International Journal of Molecular Sciences Article Recovering What Matters: High Protein Recovery after Endotoxin Removal from LPS‑Contaminated Formulations Using Novel Anti‑Lipid A Antibody Microparticle Conjugates Cristiane Casonato Melo 1,2 , Alexandra C. Fux 1,2 , Martin Himly 2 , Neus G. Bastús 3,4 , Laura Schlahsa 1 , Christiane Siewert 1 , Victor Puntes 3,4,5,6 , Albert Duschl 2 , Isabel Gessner 1, * and Jonathan A. Fauerbach 1, * 1 Chemical Biology Department, R&D Reagents, Miltenyi Biotec B.V. & Co. KG, 51429 Bergisch Gladbach, Germany; cristianec@miltenyi.com (C.C.M.); alexandraf@miltenyi.com (A.C.F.); laurasch@miltenyi.com (L.S.); christianes@miltenyi.com (C.S.) 2 Division of Allergy & Immunology, Department of Biosciences & Medical Biology, Paris Lodron University of Salzburg, 5020 Salzburg, Austria; martin.himly@plus.ac.at (M.H.); albert.duschl@plus.ac.at (A.D.) 3 Institut Català de Nanociència i Nanotecnologia (ICN2), Consejo Superior de Investigaciones Científicas (CSIC), The Barcelona Institute of Science and Technology (BIST), Campus UAB, Bellaterra, 08036 Barcelona, Spain; neus.bastus@icn2.cat (N.G.B.); victor.puntes@icn2.cat (V.P.) 4 Networking Research Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER‑BBN), 08034 Barcelona, Spain 5 Vall d’Hebron Institut de Recerca (VHIR), 08035 Barcelona, Spain 6 Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Spain * Correspondence: isabelge@miltenyi.com (I.G.); jonathanf@miltenyi.com (J.A.F.) Abstract: Endotoxins or lipopolysaccharides (LPS), found in the outer membrane of Gram‑negative bacterial cell walls, can stimulate the human innate immune system, leading to life‑threatening symp‑ Citation: Casonato Melo, C.; Fux, toms. Therefore, regulatory limits for endotoxin content apply to injectable pharmaceuticals, and ex‑ A.C.; Himly, M.; Bastús, N.G.; cess LPS must be removed before commercialization. The majority of available endotoxin removal Schlahsa, L.; Siewert, C.; Puntes, V.; systems are based on the non‑specific adsorption of LPS to charged and/or hydrophobic surfaces. Duschl, A.; Gessner, I.; Fauerbach, Albeit effective to remove endotoxins, the lack of specificity can result in the unwanted loss of es‑ J.A. Recovering What Matters: High sential proteins from the pharmaceutical formulation. In this work, we developed microparticles Protein Recovery after Endotoxin conjugated to anti‑Lipid A antibodies for selective endotoxin removal. Anti‑Lipid A particles were Removal from LPS‑Contaminated characterized using flow cytometry and microscopy techniques. These particles exhibited a deple‑ Formulations Using Novel tion capacity > 6 ×103 endotoxin units/mg particles from water, as determined with two indepen‑ Anti‑Lipid A Antibody Microparticle dent methods (Limulus Amebocyte Lysate test and nanoparticle tracking analysis). Additionally, we Conjugates. Int. J. Mol. Sci. 2023, 24, compared these particles with a non‑specific endotoxin removal system in a series of formulations 13971. https://doi.org/10.3390/ of increasing complexity: bovine serum albumin in water < insulin in buffer < birch pollen extracts. ijms241813971 We demonstrated that the specific anti‑Lipid A particles show a higher protein recovery without Academic Editors: Josef Jampilek compromising their endotoxin removal capacity. Consequently, we believe that the specificity layer and Jiri Kos integrated by the anti‑Lipid A antibody could be advantageous to enhance product yield. Received: 21 August 2023 Revised: 5 September 2023 Keywords: LPS contamination; polystyrene particles; bioconjugation; supramolecular structures; Accepted: 7 September 2023 LAL; NTA Published: 12 September 2023 1. Introduction Copyright: © 2023 by the authors. To survive in a diversity of hostile environments, bacteria have developed a complex Licensee MDPI, Basel, Switzerland. cell envelope, with selective passage of substances [1]. In Gram‑negative bacteria, a three‑ This article is an open access article layer envelope (outer membrane, peptidoglycan cell wall and inner membrane) protects distributed under the terms and conditions of the Creative Commons the inner content [1]. The outer membrane is mainly formed of lipopolysaccharides (LPS), Attribution (CC BY) license (https:// which confer structural integrity. Moreover, it limits the translocation of hydrophobic creativecommons.org/licenses/by/ molecules, such as during Gram staining [1,2]. LPS