Novel peptoid-based adsorbents for purifying IgM and IgG from polyclonal and recombinant sources

Journal of Chromatography B 1137 (2020) 121909

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb

Novel peptoid-based adsorbents for purifying IgM and IgG from polyclonal and recombinant sources

T

Hannah Reesea, Tee Bordelonb, Calvin Shanahana, Michael Crapanzanob, , Jae Slyb, , ⁎ Stefano Menegattia,c, ⁎

⁎

a

Department of Chemical and Biomolecular Engineering, North Carolina State University, Campus Box 7905, Raleigh, NC 27695, USA LigaTrap Technologies, LLC, 701 W Main St., Ste. 200, Durham, NC 27701, USA c Biomanufacturing Training and Education Center (BTEC), North Carolina State University, Raleigh, NC 27695-7928, USA b

ARTICLE INFO

ABSTRACT

Keywords: Peptoid ligand Affinity purification Protein chromatography IgG and IgM Antibody therapeutics

Polyclonal immunoglobulin therapeutics comprising dosed IgG and IgM combinations are powerful tools in fighting cancer and severe infections. The inability of protein ligands to produce polyclonal IgG- and IgMenriched formulations and recover monoclonal IgM calls for novel ligands with superior biorecognition activity. In this study, a peptoid ligand discovered by our group, and integrated into affinity adsorbents LigaTrap Technologies’ “Human IgG” and “Human IgM”, were utilized to purify IgG and IgM from complex fluids. IgG purification from human serum using LigaTrap IgG afforded 94.6% purity and 62.9% yield, on par with Protein A/G resins. When challenged with CHO and HEK cell culture harvests with low IgG titer (< 1 mg/mL), LigaTrap IgG returned values of yield and purity well above 60% and 90%. LigaTrap IgM was evaluated for purifying IgM in comparison with commercial adsorbents, and afforded a product purity of 93% from a CHO harvest (IgM titer of 1 mg/mL) and 75.1% yield from a HEK harvest (0.5 mg/mL). LigaTrap-M provided IgM enrichment up to 11fold higher than HiTrap resin. The peptoid adsorbents separated IgG-depleted human serum into IgM- and IgAenriched fractions. These results demonstrate the potential of the peptoid ligand for manufacturing polyclonal Ig formulations and monoclonal IgM therapeutics.

1. Introduction Immunoglobulin G (IgG)-based therapies have dominated the biotherapeutic market for decades. Recent developments, however, indicate that monoclonal IgG-antibodies alone cannot adequately address aggressive forms of cancer, infectious diseases, and other severe lifethreatening conditions. As a result, interest in immunoglobulin (Ig)based cocktails has been kindled. These new therapies can comprise a selection of IgG with polyclonal-like activities [1,2], alternative immunoglobulins such as immunoglobulin M (IgM) [3], or a combination of the two. Intravenous immunoglobulin (IVIG) therapies, which contain Igs of multiple isotypes, have been of interest to supplement patients with reduced immunoglobulin levels [4] and to treat immune system diseases since their first clinical trials in 1980s [5]. Recent work in this field has shown that polyclonal – rather than monoclonal - therapies

may be necessary for the treatment of several rare diseases [6,7]. Attempts have been made to create recombinant IVIG (rIVIG) therapies to treat severe viral infections [1,8,9], but the expression of these products in cell cultures has not yet been perfected. These developments, however, indicate a need to develop downstream processes for polyclonal and multi-isotype Ig formulations. In this regard, the value of IgM in human therapy has been recognized for decades. The role of IgM in microbial immunity [3,10,11] and the use of polyclonal IgM-enriched formulations for intravenous administration to treat bacterial infections and sepsis [12,13] have been long investigated, and are now established. Recent advancements in immunotherapy have also demonstrated the potential of monoclonal IgM either alone or in combination with IgG therapeutics [3]. Due to its pentameric nature and high avidity, IgM therapeutics show excellent potential to fight diseases involving low numbers of cells or a lower expression of cell surface markers [14]. Its multivalent structure

Abbreviations: IgG, immunoglobulin G; IgM, immunoglobulin M; CHO, Chinese hamster ovary cells; HEK, human embryonic kidney cells; HCP, host cell proteins; FBS, fetal bovine serum; LRV, logarithmic removal value; IVIG, IVIGIntravenous immunoglobulin; FT, flow through; El, elution ⁎ Corresponding authors at: Department of Chemical and Biomolecular Engineering, North Carolina State University, Campus Box 7905, Raleigh, NC 27695, USA (S. Menegatti). E-mail addresses: [email protected] (M. Crapanzano), [email protected] (J. Sly), [email protected] (S. Menegatti). https://doi.org/10.1016/j.jchromb.2019.121909 Received 4 October 2019; Received in revised form 24 November 2019; Accepted 27 November 2019 Available online 02 December 2019 1570-0232/ © 2019 Elsevier B.V. All rights reserved.

