In recent studies, electronegative multimodal chromatography with Eshmuno HCX was demonstrated to be a highly promising recovery step for direct immunoglobulin G (IgG) capture from undiluted cell culture fluid. In this study, the binding properties of HCX to IgG at different pH/salt combinations were systematically studied, and its purification performance was significantly enhanced by lowering the washing pH and conductivity after high capacity binding of IgG under its optimal conditions. A single polishing step gave an end-product with non-histone host cell protein (nh-HCP) below 1 ppm, DNA less than 1 ppb, which aggregates less than 0.5% and an overall IgG recovery of 86.2%. The whole non-affinity chromatography based two-column-step process supports direct feed loading without buffer adjustment, thus extraordinarily boosting the overall productivity and cost-savings. Keywords: Monoclonal antibody, Electronegative multimodal, Non-affinity purification, Cost-savings Kaleas et al. 2014). In our recent study, we demonstrated Introduction that advance chromatin extraction could significantly In biopharmaceuticals, the purification of recombinant improve the dynamic binding capacity (DBC) and IgG immunoglobulin G monoclonal antibodies (IgG mAbs) recovery of an electronegative multimodal chromatog- produced in Chinese hamster ovary (CHO) cells are usu- raphy, Eshmuno HCX. Compared with loading cell cul- ally achieved by 3–4 successive chromatographic steps ture supernatant (CCS) without chromatin extraction, (Girard et al. 2015; Shukla and Thömmes 2010). Affinity the DBC of HCX was boosted from 29 to 94 mg/mL, and chromatography of protein A is extensively used as the IgG recovery was also increased from around 80% to over primary capture and regarded as the industrial stand- 95% (Gagnon et al. 2014a). ard (Ghose et al. 2005; Tarrant et al. 2012). Nevertheless, In this study, full DBC profiles of HCX at various pH/ protein A chromatography has its inherent disadvan- salt combinations were systematically characterized. The tages, such as relatively low binding capacity, high mate- purification performance of HCX as the primary cap - rial/operational cost and ligand leachability, which add ture step was significantly enhanced by optimizing the another impurity into the process (Shukla et al. 2007; Tao column wash step. Void-exclusion anion exchange chro- et al. 2014). matography (VEAX) was further integrated with HCX to Multimodal chromatography has recently emerged form a seamless and efficient two-chromatography-step as a useful tool for antibody purification. Electronega - purification process for IgG production. tive multimodal chromatography was even considered as a potential alternative to protein A due to its lower Materials and methods cost and higher NaOH resistance (Urmann et al. 2010; Reagents and equipment All chemicals were obtained from Sigma-Aldrich (St. *Correspondence: email@example.com; firstname.lastname@example.org ™ high Louis, MO). WorkBeads 40 TREN was purchased College of Life Sciences, Jilin University, Changchun, China CAS Key Laboratory of Biobased Materials, Qingdao Institute from BioWorks (Uppsala, Sweden). Eshmuno HCX of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, was purchased from Merck Millipore (Merck KGaA, Qingdao, China © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Wang et al. AMB Expr (2018) 8:93 Page 2 of 7 bed height, at a linear flow rate of 300 cm/h, volumetric Darmstadt, Germany). UNOsphere Q was purchased flow rate of 10 mL/min). 1500 mL of c-e CCS was loaded from Bio-Rad Laboratories (Hercules, CA). Toyopearl to the column pre-equilibrated with equilibration buffer AF-rProtein A-650 was purchased from Tosoh Biosci- (EQ buffer) (50 mM MES, 100 mM NaCl, pH 6.0), fol - ence (Tokyo, Japan). Capto adhere was purchased from lowed by 10 CV of EQ buffer. The column was then GE Healthcare (Uppsala, Sweden). Chromatography washed with 10 CV of 50 mM MES, pH 6.0 or 50 mM media were packed in XK or Tricorn series columns acetic acid, pH 5.0 or 50 mM acetic acid, pH 4.0. The col - (GE Healthcare). Chromatography experiments were umn was further washed with 10 CV of EQ buffer, and conducted on an ÄKTA Explorer 100 or Avant 25 (GE IgG was eluted with a 5 CV liner gradient to 50 mM Tris, Healthcare). 