molecules are normally composed 4.0/). Int. J. Mol. Sci. 2023, 24, 13971. https://doi.org/10.3390/ijms241813971 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023, 24, 13971 2 of 15 of two different chemical components responsible for their amphiphilic nature, the hy‑ drophilic polysaccharide moiety and the hydrophobic Lipid A domain [2,3]. The latter is commonly considered the most conserved region of LPS among different bacterial species and strains and is known to be responsible for the activation of the human innate immune system when LPS enters the human bloodstream [2–4]. The intensity of the immune re‑ sponse against LPS can be different depending on the LPS source and concentration, but, in general, LPS has been described to trigger the release of inflammatory cytokines upon activation of the TLR4 pathway, with symptoms ranging from fever up to life‑threatening conditions, such as septic shock or even death [5]. To avoid contamination incidents upon drug administration into patients, all inject‑ able pharmaceuticals need to be tested for endotoxin content before being released for com‑ mercialization. With the exception of intrathecal administration, a limit of five endotoxin units (EU)/kg body weight per hour has been established for parenteral formulations [6,7]. Whenever the limit is exceeded, endotoxin inactivation or removal is required prior to safe release of the respective pharmaceutical product [8]. Nevertheless, the removal of LPS cannot be achieved through conventional steriliza‑ tion methods such as 0.22 µm filtration and/or autoclaving [9,10]. Instead, the elimination of pyrogens, including endotoxins, a process also called depyrogenation, can be accom‑ plished through specific methods such as using oxidizing agents (e.g., hydrogen peroxide), dry heat at 250 ◦ C for at least 30 min or ionizing radiation [9,11]. However, such harsh con‑ ditions, although effective to eliminate endotoxins, are not compatible with many drug products. The need for alternative endotoxin removal methods has become even more evident as several biopharmaceutical proteins are produced recombinantly in E. coli, a Gram‑negative bacterium, and endotoxin removal is therefore an integral part of their pu‑ rification processes [6,12]. In these scenarios, the downstream purification process of such proteins can account for up to 92% of manufacturing costs [6]. To date, most of the commercially available endotoxin removal methods are based on non‑specific interactions between positively charged surfaces and the negatively charged phosphate group in the LPS backbone and/or between hydrophobic surfaces and the hy‑ drophobic Lipid A [6,13,14]. While these methods are effective in removing LPS from var‑ ious aqueous solutions, their lack of specificity for LPS leads to undesirable co‑removal of other molecules, such as proteins, resulting in low recovery and higher production costs [6]. To reduce the non‑specific depletion of active components (e.g., proteins), alter‑ native systems have been developed that rely on high‑affinity interactions between LPS and a modified surface. For instance, Vagenende et al. described a system that relies on hydrogen bonding between endotoxins and crystals made of purine‑derived allantoin that reaches binding affinities in the picomolar range. In addition to a good efficiency in LPS removal, a protein recovery of over 80% for bovine serum albumin (BSA) was achieved [15]. Li et al. decorated nanoparticles with lipopolysaccharide‑binding proteins (LBP) and achieved a good LPS removal efficiency under controlled contamination con‑ ditions, with a stable protein recovery of 80% for BSA at pH values ranging from four to nine [16]. Recently, Chen et al. presented the development of silica microparticles con‑ jugated to histidine dipeptides and showed its applicability to remove LPS from buffer, protein solution and from whole blood [17]. Similar results were shown by Shi et al. using artificial peptides and a hemocompatible polymer. Furthermore, they demonstrated LPS clearance in extracorporeal in vivo studies [18]. Despite these improvements in targeting LPS, we rationalized that, by means of an antibody–antigen interaction targeting the Lipid A in LPS, we could further optimize the specific removal of LPS while achieving a high protein yield. In this work, we therefore present a new endotoxin removal platform based on the specific