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provides engineered IgM with an inherent ability to crosslink between surface-receptors on multiple cells, and induce greater immune responses [15,16]. In addition to this high avidity, IgM has been found to target difficult antigens, such as carbohydrates commonly associated with tumors [17]. Finally, IgM has been shown to direct systemic antibody response through the use of complement activation, especially in response to larger antagonists such as viruses or bacteria [12,18]. This increased complement activation also leads to substantially higher complement-dependent cytotoxicity (CDC), making IgMs more effective than IgGs in killing target cells without requiring any immune cell [19,20]. Despite its promise, polyclonal and monoclonal IgMs are not widely utilized. The low titer of IgG in biological sources, whether recombinant or bodily fluid [21], and, most importantly, the lack of affinity adsorbents for IgM capture, purification, and scale up have limited drastically the application of IgM-based formulations in clinical settings. A number of companies (e.g., IgM Biosciences [22–25], Patrys [75], and Kenta Biotech [[76]) are building strong IP portfolios surrounding monoclonal IgM-based therapeutics. This trend prompts the need for improved technology for IgM purification, which has been shown to be considerably more challenging than IgG purification. IgMs, in fact, are stable in a narrow range of salt concentration and pH values in solution, are more prone to denaturation and precipitation, and their large size hinders their transport through chromatographic media, resulting in lower yields [26,27]. Pioneering work by Gagnon et al. has accomplished IgM purification through a sequence of chromatographic steps involving ceramic hydroxyapatite and anion and cation exchange resins [28–30] or by size exclusion [31,32]. While achieving high purity, the values of yield were limited between 50 and 80%. Recently, the introduction of HiTrap IgM Purification HP resin by GE Healthcare and CaptureSelect™ IgM Affinity Matrix by POROS™ (Thermo Scientific™) has brought affinity chromatography to IgM purification, enabling high product purity and concentration in a single step [33]. While undoubtedly advantageous compared to traditional chromatographic media, the HiTrap relies on a simple mixed-mode ligand (2-mercaptopyridine) and Sepharose resin with high pore diameter [34,35]. While capable of purifying IgM from serum-free cell culture fluid, the HiTrap IgM is not recommended for applications with serum-based polyclonal or monoclonal sources. The CaptureSelect™ ligand, an IgM-binding camelid antibody, is more selective than 2-mercaptopyridine [36], but also far more expensive and less chemically stable. To advance the affinity-based separation technology for the preparation of IgM-rich polyclonal immunoglobulin formulations and pure monoclonal IgM therapeutics and diagnostic reagents, in this study we explored the use of a novel immunoglobulin-binding peptoid ligand, now integrated in affinity adsorbents commercialized by LigaTrap Technologies, LLC [37]. This ligand has been discovered in prior work for the purification of polyclonal IgG from serum and cell culture fluids [38,39], demonstrating excellent binding capacity and selectivity and biochemical stability. These benefits derive from the physicochemical nature and behavior of peptoids. Similarly to their peptide cognates, in fact, peptoids feature a polyamide backbone; however, while peptides display the side chain functional groups that define the chemical identity of their monomers (amino acids) on the α-carbon, peptoids display the side chain functional groups on the amide-nitrogen [40]. This makes their structure more flexible and improves their ability to adapt their conformation to a broader range of isoforms of their target, and endows them with resistance to proteases [41,42]. Further, their synthesis process, known as the “sub-monomer” approach, affords higher synthetic yield compared to peptides at a fraction of the cost [43]. Given its broad biorecognition activity to immunoglobulins of different subclasses and sources, we sought to evaluate the ability of peptoid-based adsorbents for purifying IgG and IgM from polyclonal and monoclonal sources. The peptoid-based LigaTrap “Human IgG” resin – henceforth “LigaTrap-G” – was able to purify IgG similarly to commercially available ligands, giving IgG purities between 55.0% (from a human

embryonic kidney (HEK) cells culture with low IgG titer) and 94.6% (from human serum). Its cognate, LigaTrap “Human IgM” resin – henceforth “LigaTrap-M” – successfully purified monoclonal IgM, affording an enriched product 11-times greater than achieved with CaptureSelectTM resin in a Chinese Hamster Ovary (CHO) cell culture fluid, and affording purities of 93.1% from CHO fluids and high product recovery from HEK cell culture fluids, where the low IgM concentration makes recovery very challenging. Collectively, these results demonstrate the potential of the peptoid ligand for the manufacturing of polyclonal Ig formulations and monoclonal IgM-based therapeutics. 2. Experimentals 2.1. Materials Fmoc-Cys(Trt)-Rink resin was purchased from Anaspec (Fremont, CA, USA). Fmoc-glycine, hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU), and diisopropylethylamine were purchased from Chem-Impex (Wood Dale, IL, USA). Fmoc-N-(3-(N-Pbf-guanidino)propyl)-glycine was from PolyPeptide (Torrance, CA, USA). Piperidine, N,N′-dimethyl-formamide (DMF), methyl-tertbutyl ether (MTBE), bromoacetic acid, 2-picolylamine, tryptamine, (3-aminopropyl) urea, 1,3diaminopropane, sodium phosphate dibasic, and potassium phosphate monobasic was purchased from Sigma-Aldrich (Saint Louis, MO, USA). N-methyl-pyrrolidone (NMP), triisopropylsilane (TIPS), ethane dithiol (EDT), methyl tertbutyl ether, sodium carbonate, β-mercaptoethanol, methanol, sodium chloride, potassium chloride, sodium acetate, acetic acid, glycine, hydrochloric acid, ammonium sulfate, potassium sulfate, bicinchonic acid protein assay, IgM, Silver Quest silver staining kit, and Easy-Titer IgM ELISA assay were purchased from Fisher Scientific (Pittsburgh, PA, USA). Adipic Acid was purchased from Acros Organics (Pittsburgh, PA, USA). Human serum was obtained from Athens Research and Technology (Athens, GA, USA). HEK cell culture supernatant was a gift from the Haugh lab at NC State University (Raleigh, NC, USA). CHO cell culture was a gift from the Biotechnology Traning and Education Center (BTEC, Raleigh, NC). The 4–20% gradient SDS PAGE gels, Coomassie blue stain, and 10–20% TRIS-Glycine HCl SDSPAGE gels were purchased from BioRad Life Sciences (Carlsbad, CA, USA). A 10 mL tricorn column, Protein A agarose column, Protein G agarose column, and GE IgM resin were from GE Healthcare (Piscataway, NJ, USA). Microbore columns were from VICI Precision Sampling (Baton Rouge, Louisiana, USA). 2.2. LigaTrap-G and LigaTrap-M affinity adsorbents The peptoid was synthesized as described in prior work [38,39] following the sub-monomer synthetic method employing bromoacetic acid and the primary amines carrying the desired functional group. Following synthesis, the peptoid was cleaved by acidolysis, precipitated in ice cold MTBE, purified by reverse-phase C-18 chromatography, and lyophilized. The peptoid was conjugated using a proprietary method developed by LigaTrap on different agarose-based resins with controlled pore size, resulting in LigaTrap-G and LigaTrap-M resins [37,44]. The resins are stored in 20% v/v methanol in water. 2.3. Purification of IgG from CHO and HEK cell culture fluids LigaTrap-G and Protein A agarose resins were packed into 0.1 mL microbore columns and initially equilibrated with PBS, pH 7.4. The feed samples were prepared by spiking (i) human monoclonal IgG2 and IgG3 antibodies at 1 mg/mL in CHO cell culture fluid containing 1% fetal bovine serum (FBS), or human polyclonal IgG at (ii) 0.1 mg/mL or (iii) 0.4 mg/mL in HEK 293 cell culture fluid containing 1% FBS. The salt composition and concentration in the four HEK feeds were adjusted to 0.1 M NaCl and 20 mM adipic acid at pH 7.4. A volume of 1 mL of feed was injected onto the LigaTrap-G column at the flow rate of 2