2.0 M NaCl, pH 8.0 and collected from the point where UV absorbance at 280 nm reached 20 mAU to the point Experimental methods where it descended below that value. The column was A biosimilar IgG mAb immunospecific for human epi - sanitized with 5 CV of 1.0 M NaOH. Before storage in dermal growth factor receptor 2 was produced by CHO 20% ethanol, the column was thoroughly washed with EQ cell using a tricistronic vector developed by Ho et al. buffer. IgG polishing step was conducted on VEAX mode (2012). Antibody was produced as described in (Gagnon as described fully in our previous publication (Nian et al. et al. 2014a, b; 2015). 2013). “Traditional harvest clarification” was performed by centrifugation at 4000×g for 20 min at room tempera- Analytical methods ture, followed by filtration through a 0.22 μm mem - IgG purity, including nh-HCP, DNA and histone, was brane (Nalgene Rapid-Flow Filters, Thermo Scientific, documented according to the methods described fully in Waltham, MA). Clarified harvest named as centrifuged/ Gagnon et al. (2014a, 2014b, 2015). microfiltered CCS (c/m CCS) was stored at 2–8 °C Aggregate content and IgG concentration were meas- for short-term usage or − 20 °C for long-term storage. ured by analytical size exclusion chromatography (SEC) Cell culture was alternatively clarified by a more effi - with a G3000SWxl column (Tosoh Bioscience) on a cient “advance chromatin extraction” method devel- Dionex Ultimate 3000 HPLC system (Thermo Scien - oped recently by the research team led by Pete Gagnon tific). Residual caprylic acid and allantoin were deter - (Gagnon et al. 2014a, b, 2015), which was based on the mined by reversed phase-HPLC (RP-HPLC) (Gagnon synergistic effect of caprylic acid and allantoin, and har - et al. 2014a, b, 2015). vest clarified by this method was named as chromatin- Reduced SDS-PAGE was performed on 4–15% Crite- extracted CCS (c-e CCS). ™ ™ rion TGX Stain-Free Gel (Bio-Rad) and stained with a IgG used for DBC study was highly purified to mini - SilverQuest Silver Staining Kit from Invitrogen (Carls- mize interference with analytical methods. Protein A bad, CA). Turbidity expressed in nephelometric turbidity affinity chromatography was performed with 20 mL units (NTU) was measured with an Orion Q4500 Hand- of Toyopearl medium, and eluted IgG was polished held Turbidity Meter (Thermo Scientific). by Capto adhere chromatography according to Gag- non et al. (2014a, b, 2015). IgG purified by this process Results contained < 1 ppm nh-HCP , < 1 ppb DNA and ≤ 0.05% DBC profiles of HCX at various pH/salt combinations aggregates. Eshmuno HCX medium is described as a multimodal DBCs (mg/mL, at 5% breakthrough) of HCX at dif- cation exchanger. It is negatively charged due to the sulfo ferent pH/salt combinations were determined by using groups (strong ionic) and carboxyl groups (weak ionic). 4 mL Tricorn 5/10 columns at a linear flow rate of It contains phenyl groups that act in hydrophobic inter- 150 cm/h (2 mL/min, 2 min residence time). The col - actions. It also contains hydroxyl and amine groups, umns were equilibrated with buffers having NaCl from 0 exhibiting hydrogen binding properties. These functional to 200 mM and pH from 6.0 to 4.0, and then stayed off groups work together and contribute to the unique DBC line. The UV detector was zeroed. Highly purified IgG profiles of HCX as a function of pH/salt (Fig. 1). The pro - with the same pH and conductivity as the equilibration tein adsorption which depends on pH, conductivity and buffer was pumped into the system until the UV signal at flow rate of the buffer, is regarded as an essential condi - the entrance of the UV monitor matched that in the feed. tion to improve the low binding capacities with protein. This UV value was seen to represent 100% breakthrough. Unlike traditional strong cation exchangers, which show The column was then put in-line and