interaction between the Lipid A moiety from LPS and an anti‑Lipid A antibody immobilized on the surface of polystyrene microparticles. We determine LPS removal in water using the well‑established and widely used Limulus Amebocyte Lysate (LAL) test and compared the results to an alternatively developed method based on nanoparticle Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 3 Int. J. Mol. Sci. 2023, 24, 13971 3 of 15 and compared the results to an alternatively developed method based on nanopa tracking analysis (NTA), which tracks the presence of supramolecular structures of While NTA is commonly used for the characterization of liposomes, lipidic nanopar tracking analysis and (NTA), which vesicles extracellular tracks the presence [19–21], of supramolecular it has, to the best of structures of LPS. not yet our knowledge, While NTA is described commonlyfor used for the characterization of liposomes, lipidic nanoparticles the evaluation of LPS supramolecular structures, with a clear adva and extracellular vesicles [19–21], it has, to the best of our knowledge, not yet been de‑ over LAL regarding sample preparation time. Additionally, we assessed the perform scribed for the evaluation of LPS supramolecular structures, with a clear advantage over of anti-Lipid A particles versus a non-specific approach regarding LPS removal effic LAL regarding sample preparation time. Additionally, we assessed the performance of and protein recovery for a series of samples with increasing complexity: BSA in w anti‑Lipid A particles versus a non‑specific approach regarding LPS removal efficiency and insulin in HEPES buffer and naturally LPS-contaminated birch pollen extracts (BPE protein recovery for a series of samples with increasing complexity: BSA in water, insulin DPBS. We demonstrate that while both particle systems maintain a similar LPS rem in HEPES buffer and naturally LPS‑contaminated birch pollen extracts (BPEs) in DPBS. performance, only the specific anti-Lipid A particles enabled a statistically signi We demonstrate that while both particle systems maintain a similar LPS removal perfor‑ higher protein recovery, consequently providing a safety and potential comm mance, only the specific anti‑Lipid A particles enabled a statistically significant higher pro‑ advantage for pharmaceutical formulations. tein recovery, consequently providing a safety and potential commercial advantage for pharmaceutical formulations. 2. Results and Discussion The objective of this study was to investigate whether the unwanted loss of pro 2. Results and Discussion can be reduced The objective of this studyby using was LPS removal to investigate systems whether that specifically the unwanted loss ofinteract proteinswith LPS therefore designed a particle platform that was based on micron-sized can be reduced by using LPS removal systems that specifically interact with LPS. We there‑ polystyrene particles that were surface-modified with anti-Lipid A antibodies. fore designed a particle platform that was based on micron‑sized polystyrene (PS) particles that were surface‑modified with anti‑Lipid A antibodies. 2.1. Anti-Lipid A Particles: Conjugation and Characterization 2.1. Anti‑Lipid A Particles: Conjugation Anti-Lipid andwere A particles Characterization prepared through a three-step conjugation as depict Anti‑Lipid A particles Figure 1a. In were short,prepared through carboxylic a three‑step acid groups on theconjugation surface of as depicted 3 µm PS particles in Figure 1a. activated In short, and carboxylic acidbound covalently groupstoon the surface of aminodextran to 3yield PS particles µm amino-PS were Subseq particles. activated and activation covalentlyof bound to aminodextran primary to yield amino‑PS amines with maleimides allowedparticles. Subsequent for the attachment of reduced activation of primary Lipid Aamines with(Ab) antibodies maleimides allowed via Michael for the attachment of reduced anti‑ addition. Lipid A antibodies (Ab) via Michael addition. Figure 1. Anti‑Lipid FigureA1.particles: Ab A Anti-Lipid conjugation andconjugation particles: Ab characterization. (a) Schematic representation and characterization. (a) Schematic represen of the anti‑LipidofAthe particles’ conjugation workflow. Anti‑Lipid A particle anti-Lipid A particles’ conjugation workflow. Anti-Lipid characterization: (b) STEM A particle characterization: (b) image at 8000× magnification (scale bar: 