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0.05 mL/min (retention time, RT: 2 min). Following injection, the resin was washed with 10 column volumes (CVs) of washing buffer at 0.1 mL/min; washing buffers were PBS at pH 7.4 for the CHO feed, and 0.1 M NaCl and 20 mM adipic acid in PBS at pH 7.4 for the HEK feeds. IgG elution from LigaTrap-G and Protein A resin was performed with 20 CVs of 0.1 M acetate buffer at pH 4.2 and 0.1 M glycine buffer pH 2.8, respectively, both at the flow rate of 0.2 mL/min. All resins were regenerated with aqueous 2% v/v acetic acid and finally cleaned with aqueous 0.5 M sodium hydroxide. The elution fractions were neutralized upon collection using phosphate buffer at pH 8.5. The effluents in all chromatographic runs were monitored by UV spectrometry at 280 nm through the entire chromatographic run. The flow-through and elution fractions were collected and analyzed by human IgG-specific ELISA and SDS-PAGE using 10–20% TRIS-Glycine HCl SDS-PAGE under reducing conditions. The gels were silver stained, and product purity was calculated by densitometry analysis of the lanes corresponding to the eluted fractions; specifically, the product purity is calculated using Eq. (1)

P=

IIg

IIg

HC

HC

+ IIg

+ IIg LC

acid at pH 7.4. A volume of 10 mL of feed was injected onto the columns at the flow rate of 0.5 mL/min (RT: 2 min). Following injection, the resin was washed with 10 CVs of washing buffer at 1 mL/min; washing buffers were 0.35 M NaCl and 20 mM adipic acid in PBS at pH 7.4 for the CHO feed, and 0.1 M NaCl and 20 mM adipic acid in PBS at pH 7.4 for both HEK feeds. IgM elution from LigaTrap-G was performed with 20 CVs of 0.1 M acetate buffer at pH 4.2; IgM elution from the HiTrap resin was performed with 20 CVs of either 0.8 M ammonium sulfate, or 1.0 M ammonium sulfate, or 0.5 M potassium sulfate, all at pH 7.4; IgM elution from the CaptureSelect™ IgM resin was performed with 0.1 M glycine buffer pH 2.8; all elution steps were performed at the flow rate of 2 mL/min. All resins were regenerated with aqueous 2% v/v acetic acid and finally cleaned with aqueous 0.5 M sodium hydroxide. The elution fractions were neutralized upon collection using phosphate buffer at pH 8.5. The effluents in all chromatographic runs were monitored by UV spectrometry at 280 nm through the entire chromatographic run. The flow-through and elution fractions were collected and analyzed by human IgM-specific ELISA and SDS-PAGE using 10–20% TRIS-Glycine HCl SDS-PAGE under reducing conditions. The gels were silver stained and product purity was calculated by densitometry analysis of the lanes corresponding to the eluted fractions.

LC

+ IOP

(1)

wherein IIg-HC is the intensity of the band associated to the heavy chain of the target immunoglobulin (i.e., IgG or IgM), IIg-LC is the intensity of the band associated to the light chain of the target immunoglobulin (i.e., IgG or IgM), and IOP is sum of the intensities of the bands associated to all the other proteins detected in the lane.

2.6. Purification of IgM and IgA from IgG-depleted human serum LigaTrap-M resin was packed into 10 mL Tricorn column and initially equilibrated with 0.35 M NaCl in PBS at pH 7.4. IgG-depleted human serum obtained in Section 2.4 adjusted to a final salt composition of 0.35 M NaCl and 20 mM adipic acid, pH of 7.4, and concentration of IgG at 0.112 mg/mL and IgA at 0.274 mg/mL. A volume of 600 mL of feed was injected onto the columns at the flow rate of 2 mL/ min (RT: 5 min). Following injection, the LigaTrap-M resin was washed with 10 CVs of 0.35 M NaCl and 20 mM adipic acid in PBS at 40 mL/ min. IgM and IgA elution was performed using 0.1 M acetate buffer at pH 4.0, at the flow rate of 20 mL/min. The resin was regenerated with aqueous 2% v/v acetic acid and finally cleaned with aqueous 0.5 M sodium hydroxide. The elution fractions were neutralized upon collection using phosphate buffer at pH 8.5. The effluents in all chromatographic runs were monitored by UV spectrometry at 280 nm through the entire chromatographic run. The flow-through and elution fractions were collected and analyzed by size exclusion chromatography (SEC) using a HiLoad Superdex 200 SEC column at the flow rate of 10 mL/ min. Fractions from the SEC column were collected and analyzed using 10–20% TRIS-Glycine HCl SDS-PAGE under reducing conditions. The gels were stained using Coomassie Blue and product purity was calculated by densitometry analysis of the lanes corresponding to the eluted fractions.