monitored until UV the strongest binding at low pH and low conductiv- signal indicated 5% breakthrough. ity (Urmann et al. 2010), HCX demonstrated the high- IgG capture step was directly performed on HCX chro- est binding capacity for IgG at pH 6.0 in the presence of matography (20 mL medium packed in XK 16/20, 10 cm Wang et al. AMB Expr (2018) 8:93 Page 3 of 7 Fig. 1 DBC of HCX in response to different pH/salt combinations 100 mM NaCl. 95 mg/mL DBC was achieved under this optimal condition, which is significantly higher than pro - tein A resins with ≤ 40 mg/mL DBC commercially avail- able on the market (Nian et al. 2016; Urmann et al. 2010) and equivalent to most cation exchangers (Nian and Gag- non 2016). pH played an essential role in HCX binding capac- ity to IgG, which should be modulated by the weak ion Fig. 2 SDS-PAGE in comparison of the NaOH cleaning peak from HCX exchange matrices. The pH had an influence on the and protein A chromatography. Lane 1. Molecular weight marker. ligand density of protein on adsorbent matrixs. At opti- Lane 2. Supernatant fraction of neutralized 1.0 M NaOH cleaning mal pH value, protein massively utilized bonding site peak from HCX. Lane 3. Precipitate fraction of neutralized 1.0 M NaOH reducing electronic repulsion between different mol - cleaning peak from HCX. Lane 4. Supernatant fraction of neutralized ecules or unsaturation, which made HCX more effective 0.1 M NaOH cleaning peak from protein A. Lane 5. Precipitate fraction of neutralized 0.1 M NaOH cleaning peak from protein A and more flexible to achieve the highest binding capacity of IgG. Both increase and decrease of optimal pH dra- matically reduced the DBC of HCX. Notably, at all NaCl concentrations tested, the lowest binding capacity hap- As shown in Fig. 2, significant amount of intact IgG was pened at pH 5.0, and it recovered slightly when pH was found in protein A cleaning fraction (Lane 4 and Lane lower than 5.0. It was also found that, under high-salt 5), which was supposed to be mediated by nonspecific conditions, the relative position of the aromatic group interactions of chromatin with IgG and protein A (Gag- was critical to improve the breakthrough capacity, and an non et al. 2014b, 2015). Contaminant molecules occupied amide group on the α-carbon was essential for capturing limited bead pools to constrain and hinder IgG bonding proteins (Johansson et al. 2003). with resins. While there was mainly free IgG light chain (LC) and histone components for HCX, compared with Reducing contaminants binding profiles of HCX protein A, HCX can preferably remove contaminants out It is DNA, histone proteins and nh-HCP that constituted of CCS, which found a more high-efficiency methods to main contaminants in capturing of IgG from CHO. Dur- capture proteins. ing the whole capture step, IgG was considerably acces- sible to other dispersive contaminants through elution Optimization of HCX washing conditions to elevate the step even washing step no doubt giving rise to decrease purity of IgG the recovery of IgG. c/m CCS without advance chro- In our previous study, advance chromatin extraction ena- matin extraction, which contained all of the impurities bled HCX to achieve 94 mg/mL DBC and recover 95% mentioned above, was loaded onto HCX and protein A IgG in a sharp peak (Gagnon et al. 2014a, b). We ana- resins separately, and NaOH cleaning fractions of these lyzed the influence parameters of elution step above, but two chromatographies were compared by SDS-PAGE. Wang et al. AMB Expr (2018) 8:93 Page 4 of 7 not mentioned another crucial step, washing step. In this binding properties of HCX, other reported methods to study, we further optimized the washing steps with buff - maximize HCP clearance (Ishihara and Hosono 2015; ers at different pH. Interestingly, once IgG was bound Shukla and Hinckley 2008) may also be worthy to be onto HCX resin under optimal conditions of pH 6.0 and investigated with HCX in order to achieve more contami- 100 mM NaCl, lowering pH and salt concentration would nant removal in a single purification step. not detach bound IgG from