10 µm); (c) quantification of anti‑Lipid A antibody loading image at 8000× magnification (scale bar: 10 µm); (c) quantification of anti-Lipid A antibody lo × 108 particles normalized to 1normalized to 1 (n × 108 particles (n = 3); and (d) flow cytometry median fluorescence intensity = 3); and (d) flow cytometry median fluorescence intensity (MFI) of PE‑secondary Ab staining of anti‑Lipid A of of PE-secondary Ab staining Abanti-Lipid A Ab on particles. on particles. After conjugation, anti‑Lipid A particles were imaged using scanning transmission electron microscopy (STEM) and scanning electron microscopy (SEM) (Figures 1 and S1), which revealed spherical particles with a narrow size distribution (2.68 ± 0.05 µm, Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 of 15 After conjugation, anti-Lipid A particles were imaged using scanning transmission Int. J. Mol. Sci. 2023, 24, 13971 electron microscopy (STEM) and scanning electron microscopy (SEM) (Figures 1b and4 of S1), 15 which revealed spherical particles with a narrow size distribution (2.68 ± 0.05 µm, Figure S2). Optical images obtained using Countess II Cell counter indicated a high dispersibility of the conjugated Figure S2). Opticalparticles images in phosphate-buffered obtained using Countess saline (PBS)-0.03% II Cell PluronicaF-68 counter indicated highwith dis‑ no visual signs of aggregation (Figure S3). This was further substantiated persibility of the conjugated particles in phosphate‑buffered saline (PBS)‑0.03% Pluronic by a high percentage F‑68 with no value of single visual particles signs of obtained aggregation usingS3). (Figure flowThis cytometry measurements was further (Figure substantiated by aS4). The high supernatant percentage value(SN) obtained of single after Ab particles conjugation obtained with cytometry using flow maleimidemeasurements particles was subjected (Figure to The S4). protein quantification supernatant via a bicinchoninic (SN) obtained acid (BCA)with after Ab conjugation assay, indicating maleimide that an particles average was of 5.1 to subjected ± 0.6 µg (nquantification protein = 3) of polyclonal via a anti-Lipid bicinchoninicA Ab was acid conjugated (BCA) to 1 × 108 assay, indicating particles that an average 5.1 ± (Figureof1c). Upon 0.6 µgthe(nincubation of anti-Lipid = 3) of polyclonal A particles anti‑Lipid A Ab was with a specific conjugated to 1 × 10 particles (Figure 1c). Upon the incubation of anti‑Lipid A particles withsurface 8 secondary Ab (anti-Goat-PE), it was possible to stain the conjugated Ab on the of a specific the particles. secondary AbAn increase in the (anti‑Goat‑PE), PE median it was possiblefluorescence intensity (MFI) to stain the conjugated Ab oncompared the surfacewith of controls the indicated particles. the presence An increase in theofPE anti-Lipid median A Ab on the particle fluorescence surface, intensity (MFI) which comparedprovides with an independent controls indicated determination the presence of of Ab conjugation anti‑Lipid A Ab on the toparticle that ofsurface, the BCAwhichassay. No provides fluorescence an independent was observed forof determination Ab-conjugated Ab conjugation particles to that ofwithout the BCAsecondary assay. NoAb (negative fluorescence control) was nor forfor observed Ab-conjugated Ab‑conjugated particles incubated particles withoutwith an unspecific secondary secondary Ab (negative Ab (anti- control) nor for Ab‑conjugated Human-PE, Figuresparticles incubated with an unspecific secondary Ab (anti‑Human‑PE, 1d and S4). Figures 1d and S4). 2.2. Anti-Lipid A Particles’ Functionality and Stability 2.2. Anti‑Lipid A Particles’ Functionality and Stability 2.2.1. LPS Removal Capacity of Anti-Lipid A Particles 2.2.1. LPS Removal Capacity of Anti‑Lipid A Particles Next, the LPS removal capacity of anti-Lipid A particles was determined. In view of Next, the LPS removal capacity of anti‑Lipid A particles was determined. In view of the ubiquity of E. coli [22], a solution of 1000 ng/mL ≈ 10,000 endotoxin units (EU)/mL of the ubiquity of E. coli [22], a solution of 1000 ng/mL ≈ 10,000 endotoxin units (EU)/mL of LPS from E. coli O111:B4 in water was used first. Therefore, anti-Lipid A particles were LPS from E. coli O111:B4 in water was used first. Therefore, anti‑Lipid A particles were incubated with 10,000 EU/mL of LPS in water for 20 min at RT, followed by centrifugation. incubated with 10,000 EU/mL of LPS