2.4. Purification of IgG from human serum LigaTrap-G resin, Protein A agarose, and Protein G agarose resins were packed in a 1 mL Tricorn column and initially equilibrated with PBS, pH 7.4. The feed sample was prepared by 1:1 dilution of human serum with 0.85 M NaCl and 40 mM adipic acid in PBS to a total protein concentration of 33.5 mg/mL, followed by centrifugation at 10,000g and filtration using a 0.22 µm cellulose filter. A volume of 5 mL of diluted serum was injected on the columns at the flow rate of 0.2 mL/ min (RT: 5 min). The flow-through fraction from LigaTrap-G resin was collected as IgG-depleted human serum. Following injection, the LigaTrap-G resin was washed with 10 CVs of 0.35 M NaCl and 20 mM adipic acid in PBS at pH 7.4, at 1 mL/min, whereas Protein A and Protein G agarose resins were washed with PBS at pH 7.4. IgG elution from LigaTrap-G was performed with 20 CVs of 0.1 M acetate buffer at pH 4.2, while elution from Protein A and G resins was performed with 20 CVs of 0.1 M glycine buffer pH 2.8, both at the flow rate of 2 mL/ min. All resins were regenerated with aqueous 2% v/v acetic acid and finally cleaned with aqueous 0.35 M sodium hydroxide. The elution fractions were neutralized upon collection using phosphate buffer at pH 8.5. The effluents in all chromatographic runs were monitored by UV spectrometry at 280 nm through the entire chromatographic run. The flow-through and elution fractions were collected and analyzed by human IgG-specific ELISA and SDS-PAGE using 10–20% TRIS-Glycine HCl SDS-PAGE under reducing conditions. The gels were stained using Coomassie Blue and product purity was calculated by densitometry analysis of the lanes corresponding to the eluted fractions.

3. Results 3.1. LigaTrap-G and LigaTrap-M affinity adsorbents The immunoglobulin-binding peptoid ligand utilized in this study has been discovered in prior work by screening a designed ensemble of peptoid variants [38]. Concerted experimental evaluation and in silico modeling by molecular docking demonstrated that the peptoid ligand binds the Fc fragment of γ-globulins with high affinity and selectivity, and can therefore be utilized as affinity ligands for their purification. The peptoid can be conjugated onto agarose-based resins with controlled pore size, resulting in a number of affinity adsorbents that are customized for specific Ig purifications. LigaTrap “Human IgG” (herein, LigaTrap-G) and “Chicken immunoglobulin Y (IgY)” [37,44,45] have been evaluated for their ability to purify polyclonal IgG from the serum of different non-human mammals (mouse, rat, rabbit, sheep and goat, donkey, and llama), avian IgY from chicken serum and yolk, as well as monoclonal antibodies from ascites fluids [39], demonstrating excellent binding capacity and selectivity, and biochemical stability.

2.5. Purification of IgM from CHO and HEK cell culture fluids LigaTrap-M resin, HiTrap IgM Purification HP resin, and POROS™ CaptureSelect™ IgM Affinity Matrix were packed into 1 mL Tricorn column and initially equilibrated with PBS, pH 7.4. The feed samples were prepared by spiking human IgM (i) at 1 mg/mL in CHO cell culture fluid containing 5% FBS, (ii) at 0.1 mg/mL in HEK 293 cell culture fluid containing 0.1% FBS, and (iii) at 0.1 mg/mL in HEK 293 cell culture fluid containing 1% FBS. The salt composition and concentration in the HEK feeds were adjusted to 0.1 M NaCl and 20 mM adipic 3

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This work presents for the first time the use of peptoid-based adsorbents LigaTrap-G and LigaTrap-M [37,44] for the purification of IgG and IgM respectively from a variety of complex sources, including recombinant CHO and HEK cell culture harvests. The model fluids utilized in this study are particularly challenging due to the variety and abundance of protein impurities, coming from the use of fetal bovine serum (FBS) in the cell culture, as well as their low product titer, varying between 0.1 and 1 mg/mL.