the resin, and IgG recovery was around 95% under all testing conditions (Fig. 3a–d). Integrated purification process with two‑column‑step However, nh-HCP was respectively reduced to 1950, 660, containing capture and polishing steps 296 and 453 ppm, which proved that the optimized wash- VEAX stands for a new mode of anion-exchange chro- ing strategy could significantly enhance the purification matography and has been successfully applied as a pol- performance of HCX. Contaminate clearance is a signifi - ishing step for IgG purification with precipitation as the cant problem that can’t be ignored through IgG capture. primary capture (Chen et al. 2016). Table 1 summarizes Moreover, the weaker interaction between mAbs and a complete process for IgG purification beginning with HCX contributed to sufficiently elute proteins avoiding advance chromatin extraction, continuing to enhance recombination and obstraction. Considering the unique HCX capture, then a single polishing step with VEAX for Fig. 3 Chromatographic profiles of HCX washed with buffers of different pH and salt concentrations. a 200 mM NaCl pH 6. b 0 mM NaCl pH 6. c 0 mM NaCl pH 5. d 0 mM NaCl pH 4 Wang et al. AMB Expr (2018) 8:93 Page 5 of 7 Table 1 Summary of two-column-step IgG purification process CCS c‑ e CCS c‑ e CCS > enhanced HCX c‑ e CCS > enhanced HCX > VEAX IgG (mg/mL) 1.45 1.12 15.9 10.6 Stepwise IgG recovery (%) 100 91.8 94.6 99.3 DNA (ppm) 10,600 0.05 0.006 0.0004 Histone HCP (ppm) 28,560 < LOD < LOD < LOD nh-HCP (ppm) 224,800 8380 296 0.6 Aggregates (%) 18.65 0.45 0.42 0.40 LC (%) 12.3 1.2 < 0.05 < 0.05 Turbidity (NTU) 25.6 3.11 2.15 1.88 Caprylic acid (μg/mL) NA 5.67 2.32 0.14 Allantoin (μg/mL) NA 1119.05 < LOD < LOD NA not applicable Fig. 4 SEC chromatograms for each step of the proposed two-column-step IgG purification platform further contaminant clearance. Figure 4 illustrates ana- below the limit of detection (LOD, 0.05 μg/mL) respec- lytical SEC profiles for each process step which included tively. IgG monomer was increased to 99.99% (Fig. 4) c/m CCS, c-e CCS, enhanced HCX for IgG capture and with an overall recovery of 86.2%. VEAX for polishing. From the data, we can conclude that many impurities sharp peaks beside IgG peak when Discussion using c/m CCS or c-e CCS methods to capture protein. Many platform technologies have been developed for The recovery and purity of IgG can’t have a good per - IgG mAbs purification. Ion-exchange chromatography formance. While for enhanced HCX for IgG capture (IEX) as one of them was extensively used as a purifica - and VEAX for polishing methods, they both had a sin- tion technology for mAbs in biochemistry (Ahamed et al. gle sharp peak belonged to IgG and the latter one had a 2007). Interactions between protein and charged sub- higher recovery efficiency of IgG. stances in IEX were influenced by oppositely charged In the end-product, nh-HCP was reduced to 0.6 ppm, surfaces of porous chromatographic media. The power DNA to below 1 ppb and aggregated to less than 0.5%. of such interactions was supposed to be related to con- Residual caprylic acid and allantoin were 0.14 μg/mL and ductivity (Harinarayan et al. 2006). An electronegative Wang et al. AMB Expr (2018) 8:93 Page 6 of 7 multimodal chromatography, a significant branch of IEX, could reduce impurities. Enhanced washing steps were was deeply involved in the proceeding of IgG purification thus developed and demonstrated to be effective to ele - throughout the research. vate HCP removal. Eluted IgG was directly loaded onto Traditional harvest clarification in upstream process VEAX to further remove HCP to below 1 ppm, DNA needs to experience a series of complicated operations, below 1 ppb and aggregates less than 0.5% with an over- such as centrifugation and filtration, consuming a long all IgG recovery of 86.2%. This new two-column-step period of time. A cutting edge method called advance purification platform supports direct IgG capture from chromatin extraction plays the same clarified role as c-e CCS and polishing with VEAX without buffer adjust - traditional harvest