in water for 20 min at RT, followed by centrifugation. The particle pellet was resuspended with a fresh LPS solution, exposing particles to The particle pellet was resuspended with a fresh LPS solution, exposing particles to another another round of 10,000 EU/mL of LPS in water. A total of 12 consecutive cycles were round of 10,000 EU/mL of LPS in water. A total of 12 consecutive cycles were performed, performed, as shown in the scheme in Figure 2a. The remaining amount of endotoxin in as shown in the scheme in Figure 2a. The remaining amount of endotoxin in each SN was each SN was quantified using the traditional LAL test based on calibration curves (Figure quantified using the traditional LAL test based on calibration curves (Figure S5), as well S5), as well as with NTA (Figure 2b). as with NTA (Figure 2b). Figure 2. LPS LPS removal removal capacity and longitudinal stability of anti‑Lipid anti-Lipid A A particles. particles. (a) Schematic representation of the the LPS LPSremoval removalprocedure procedurebased basedononrepeating repeatingremoval removalcycles, and cycles, thethe and analysis of analysis of SN using LAL and NTA. (b) LPS saturation curve obtained by exposing 5 mg of anti‑Lipid A SN using LAL and NTA. (b) LPS saturation curve obtained by exposing 5 mg of anti-Lipid A particles to repeated cycles of 10,000 EU of LPS from E. coli O111:B4 in water (mean ± sem, n ≥ 3). (c) LPS removal performance of anti‑Lipid A particles when incubated with 10,000 EU/mL of LPS from E. coli O111:B4 measured over a period of 240 days and stored at 4 ◦ C. Int. J. Mol. Sci. 2023, 24, 13971 5 of 15 The amount of LPS detected in the SN after the first removal cycle was under the limit of detection for the LAL test (<0.1 EU/mL), which translates to a removal of at least 99.999%. From cycles 2 to 5, the removal was ≥99.99% according to LAL. In subsequent incubation cycles, the LPS removal performance of anti‑Lipid A particles decreased continuously, in‑ dicating the stepwise saturation of the particle surface with LPS and their limit in effective further LPS removal (Figure 2b, blue curve). Similarly to other amphiphilic molecules, LPS can form supramolecular structures in aqueous solutions [23–25]. The presence of pre‑micelle LPS oligomers were already described in the literature by Santos et al. using light‑scattering spectroscopy in LPS eval‑ uation below the critical aggregation concentration [25]. To determine the sensitivity of NTA for LPS detection in the SN, a dilution series of LPS in water ranging from 1 to 1 × 106 EU/mL was prepared (Figure S6). Unexpectedly, the results indicated that even at the lowest tested concentration (1 EU/mL ≈ 0.1 ng/mL), supramolecular structures were observed by NTA, providing enough sensitivity to determine the LPS removal efficiency of anti‑Lipid A particles. The SN was then analyzed using NTA, measuring the number of particles/frame (Figures S7 and S8) and calculating the endotoxin removal for each cycle relative to the LPS starting concentration A (Figures 2b and S8). While almost no LPS was detected in the SN during the first three cycles, a decrease in LPS removal performance was observed above 30,000 EU, followed by a continuous decrease during subsequent cycles (Figure 2b, black curve). The curves obtained with LAL (enzymatic method) and NTA (physical method) are in close correspondence between cycles 1 and 8, demonstrating that both methods are well‑ suited for quantifying LPS removal from water. Starting at cycle 9, the results measured between the two methods started diverging. This can be explained considering that the NTA detection method is based on optical detection of supramolecular structures, while LAL is not limited to these aggregates. Nevertheless, NTA provided a faster and less ex‑ pensive initial analysis of LPS solutions without the requirement of extensive sample dilu‑ tion, as is common practice for LAL. Therefore, this technique can potentially be explored, complementary to LAL, in samples without light‑scattering molecules. Overall, our data showed a quantitative removal of more than 98% during the first three cycles evidenced by NTA and the first five cycles by LAL (Figures 2b and S9). This indicates a saturation of 5 mg of particles with LPS between 30,000 and 50,000 EU; thus, at least 6000 to 10,000 EU can be removed with only 1 mg of anti‑Lipid A particles (which corresponds to roughly 6.7 × 107 particles). 