molecule C, enabling higher separation resolution between large LMWs and the target monomers.\u003c/p\u003e\n\u003cp\u003eThe C-site cooperation also well explained better large LMW separation by CEX than CHT-NaCl for Molecule A and B. As CEX did not have C-sites to retard elution of large LMWs at high NaCl concentration, LMW species were likely to elute earlier by CEX than CHT-NaCl, resulting in better separation resolution. Impressively, CHT-P could even outperform CEX for Molecule C to remove large LMW species. This evidence was also driven by larger molecular size of Molecule C, allowing the protein to make the most use of C-site cooperation for the best performance on LMW removal efficiency among all tested chromatographic runs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003eCHT-NaCl showed great performance on process-related impurity removal\u003c/h2\u003e\n\u003cp\u003eConsistently low HCP contents were observed in post CHT-NaCl products for all three bsAbs (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), indicating that the sodium chloride elution mechanism can efficiently remove HCPs from the product pools. Other previous studies also demonstrated similar observations (Gagnon, \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e). This could be explained by pI values of HCPs, along with purification process conditions. The CHO HCPs have a wide range of pI values (pI\u0026thinsp;=\u0026thinsp;2\u0026ndash;11), majority of which are between pI of 4.5\u0026ndash;7.0 (Chollangi et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). After a neutralization step (post Protein A, at pH of 6.5), some HCPs with neutral pI values could potentially be eliminated via precipitation, leaving majority of the acidic HCPs in CHT and CEX loads. As acidic proteins were well-coordinated with the C-sites on the CHT medium, products eluted via sodium chloride gradient contained less HCPs. This was because those acidic HCPs still bound to the C-sites during elution with sodium chloride, whereas application of phosphate solutes totally abolished this acidic HCP holding mechanism. Obviously, the CEX chromatography had no such mechanism to hold these acidic HCPs, resulting in co-elution of the acidic HCPs with the target proteins.\u003c/p\u003e\n\u003cp\u003eAlthough HCDNA contents in both post CHT and post CEX products were undetectable in our study (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), other previous work demonstrated that CHT-NaCl provided not only lower HCP contents than CHT-P performed, but it also yielded lower contents of other process-related impurities, including HCDNA and endotoxins (Gagnon, \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003eCHT-NaCl and CHT-P significantly mitigated \u0026ldquo;chromatography-induced aggregation\u0026rdquo; against highly aggregation-prone bsAbs\u003c/h2\u003e\n\u003cp\u003eIn general, bsAbs are highly aggregation-prone proteins, especially for the scFv-containing molecules (Chen et al., \u003cspan class=\"CitationRef\"\u003e2022a\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2022b\u003c/span\u003e; Li et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Chromatography-induced aggregation for both Molecule A and B have been previously reported, when performing a bind-elute mode for both protein A and CEX chromatography (Chen et al., \u003cspan class=\"CitationRef\"\u003e2022a\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003eb\u003c/span\u003e). Our study further confirmed the phenomenon. In addition, we observed that symmetric bsAb Molecule C is more prone to aggregation than the other two asymmetric bsAbs Molecule A and B. This is potentially because Molecule C contains two scFv domains, which are known to be less folded, more hydrophobic and with higher aggregation propensity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). One of the most impactful advantages of CHT is that it mitigated aggregate formation during purification, while significantly higher aggregate contents were generated during CEX chromatography (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e (right)). A possible mechanistic explanation for minimal aggregate formation by CHT is likely due to the presence of calcium ions at C-sites. During column chromatography, proteins are bound on the medium at high concentrations, making intermolecular interactions more favorable than intramolecular interactions (Baek \u0026amp; Zydney, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Thus, more aggregate formation could be expected during the purification process given hydrophobic interactions as key driving forces. Chaotropic salts, such as calcium chloride and arginine hydrochloride, have been commonly used as additives to suppress \u0026ldquo;chromatography-induced aggregation\u0026rdquo; in protein purification (Chen et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Luo et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e; Song et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). Based on this