IgG-specific ELISA (all analyses were performed in triplicate) to determine product yield (SI Table 1) and SDS-PAGE (Fig. 1) followed by densitometry analysis to calculate product purity. As expected, Protein A agarose resin was capable of binding human IgG2, but not IgG3, whereas both LigaTrap-G and Protein G agarose resin were capable of binding both target antibodies. Both LigaTrap-G and Protein A resins afforded remarkable values of product yield and purity (> 67% and > 92%, respectively). Of note was the ability of the LigaTrap-G to clear the protein impurities derived from both the FBS, albumin in particular, and the CHO cells. The values of logarithmic removal of CHO host cell proteins (HCP LRV) obtained with LigaTrap-G resin (0.44) were in fact on par with those obtained with Protein A and G resins (0.51 and 0.49, respectively). This suggests that the few residual protein contaminants in the elution fractions from the LigaTrapG resin comprised mostly serum proteins, rather than CHO HCPs. Following IgG purification from CHO fluids, the LigaTrap-G resin was further evaluated using HEK-derived feed fluids. The lower product titer in these feed samples (0.1 mg/mL and 0.4 mg/mL) makes IgG purification considerably more challenging. As seen before, resin equilibration and post-injection washing with PBS added with 0.1 M NaCl and 20 mM adipic acid were performed to minimize non-specific binding of serum-derived and HEK proteins. Protein elution from LigaTrap-G resin and the determination of product yield and purity were performed as described above. The SDS-PAGE analysis of the chromatographic fractions (Fig. 2) shows that, while initially captured by the LigaTrap-G resin, the serum albumin was removed in the washing step. Unlike what observed with CHO fluids, the binding of albumin from HEK-derived feed samples is rather conspicuous. This can be attributed to the much lower ratio of IgG vs. BSA in the HEK fluids compared to that of CHO fluids. With little IgG available in the feed to saturate the peptoid binding sites, BSA and other plasma proteins can be adsorbed by electrostatic interactions; accordingly, washing with a buffer at higher ionic strength obliterates electrostatic binding, as shown by the remarkable amount of BSA found in the wash fractions, ultimately enabling the recovery of pure IgG. The values of IgG yield (determined by analyzing the chromatographic fractions by human IgGspecific ELISA) and purity (determined by densitometric analysis of the lanes corresponding to the eluted fractions) of 46.3% and 57.1% were obtained from the HEK fluid containing 0.1 mg/mL IgG, corresponding to a 8-fold product enrichment. The corresponding values of yield and purity obtained from the HEK fluid containing 0.4 mg/mL IgG were of approximately 54.5% and 77.9%. Given the low concentration and purity (5–8%) of the target IgG in the fed mixture, the 10-fold product enrichment that the LigaTrap-G resin to affords is remarkable. While post-capture polishing of the IgG product would be necessary, for

3.2. Purification of IgG from CHO and HEK cell culture fluids Chinese Hamster Ovary (CHO) [46] and Human Embrionic Kidney (HEK 293) [47] cells are the expression systems of choice in the development and large-scale expression of monoclonal antibodies (mAbs). They provide the correct glycosylation and post-transcriptional modifications that ensure high therapeutic activity and safety in human patients [48,49]. The titers of secreted IgG in HEK cell culture fluids typically fall within the range of 0.05–0.1 mg/mL [50], although higher titers of ~1.0 mg/mL have been reported, though they are difficult to achieve [51]. Current engineered CHO-K1 cells can express human mAbs with titer well above 1 mg/mL [52]. Together with titer, the ability to achieve specific glycosylation profiles and post-translational modifications, which determine the anticancer activity and serum halflife of the mAb are important considerations in choosing an expression cell line [53]. Feed samples mimicking industrial cell culture harvests were prepared. A first set of two samples was prepared by spiking monoclonal IgG2 and IgG3 antibodies (both at 1 mg/mL) in a CHO-S cell culture supernatant added with 1% fetal bovine serum (FBS). IgG2 and IgG3 were chosen in place of IgG1 and IgG4, more commonly utilized in the biomanufacturing industry, to show the potential of the peptoid-based resin to enable purification of antibodies that are either less common or employed in niche applications (IgG2) or cannot be processed by Protein A (IgG3). Two more samples were prepared by spiking human polyclonal IgG at either 0.1 or 0.4 mg/mL in a HEK 293 cell culture supernatant added with 0.1% fetal bovine serum (FBS). Prior to injecting the sample, the LigaTrap-G resin was equilibrated with 0.1 M NaCl and 20 mM adipic acid in PBS at pH 7.4, as done in prior work, to prevent non-specific binding of bovine serum albumin (BSA) to the adsorbent. Protein A and Protein G agarose resin, utilized herein as control adsorbents, were equilibrated in PBS. Following sample injection and wash, the bound IgG was eluted from the LigaTrap-G resin using 0.1 M acetate buffer at pH 4.2, and from the Protein A/G resins using 0.1 M glycine buffer pH 2.8, as commonly found in literature [54,55]. The eluted fractions were collected and analyzed by human

Fig. 1. SDS-PAGE analysis (reducing conditions, Coomassie Blue staining) of (A) elution and (B) washing fractions obtained from the purification of monoclonal IgG2 antibody, and (C) elution fractions obtained from the purification of monoclonal IgG3 antibody from CHO cell culture fluids using LigaTrap-G resin, Protein Aagarose, and Protein G-agarose. Labels: MW, molecular weight ladder; Feed, CHO cell culture fluid added with 0.1% FBS and spiked with monoclonal IgG; W1 LTG, W2 LTG, and W3 LTG, subsequent collections during the washing of LigaTrap-G resin with 0.35 M NaCl and 20 mM adipic acid in PBS; El LTG, elution at pH 4.2 from LigaTrap-G resin; El PrA, elution at pH 2.8 from Protein A-agarose; El PrG, elution at pH 2.8 from Protein G-agarose; R LTG, regeneration of LigaTrap-G resin at pH 2.5; BSA, bovine serum albumin; IgG HC, heavy chain of IgG; IgG LC, light chain of IgG. 4

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Fig. 2. SDS-PAGE analysis (reducing conditions, silver staining) of flow-through, washing, and elution fractions obtained from the purification of human IgG from HEK 293 cell culture fluids added with either 0.1% FBS or 1% FBS using LigaTrap-G resin. Labels: MW, molecular weight ladder; Feed, HEK 293 cell culture fluid added with 1% FBS and spiked with monoclonal IgG; FT, flow-through; W, wash; El elution at pH 4.2; R, regeneration with 2% v/v acetic acid; BSA, bovine serum albumin; IgG HC, heavy chain of IgG; IgG LC, light chain of IgG. The red arrows are added to facilitate the visualization of the heavy and light chains of IgG. Note: purification with Protein G was attempted but did not return any appreciable data of IgG concentration in either FT or El fractions; accordingly, these fractions were not subjected to SDS-PAGE analysis, nor analyzed any further.