clarification with more efficient har - ment. It overcomes the traditional limitations of protein vest. Antibodies obtained above were directly captured A chromatography, and can help boost productivity and through electronegative multimodal resin, Eshmuno cost-savings. HCX, with different pH/salt combinations to analyze Protein A chromatography as a highly robust technol- DBCs parameter of HCX. The results presented in this ogy was also significantly deficient in high cost and low study demonstrated a satisfactory performance of HCX productivity (Dutta et al. 2015). Developing technolo- to utilize for IgG purification. It was further systemati - gies for overcoming the drawbacks of existing methods cally characterized for its IgG binding capacity at various was of great importance to purify IgG mAbs which were pH/salt combinations. Protein DBCs which were dem- produced for commercial products interested in thera- onstrated as a function of pH and conductivity on resin peutic treatments for numerous diseases. Ion-exchange were expected to decrease with increasing conductivity chromatography was currently used as a part of mAbs and decreasing protein charge (Harinarayan et al. 2006). purification process to effectively remove out of contami - Although the resin supports the highest binding capacity nants within CCS. In this study, it illustrated that cost- at pH 6.0 with 100 mM NaCl, lowering pH and salt con- saving ion-exchange chromatography had the potential centration would not detach the bound IgG. to replace protein A chromatography as the first step in The current process supports direct feed loading for capturing IgG. both capture and polishing steps without buffer adjust - ment called VEAX. Both HCX and VEAX resins are able Abbreviations to withstand the exposure to 1.0 M NaOH which leads IgG: immunoglobulin G; nh-HCP: non-histone host cell protein; IgG mAbs: to lower bioburden, longer cycle life and decreased vali- immunoglobulin G monoclonal antibodies; CHO: Chinese hamster ovary; DBC: dynamic binding capacity; CCS: cell culture supernatant; VEAX: void-exclusion dation cost compared to other purification platforms anion exchange chromatography; c/m CCS: centrifuged/microfiltered CCS; c-e based on protein A or the combination of ion exchang- CCS: chromatin-extracted CCS; EQ buffer: equilibration buffer; SEC: size exclu- ers (Ahamed et al. 2007; Kröner et al. 2013) and hydro- sion chromatography; RP-HPLC: reversed phase-HPLC; LOD: limit of detection; NTU: nephelometric turbidity units; HIC: hydrophobic interaction chromatog- phobic interaction chromatography (HIC) (Queiroz et al. raphy; IEX: ion exchange chromatography. 2001; Baumann et al. 2015). Therefore the two-column- step process proposed in this study provides a promising Authors’ contributions FX, YW and RN: designing the experiment. YW and QC: carrying out the exper- alternative for IgG purification at first step. The current imentation. All authors participated in analysis and interpretation of data and study had explored the effect of pH/salt combinations on drafting the manuscript. All authors read and approved the final manuscript. the DBC of IgG. It was seen to be related to conductivity and resin ligand density. Different pH/salt components Acknowledgements could adjust the ligand density with proteins. The optimal The research was supported by both of Jilin University of College of Life Sci- condition can make full use of space on the resin to keep ences and CAS Key Laboratory of Biobased Materials in Qingdao Institute of Bioenergy and Bioprocess Technology. the protein molecules on balance without hindering and repelling of protein charge. Competing interests In this work, nh-HCP, DNA and histone proteins The authors declare that they have no competing interests. regarded as primary impurities should be carefully Availability of data and materials focused to be removed out of purified IgG. Compared All datasets supporting the conclusions of this article are available in the with protein A resins, HCX made a better performance manuscript. in detaching histone from IgG