evidence, we hypothesized that calcium structures, installed on CHT as the C-sites, could potentially mitigate the issue as well. Chaotropic calcium salts can disrupt hydrogen bonding among water molecules, leading to increase in solution hydrophobicity (Jacob, \u003cspan class=\"CitationRef\"\u003e2000\u003c/span\u003e). This eventually reduces intermolecular hydrophobic interactions among protein molecules, leading to lesser protein aggregation (Pham \u0026amp; Meng, \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). This phenomenon explained why CHT could mitigate \u0026ldquo;on-column aggregation\u0026rdquo; whereas CEX lacked this capability. The potential of CHT to minimize process-induced aggregates accentuates its suitability for bsAb purification in a commercial scale to achieve high productivity.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study demonstrated the effectiveness of using CHT Type II medium for bsAb purification, covering both asymmetric and symmetric IgG-like bispecific formats. Utilizing CHT as a polishing step yielded bsAb products with at least 97% purity. Dual binding mechanisms by CHT offered such great advantages to remove both product- and process-related impurities.\u003c/p\u003e \u003cp\u003eExcellent HMW removal capability of CHT makes it a suitable medium for bsAb purification, as bsAbs are commonly associated with high levels of aggregates due to their high aggregation propensity. HMWs are also able to trigger severe immunogenicity, which poses more concerns to drug regulatory authorities (Lundahl et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ratanji et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Great aggregate removal efficiency by CHT was observed across all tested bsAb molecules. In general, CHT-NaCl showed the best aggregate removal performance than both CEX and CHT-P, but CHT-P could express similar HMW removal efficiency when applied for large bsAbs, where C-site cooperation was more beneficial for better separation. For large bsAbs, CHT-P may also render good large LMWs removal provided that there is divergent binding preference between monomers and large LMW species towards C-sites. Therefore, for bsAbs with similar size to IgG (e.g. Molecule A and B), CHT-NaCl may be selected as the elution strategy to achieve the best HMW removal, whereas CHT-P elution strategy can be explored for larger bsAbs (e.g. Molecule C) to potentially remove both HMW and large LMW impurities. More impressively, \u0026ldquo;chromatography-induced aggregation\u0026rdquo;, which was previously reported on the same bsAb molecules during Protein A and CEX (Chen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e), was barely detected in post CHT products, especially, those from CHT-NaCl runs. Finally, CHT-NaCl offered a unique mechanism to yield bsAb products with the lowest HCP contents among all tested runs. Among all benefits, which CHT provided to serve efficient purification, C-site cooperation was a key player behind the success.\u003c/p\u003e \u003cp\u003eLastly, there are other possible solutions to resolve certain imperfections of CHT chromatography. CHT-NaCl might not be able to efficiently remove large LMW species for certain bsAb molecules (e.g. Molecule A and B). However, other resin types could be used in conjunction with CHT to assist in separating those impurities. The previous study showed that great LMW removal (including half antibodies and homodimers) from asymmetric bsAbs could be achieved during Protein A affinity purification with an implementation of low pH intermediate washes (Chen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). Therefore, CHT-NaCl can potentially be coupled with Protein A chromatography and the low pH wash strategy to achieve even higher product purity with minimal LMWs and HMWs. Although CHT-P was less effective to remove process-related contaminants (e.g. HCPs and HCDNA), this elution mechanism can still be utilized, mainly for the challenging large LMW and aggregate removal applications. An orthogonal polishing strategy (e.g. anion exchange chromatography (Li, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)) can always be coupled with CHT-P to further remove these process-related impurities in order to meet drug safety compliance.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ebsAbs: bispecific antibodies\u003c/p\u003e\n\u003cp\u003eCEX: cation exchange chromatography\u003c/p\u003e\n\u003cp\u003eCHT: ceramic hydroxyapatite\u003c/p\u003e\n\u003cp\u003eCHT-NaCl: ceramic hydroxyapatite with sodium chloride linear gradient elution\u003c/p\u003e\n\u003cp\u003eCHT-P: ceramic hydroxyapatite with sodium phosphate linear gradient elution\u003c/p\u003e\n\u003cp\u003eHCCF: harvest cell culture fluid\u003c/p\u003e\n\u003cp\u003eHCP: host cell proteins\u003c/p\u003e\n\u003cp\u003eHMW: high molecular weight\u003c/p\u003e\n\u003cp\u003eLMW: low molecular weight\u003c/p\u003e\n\u003cp\u003emAbs: monoclonal antibodies\u003c/p\u003e\n\u003cp\u003ePLW: post-load-wash\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by HOYA Technosurgical Corporation, Singapore Branch and the Agency for Science, Technology and Research of Singapore.