removal of the myriad of plasma proteins in the washing step (SI Fig. 1) and secures the elution of an IgG product whose purity (94.6%) is on par with the values obtained with commercial Protein A (96.7%) and Protein G (96.9%) resins (Fig. 3). LigaTrap-G afforded a product yield (62.9%) comparable with that given by Protein A agarose resin (68.1%), and both were decidedly higher than that obtained with Protein G agarose (40.6%). This is likely due to the higher binding capacity and strength of Protein A, which enabled a better retention of IgG throughout the adsorption and washing steps; on the other hand, a considerable fraction of the injected IgG (33.2%) was lost during loading onto the resins. The load of ~30 mg of IgG per mL of resin is within the dynamic binding capacity of LigaTrap-G; the latter, however, is measured with pure IgG, and does not account for competing ligand binding phenomena that occur – with peptoid and protein ligands alike - when IgG is co-injected with all the other serum proteins. On the other hand, the milder IgG:peptoid affinity enables elution at pH 4.2, whereas Protein A requires much harsher conditions, which can degrade and denature the product, as noted in published literature [62–64].

example by ion exchange or mixed-mode chromatography, the efficiency of this additional step would be magnified owing to the affinitybased product enrichment achieved by the peptoid-based resin. 3.3. Purification of IgG from human serum Intravenous immunoglobulin (IVIG) is a therapeutic formulation obtained by pooling polyclonal IgG from the plasma of healthy individuals [56,57]. Large amounts of IVIG are clinically utilized in myriad applications, including immunology and rheumatology, haematology, nephrology, dermatology, and neurology [58]. Traditionally, the mass manufacturing of IVIG formulations has relied on the CohnOncley process, which relies on the separation of plasma into albuminrich, Ig-rich, and clotting factor-rich fractions through a series of precipitation using cold ethanol, addition of sodium chloride and caprylate, and by controlling the pH of the protein solution. More recently, however, a stronger interest has arisen on the use of chromatography for plasma separation and extraction of IgG-rich fractions [59]. In prior work, our group has utilized peptide-based resins to recover polyclonal IgG from the Ig-rich Cohn fractions II + III paste of human plasma with high values of yield (84%) and purity (95%) [60,61]. In this work, we sought to utilize the LigaTrap-G resin for purifying IgG directly from whole human serum. Specifically, a volume of 5 mL of diluted human serum at ~33.5 mg/mL total protein concentration was injected in a 1 mL column packed with LigaTrap-G resin, corresponding to a load of ~30 mg of IgG per mL of resin. Resin equilibration prior to load and post-load washing were both performed with a buffer at high ionic strength (0.35 M NaCl in PBS at pH 7.4), to prevent or disrupt non-specific binding of serum albumin by electrostatic interaction. Unwanted binding of plasma proteins can also derive from clustering of multiple albumins or the complexing of other plasma proteins to albumin. To reduce this effect, 20 mM adipic acid was also added to the washing buffer. Albumin features in fact a number of surface pockets that can be readily docked by small fatty acids, reducing albuminmediated interactions. After washing, elution was performed at mild conditions using 0.1 M acetate buffer at pH 4. Protein A and Protein G agarose resins were utilized as controls and operated according to the instruction provided by the manufacturer, namely adsorption and wash with PBS at pH 7.4, and elution in 0.1 M glycine pH 2.8. The eluted fractions were analyzed by IgG-specific ELISA assay and SDS-PAGE (Fig. 3) to determine product yield and purity (SI Table 2), respectively. As expected, the adoption of optimized equilibration and washing conditions on LigaTrap-G resin prevents the binding and favors the

3.4. Purification of IgM from CHO and HEK cell culture fluids Recent developments in expression systems and purification methods tailored to IgM have rekindled the interest in this class of immunoglobulins [65]. In particular, novel strains of engineered mammalian (e.g., CHO DG44 [50]) and human (HEK 293 [50]; PER.C6® [66]) cell lines have managed to increase the IgM titer from about 0.01 mg/mL to above 0.1 mg/mL. New cell lines and purification tools tailored to IgM have been introduced, which have rekindled the interest in this class of molecules. CHO strains CHO-DUKX-B11, K1, and DG44 have been developed for the expression of both pentameric and hexameric forms of IgM at titers up to0.14 g/L [67]. PER.C6 cells, generated by immortalizing human retina cells, have been shown to express IgM at titers as high as 2 g/L [66]. These cells are particularly interesting for bioprocessing, having the ability of growing at high density in suspension and serum-free conditions [68]. The lack of adequate affinity-based tools for IgM purification offering equivalent performance to established protein-based ligands continues to slow the large scale manufacturing of IgM. Protein A and Protein G, in fact, do not reliably bind IgM [69,70]. Protein L, on the other hand, binds the Fab portion of IgM, but is expensive, lacks significant cycles of use, is biochemically labile, and requires harsh elution conditions (pH 2.8 – 3), and does not bind all varieties of Fab [71]. Further, unlike IgG, which feature high stability and resist to low-pH 5