illustrated in SDS-PAGE Consent for publication (Fig. 2). Once IgG mAbs bound with HCX matrixs in Not applicable. the optimal pH no matter how to change the pH values Ethics approval and consent to participate of washing buffer, IgG still combined with resins. While Not applicable. alter washing step not only limited in pH but also con- tained components and washing methods, all of them Wang et al. AMB Expr (2018) 8:93 Page 7 of 7 Funding Ho SC, Bardor M, Feng H, Mariati Tong YW, Song Z, Yap MG, Yang Y (2012) IRES- This work was financially supported by the National Natural Science Founda- mediated tricistronic vectors for enhancing generation of high mono- tion of China (No. 21676286). clonal antibody expressing CHO cell lines. J Biotechnol 157(1):130–139. https ://doi.org/10.1016/j.jbiot ec.2011.09.023 Ishihara T, Hosono M (2015) Improving impurities clearance by amino acids Publisher’s Note addition to buffer solutions for chromatographic purifications of mono - Springer Nature remains neutral with regard to jurisdictional claims in pub- clonal antibodies. J Chromatogr B Analyt Biomed Life Sci 995–996:107– lished maps and institutional affiliations. 114. https ://doi.org/10.1016/j.jchro mb.2015.05.018 Johansson BL, Belew M, Eriksson S, Glad G, Lind O, Maloisel JL, Norrman N Received: 31 January 2018 Accepted: 24 May 2018 (2003) Preparation and characterization of prototypes for multi-modal separation aimed for capture of positively charged biomolecules at high- salt conditions. J Chromatogr A 1016(1):35–49 Kaleas KA, Tripodi M, Revelli S, Sharma V, Pizarro SA (2014) Evaluation of a multimodal resin for selective capture of CHO-derived monoclonal antibodies directly from harvested cell culture fluid. J Chromatogr B References Analyt Technol Biomed Life Sci 969:256–263. https ://doi.org/10.1016/j. Ahamed T, Nfor BK, Verhaert PD, van Dedem GW, van der Wielen LA, Eppink jchro mb.2014.08.026 MH, van de Sandt EJ, Ottens M (2007) pH-gradient ion-exchange chro- Kröner F, Hanke AT, Nfor BK, Pinkse MW, Verhaert PD, Ottens M, Hubbuch J matography: an analytical tool for design and optimization of protein (2013) Analytical characterization of complex, biotechnological feed- separations. J Chromatogr A 1164(1–2):181–188 stocks by pH gradient ion exchange chromatography for purification pro - Baumann P, Baumgartner K, Hubbuch J (2015) Influence of binding pH and cess development. J Chromatogr A 1311:55–64. https ://doi.org/10.1016/j. protein solubility on the dynamic binding capacity in hydrophobic chrom a.2013.08.034 interaction chromatography. J Chromatogr A 1396:77–85. https ://doi. Nian R, Gagnon P (2016) Advance chromatin extraction enhances perfor- org/10.1016/j.chrom a.2015.04.001 mance and productivity of cation exchange chromatography-based Chen Q, Abdul Latiff SM, Toh P, Peng X, Hoi A, Xian M, Zhang H, Nian R, Zhang capture of immunoglobulin G monoclonal antibodies. J Chromatogr A W, Gagnon P (2016) A simple and efficient purification platform for 1453:54–61. https ://doi.org/10.1016/j.chrom a.2016.05.029 monoclonal antibody production based on chromatin-directed cell Nian R, Chuah C, Lee J, Gan HT, Latiff SM, Lee WY, Vagenende V, Yang YS, culture clarification integrated with precipitation and void-exclusion Gagnon P (2013) Void exclusion of antibodies by grafted-ligand porous anion exchange chromatography. J Biotechnol 236:128–140. https ://doi. particle anion exchangers. J Chromatogr A 1282:127–132. https ://doi. org/10.1016/j.jbiot ec.2016.08.014 org/10.1016/j.chrom a.2013.01.065 Dutta AK, Tran T, Napadensky B, Teella A, Brookhart G, Ropp PA, Zhang AW, Nian R, Zhang W, Tan L, Lee J, Bi X, Yang Y, Gan HT, Gagnon P (2016) Advance Tustian AD, Zydney AL, Shinkazh O (2015) Purification of monoclonal chromatin extraction improves capture performance of protein A affinity antibodies from clarified cell culture fluid using Protein A capture chromatography. J Chromatogr A 1431:1–7. https ://doi.org/10.1016/j. continuous countercurrent tangential chromatography. J Biotechnol chrom a.2015.12.044 213:54–64. https ://doi.org/10.1016/j.jbiot ec.2015.02.026 