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNI designed experiments, analysed results, and wrote the manuscript draft; WX did experiments; LJM did experiments and assisted reviewing the manuscript draft; FBM did cell culture; BKP did MS experiments; KYJ did MS analysis; XB oversaw the MS analysis; YY oversaw the cell culture; KS and TM designed experiments and reviewed the manuscript draft; ZW conceived, oversaw the project, and revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by HOYA Technosurgical Corporation, Singapore Branch and the Agency for Science, Technology and Research, Singapore.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAoyama, K., \u0026amp; Chiba, J. 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T cell engaging bispecific antibody (T-BsAb): From technology to therapeutics. \u003cem\u003ePharmacol Ther\u003c/em\u003e,\u003cem\u003e 182\u003c/em\u003e, 161-175. https://doi.org/10.1016/j.pharmthera.2017.08.005\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 2 is available in the Supplementary Files section.\u003c/p\u003e "}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"bispecific antibody, mixed-mode chromatography, product- and process-related impurity removal, chromatography-induced aggregation","lastPublishedDoi":"10.21203/rs.3.rs-3402362/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3402362/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBispecific antibody (bsAb), a novel therapeutic modality, provides excellent treatment efficacy, yet poses numerous challenges to downstream process development, which are mainly due to high diversity and complexity of bsAb structures and impurity profiles. The mixed-mode medium, ceramic hydroxyapatite (CHT), allows proteins to interact with its calcium sites (C-sites) through metal affinity and/or its phosphate sites (P-sites) through cation exchange interactions. This dual binding capability potentially offers unique bind and elute behaviours for different proteins of interest, resulting in optimal product purity when suitable elution conditions are employed. In this study, the effectiveness of CHT as a polishing step for bsAb purification was investigated across three model molecules and benchmarked against the traditional cation exchange chromatography (CEX). For both asymmetric and symmetric IgG-like bsAb post Protein A eluates, at least 97% product purity was achieved after CHT polishing. CHT delivered a superior aggregate clearance to CEX, resulting in low high molecular weight (HMW) impurities (0.5%) and low process-related impurities in the product pools. CHT also yielded significantly less \u0026ldquo;chromatography-induced aggregation\u0026rdquo; for all aggregation-prone bsAb molecules. Developability of CHT for more efficient low molecular weight (LMW) impurity removal was further demonstrated via post-load-wash (PLW) optimization with the selected bsAbs, resulting in up to 48% additional LMW reduction. Furthermore, possible mechanistic explanation about CHT performance on both process- and product-related impurity removal in bsAb polishing was proposed. Positive CHT C-site cooperation mediated effective impurity removal and mitigated \u0026ldquo;chromatography-induced aggregation\u0026rdquo;, and domain composition and size of bsAbs molecules may determine the effectiveness of such C-site cooperation.\u003c/p\u003e","manuscriptTitle":"Harnessing Ceramic Hydroxyapatite as an Effective Polishing Strategy to Remove Product- and Process-related Impurities in Bispecific Antibody Purification","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-10 18:58:09","doi":"10.21203/rs.3.rs-3402362/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-10-29T21:25:37+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2023-10-05T04:03:33+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-10-05T01:01:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-10-04T07:27:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioresources and Bioprocessing","date":"2023-10-01T06:04:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f5b1a2b5-7e8f-4870-bd72-769ac5724560","owner":[],"postedDate":"October 10th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-12-18T15:03:48+00:00","versionOfRecord":{"articleIdentity":"rs-3402362","link":"https://doi.org/10.1186/s40643-023-00713-9","journal":{"identity":"bioresources-and-bioprocessing","isVorOnly":false,"title":"Bioresources and Bioprocessing"},"publishedOn":"2023-12-13 15:01:16","publishedOnDateReadable":"December 13th, 2023"},"versionCreatedAt":"2023-10-10 18:58:09","video":"","vorDoi":"10.1186/s40643-023-00713-9","vorDoiUrl":"https://doi.org/10.1186/s40643-023-00713-9","workflowStages":[]},"version":"v1","identity":"rs-3402362","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3402362","identity":"rs-3402362","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}