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Fig. 3. SDS-PAGE analysis (reducing conditions, Coomassie staining) of the (A) elution and (B) flowthrough and wash fractions obtained from the purification of IgG from human serum using LigaTrap-G, Protein A agarose, and Protein G agarose resin. Labels: MW, molecular weight ladder; Feed, 10-fold diluted human serum; El LTG, elution at pH 4.2 from LigaTrap-G resin; El PrA, elution at pH 2.8 from Protein A-agarose; and El PrG, elution at pH 2.8 from Protein G-agarose; FT, flow through from LigaTrap-G resin; W1, W2, and W3, wash fractions from LigaTrap-G resin; BSA, bovine serum albumin; IgG HC, heavy chain of IgG; IgG LC, light chain of IgG. Note: prior to injection in the gel, the Feed sample underwent a 33-fold dilution to a total protein concentration of 2 mg/mL; the flow-through fraction was not further diluted after collection and was injected directly at the concentration of ~ ~ 8.25 mg/mL, to enable appreciating the complexity of the profile of protein impurities that the ligands allows flowing through owing to its binding selectivity.

elution conditions, IgMs are less biochemically stable and require processing under milder conditions. Without adsorbents enabling an affinity-based capture step, IgMs have been paid much less attention than their therapeutic value would warrant. Furthermore, the commercialization of HiTrap IgM Purification HP resin by GE Healthcare and CaptureSelect™ IgM Affinity Matrix by POROS™ (Thermo Scientific™) has introduced affinity chromatography to IgM purification, enabling high product purity and concentration in a single step [72]. The LigaTrap-M resin, demonstrated for the first time in this work, has been developed specifically for IgM purification. To this end, crosslinked agarose resins with high pore diameter were utilized as a substrate for ligand conjugation, using a proprietary method developed by LigaTrap Technologies, LLC. The resin was first challenged with a CHO cell culture fluid containing 5% FBS, 0.1 mg/mL of CHO HCPs, and IgM at 1 mg/mL. This corresponds to an initial IgM purity in the feed sample of 51.2%. A Tricorn column packed with 1 mL of LigaTrapM resin was equilibrated with 0.1 M NaCl and 20 mM adipic acid at pH 7.4, loaded with 10 mL of feed, and washed with loading buffer to remove non-specifically bound proteins. Elution was then performed with 0.1 M acetate buffer at pH 4.2. HiTrap IgM HP resin and CaptureSelect™ IgM affinity matrix were utilized as controls and operated following manufacturer’s instruction; specifically, elution from the HiTrap resin was performed with either 0.8 M ammonium sulfate, or 1.0 M ammonium sulfate, or 0.5 M potassium sulfate, all at pH 7.4; elution from the CaptureSelect™ IgM resin was performed with 0.1 M glycine buffer pH 2.8. The eluted fractions were analyzed by IgM-specific ELISA assay and SDS-PAGE (Fig. 4) to determine product yield and purity (SI Table 3), respectively. Notably, the LigaTrap-M resin outperformed the CaptureSelect™ IgM resin in terms of both product purity (93.1% vs. 76.2%) and yield. Poor binding selectivity by the 2-mercaptopyridine, in fact, resulted in a conspicuous amount of albumin eluted in 1 M ammonium sulfate. The peptoid ligand, on the other hand, featured excellent binding capacity and selectivity. Similar comparison was obtained with CaptureSelect™ IgM affinity resin, which offered particularly low yield, likely an effect of the low IgM vs. serum protein ratio. Finally, the IgM enrichment afforded by LigaTrap-M was found to be 11-fold compared to that provided by the CaptureSelect™ IgM resin. Together with the binding capacity and selectivity of the peptoid ligand, the pore size of the substrate resins plays a crucial role in determining IgM binding and yield. The manufacturers disclose a pore size of 160 nm for CaptureSelect™ IgM resin and 100 – 360 nm for CaptureSelect™ IgM matrix. The larger pore size featured by the crosslinked agarose material utilized to produce the LigaTrap-M resin

Fig. 4. SDS-PAGE analysis (reducing conditions, silver staining) of the elution fractions obtained from the purification of IgM from a CHO cell culture fluid added with 5% FBS using LigaTrap-M and HiTrap IgM HP resins. Labels: MW, molecular weight ladder; Feed, CHO cell culture fluid added with 5% FBS; D-El LTM, elution at pH 5.5 from LigaTrap-M resin; S-El LTM, elution at pH 4.2 from LigaTrap-M resin; El1 HT, elution with 0.8 M ammonium sulfate from HiTrap IgM HP resin; El2 HT, elution with 1 M ammonium sulfate from HiTrap IgM HP resin; El3 HT, elution with 0.5 M potassium sulfate from HiTrap IgM HP resin; BSA, bovine serum albumin; IgM HC, heavy chain of IgM; IgM LC, light chain of IgM.

facilitates the access and transport of IgM through the chromatographic bead even at short residence times. The flow rate of 0.5 mL/min utilized in this work, corresponding to a residence time of 2 min, is likely insufficient to enable access of IgM to the pores of the commercial IgMbinding affinity adsorbents. The LigaTrap-M resin was then utilized for IgM purification from a HEK cell culture fluid added with 1% FBS and containing IgM at 0.1 mg/mL. The Tricorn column packed with 1 mL of LigaTrap-M resin was equilibrated with 0.35 M NaCl and 20 mM adipic acid in PBS at pH 7.4, loaded with 10 mL of feed, and washed with loading buffer to remove non-specifically bound proteins. Elution was then performed with 0.1 M acetate buffer at pH 4.2. All chromatographic fractions were analyzed by SDS-PAGE (Fig. 5) and IgM-specific ELISA assay to measure product yield. Due to its extremely low concentration, the target IgM is almost invisible in the silver stained SDS-PAGE; nonetheless, the removal of BSA by the flow-through and wash fractions is appreciable and 6

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Fig. 5. SDS-PAGE analysis (reducing conditions, silver staining) of the chromatographic fractions obtained from the purification of IgM from a HEK cell culture fluid added with 1% FBS using LigaTrap-G resin. Labels: MW, molecular weight ladder; Feed, HEK cell culture fluid added with 1% FBS and containing 1 mg/mL IgM; FT, flowthrough; W, wash; El, elution at pH 4.2 from LigaTrap-G resin; BSA, bovine serum albumin; IgM HC, heavy chain of IgM; IgM LC, light chain of IgM. The red arrows are added to facilitate the visualization of the heavy and light chains of IgG.