Queiroz JA, Tomaz CT, Cabral JMS (2001) Hydrophobic interaction chroma- Gagnon P, Nian R, Tan L, Cheong J, Yeo V, Yang Y, Gan HT (2014a) Chromatin- tography of proteins. J Biotechnol 87:143–159. https ://doi.org/10.1016/ mediated depression of fractionation performance on electronegative S0168 -1656(01)00237 -1 multimodal chromatography media, its prevention, and ramifications for Shukla AA, Hinckley P (2008) Host cell protein clearance during protein A purification of immunoglobulin G. J Chromatogr A 1374:145–155. https :// chromatography: development of an improved column wash step. doi.org/10.1016/j.chrom a.2014.11.052 Biotechnol Prog 24(5):1115–1121. https ://doi.org/10.1002/btpr.50 Gagnon P, Nian R, Lee J, Tan L, Latiff SM, Lim CL, Chuah C, Bi X, Yang Y, Zhang W, Shukla AA, Thömmes J (2010) Recent advances in large-scale production Gan HT (2014b) Nonspecific interactions of chromatin with immuno - of monoclonal antibodies and related proteins. Trends Biotechnol globulin G and protein A, and their impact on purification performance. J 28(5):253–261. https ://doi.org/10.1016/j.tibte ch.2010.02.001 Chromatogr A 1340:68–78. https ://doi.org/10.1016/j.chrom a.2014.03.010 Shukla AA, Hubbard B, Tressel T, Guhan S, Low D (2007) Downstream Gagnon P, Nian R, Yang Y, Yang Q, Lim CL (2015) Non-immunospecific processing of monoclonal antibodies—application of platform association of immunoglobulin G with chromatin during elution from approaches. J Chromatogr B 848(1):28–39. https ://doi.org/10.1016/j.jchro protein A inflates host contamination, aggregate content, and antibody mb.2006.09.026 loss. J Chromatogr A 1408:151–160. https ://doi.org/10.1016/j.chrom Tao Y, Ibraheem A, Conley L, Cecchini D, Ghose S (2014) Evaluation of a.2015.07.017 high-capacity cation exchange chromatography for direct capture of Ghose S, Allen M, Hubbard B, Brooks C, Cramer SM (2005) Antibody variable monoclonal antibodies from high-titer cell culture processes. Biotechnol region interactions with Protein A: implications for the development of Bioeng 111(7):1354–1364. https ://doi.org/10.1002/bit.25192 generic purification processes. Biotechnol Bioeng 92(2):665–673. https :// Tarrant RD, Velez-Suberbie ML, Tait AS, Smales CM, Bracewell DG (2012) Host doi.org/10.1002/bit.20729 cell protein adsorption characteristics during protein A chromatography. Girard V, Hilbold NJ, Ng CK, Pegon L, Chahim W, Rousset F, Monchois V (2015) Biotechnol Prog 28(4):1037–1044. https ://doi.org/10.1002/btpr.1581 Large-scale monoclonal antibody purification by continuous chromatog- Urmann M, Graalfs H, Joehnck M, Jacob LR, Frech C (2010) Cation-exchange raphy, from process design to scale-up. J Biotechnol 213:65–73. https :// chromatography of monoclonal antibodies: characterisation of a novel doi.org/10.1016/j.jbiot ec.2015.04.026 stationary phase designed for production-scale purification. MAbs Harinarayan C, Mueller J, Ljunglöf A, Fahrner R, Van Alstine J, van Reis R (2006) 2(4):395–404 An exclusion mechanism in ion exchange chromatography. Biotechnol Bioeng 95(5):775–787. https ://doi.org/10.1002/bit.21080
AMB Express – Springer Journals
Published: Jun 1, 2018
It’s your single place to instantly
discover and read the research
that matters to you.
Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.
All for just $49/month
Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly
Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.
Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.
Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.
All the latest content is available, no embargo periods.
“Hi guys, I cannot tell you how much I love this resource. Incredible. I really believe you've hit the nail on the head with this site in regards to solving the research-purchase issue.”Daniel C.
“Whoa! It’s like Spotify but for academic articles.”@Phil_Robichaud
“I must say, @deepdyve is a fabulous solution to the independent researcher's problem of #access to #information.”@deepthiw
“My last article couldn't be possible without the platform @deepdyve that makes journal papers cheaper.”@JoseServera