4. Conclusions

speaks of the high IgM vs. BSA binding selectivity of the peptoid ligand. Similarly, the IgM yield of 75.1%, as measured by IgG-specific ELISA, indicates that, despite the low concentration of IgM in the feed (0.1 mg/ mL, 0.13 μM, corresponding to 1 – 3% of the total protein content in the feed), the peptoid ligand was successful in recovering the product. The recovery of IgM from this source and the evaluation of product yield and purity in the eluted fraction are particularly challenging, due to the highly diluted character of these samples. Increasing the titer in the cell culture harvests is paramount to improve the performance of the separation process, even with the most advanced affinity-based adsorbents.

IgG- and IgM-rich polyclonal formulations and emerging monoclonal IgM-based therapeutics are set to strengthen the therapeutic weaponry available in clinical settings to fight drug-resistant infectious diseases and other life-threatening conditions. Chromatography is a key technology to enable the production of clinically relevant amount of these biotherapeutics, and the availability of robust and affordable adsorbents providing selective binding and long lifetime is paramount. The recent introduction of mixed-mode and protein-based ligands has demonstrated the usefulness of affinity chromatography in IgM purification. On the other hand, concerns on the cost and stability of the ligands and pore size of the adsorbents, limit the effectiveness of these adsorbents under the conditions, in particular residence time, that are representative of industrial processes. To obviate these limitations, we propose the use of a synthetic peptoid as ligand for the purification of IgG and IgM from complex sources. Key to the development of the adsorbents is the use of chromatographic substrates with select pore diameter and the implementation of LigaTrap's proprietary conjugation chemistries that afford optimal density and display of the ligands on the surface of the pores. The resulting peptoid-based adsorbents LigaTrap-G and LigaTrap-M were evaluated by purifying human polyclonal and monoclonal IgG and IgM from a variety of sources, including cell culture harvests and serum. Of particular notice is the ability of both adsorbents to recover the target immunoglobulins even when present at very low concentration, or separate IgM-rich and IgG-rich fractions of serum. Should such endeavor be attempted with commercial proteinbased ligands, it would require the combination of multiple expensive adsorbents. LigaTrap adsorbents, on the other hand, provide facile operation and the recovery of the target immunoglobulins with high yield and purity. Collectively, these results demonstrate that synthetic ligands, and peptide mimetics in particular, represent the future of affinity-based bioprocess technologies and the key to increase the access to biotherapeutics that greatly increase the quality of life and survival rate of patients worldwide.

3.5. Purification of IgM from IgG-depleted serum IgM-enriched polyclonal formulations are utilized widely in the clinics for the treatment of bacterial sepsis [73]. In particular, IVIG enriched with IgA and IgM (ivIg-GAM) seem to have higher efficacy compared to traditional IVIG formulations in treating patients undergoing septic shock caused by drug-resistant pathogens. Commercial ion exchange resins have been utilized to produce ivIg-GAM from Cohn II and III fractions of human plasma [74]. In this work, we utilized the LigaTrap-M resin to enrich an IgGdepleted human serum of the IgA and IgM contents. The IgG-depleted fluid was obtained as flow-through fraction from the LigaTrap-G and Protein A resins in Section 3.3. A volume of 600 mL of feed fluid at 0.11 mg/mL of IgM and 0.27 mg/mL of IgA was injected in a 10 mL column packed with LigaTrap-M resin. As seen above, resin equilibration and post-load washing were performed with 0.35 M NaCl and 20 mM adipic acid in PBS at pH 7.4 to prevent non-specific capture of the serum proteins that, being not bound by LigaTrap-G resin, are present in the IgG-depleted fluid. After washing, elution was performed using 0.1 M acetate buffer at pH 4. The LigaTrap-M eluate, initially at 2.34 mg/mL, was fractionated by size exclusion chromatography (SEC). The resulting fractions were finally analyzed by SDS-PAGE (Fig. 6) to evaluate the content of IgA and IgM. The electrophoretic analysis of the concentrated eluate indicates that IgA and IgM are both present and at high purity (91.7%). Subsequent SEC fractionation returned separate IgM-enriched (lanes 1–6) and IgA-enriched (lanes 18–24). Collectively these results suggest a 3-step method, comprising of the train of LigaTrap-G, LigaTrap-M, and size exclusion chromatographic steps to separate human serum in IgG-rich, IgA-rich, and IgM-rich fractions to be utilized in formulating ivIg-GAM preparations for anti-sepsis treatment.

CRediT authorship contribution statement Hannah Reese: Conceptualization, Data curation, Formal analysis, Writing - original draft. Tee Bordelon: Conceptualization, Data curation, Validation. Calvin Shanahan: Data curation, Writing - review & editing. Michael Crapanzano: Funding acquisition, Writing - review & editing. Jae Sly: Funding acquisition, Writing - review & editing. 7

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Fig. 6. SDS-PAGE analysis (reducing conditions, Coomassie staining) of the fractions obtained by size exclusion chromatography of the eluate obtained using LigaTrap-M resin from IgG-depleted human serum. Labels: MW, molecular weight ladder; Feed, LigaTrap-M eluate; IgM HC, heavy chain of IgM; IgA HC, heavy chain of IgA; Ig LC, light chain of IgA and IgM.

Stefano Menegatti: Conceptualization, Funding acquisition, Project administration, Writing - review & editing.

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