2.2.2. Longitudinal Stability In order to evaluate the stability of the Ab‑particle conjugates, anti‑Lipid A particles were stored at 2–8 ◦ C and LPS removal performance was tested at specific time points between 8 and 251 days. As described for previous experiments, 5 mg of particles was incubated with a fresh solution of 10,000 EU/mL LPS in water at the indicated time points, and the corresponding SN was analyzed for traces of remaining LPS. Endotoxin levels for all SNs were below the limit of detection of the LAL assay, presenting less than 0.1 EU/mL even after more than 250 days of particle storage (Figure 2c). With that, we conclude that particles can be stored for at least 8 months at 2–8 ◦ C (and potentially longer) without any measurable loss in functionality. 2.3. LPS Removal and Protein Recovery Furthermore, the LPS removal performance of anti‑Lipid A particles was compared with commercially available Endotoxin Removal Beads from Miltenyi Biotec [26]. Since these particles remove endotoxins through electrostatic interactions [6,26], in contrast to antibody–antigen interactions, they are here referred to as non‑specific particles. Both particle types were tested in direct comparison in LPS solutions that additionally con‑ tained proteins ranging from low complexity (BSA in water) to medium (insulin in HEPES these particles remove endotoxins through electrostatic interactions [6,26], in contrast to antibody–antigen interactions, they are here referred to as non-specific particles. Both Int. J. Mol. Sci. 2023, 24, 13971 particle types were tested in direct comparison in LPS solutions that additionally 6 of 15 contained proteins ranging from low complexity (BSA in water) to medium (insulin in HEPES 25 mM pH 7.1) and high (BPE, naturally contaminated with unknown LPS strain in Dulbecco′s Phosphate-Buffered Saline, or DPBS). To evaluate differences in the 25 mM pH 7.1) and high (BPE, naturally contaminated with unknown LPS strain in Dul‑ performance of the two particle platforms, LPS removal efficiency was evaluated with becco’s Phosphate‑Buffered Saline, or DPBS). To evaluate differences in the performance LAL (BSA and insulin formulations) or with HEK-Blue™-hTLR4 cells (BPE formulation) of the two particle platforms, LPS removal efficiency was evaluated with LAL (BSA and and protein recovery was assessed with the BCA assay (BSA and insulin formulations) or insulin formulations) or with HEK‑Blue™‑hTLR4 cells (BPE formulation) and protein re‑ Bradford covery assay was (BPEwith assessed formulation). the BCA assay (BSA and insulin formulations) or Bradford assay (BPE formulation). 2.3.1. LPS Removal and Protein Recovery in 1 mg/mL BSA in Water 2.3.1.AsLPS Removal BSA and Protein is excipient Recovery for several in 1 mg/mL formulations and BSA in Water pharmaceutical products [27,28] and, As therefore, is a widely used model protein for the BSA is excipient for several formulations and pharmaceutical products evaluation of new[27,28] endotoxin and, removal methods therefore, is a widely[13,15,16], the LPS used model removal protein for performance the evaluation ofof anti-Lipid A particles new endotoxin and removal non-specific methods particles [13,15,16], thewas LPSfirst evaluated removal in the presence performance of BSA of anti‑Lipid in water.and non‑specific A particles Here, particles wasdifferent stock solutions first evaluated of LPS of in the presence from BSAE.incoli O111:B4 (100 to 10,000 EU/mL) water. containing 1 mg/mLstock Here, different BSA were prepared. solutions of LPSAfromtotalE. ofcoli 5 mg of either O111:B4 anti-Lipid (100 to 10,000AEU/mL) particles or con‑ non-specific particles were used to resuspend 1 mL of such taining 1 mg/mL BSA were prepared. A total of 5 mg of either anti‑Lipid A particles or LPS-BSA solutions. After incubation, particles non‑specific particleswerewerecentrifuged, used to resuspend and the1SNs mL were of such evaluated LPS‑BSAusing LAL and solutions. BCA After in‑ for LPS and cubation, proteinwere particles content, respectively. centrifuged, OurSNs and the results wereshowed evaluatedno using significant LAL difference and BCA for in LPS and removal the LPS protein content, respectively. performance betweenOur bothresults showed particle no significant platforms, independentdifference of theinLPS the LPS removal content performance (Figure between both 3a). Nevertheless, particle platforms, the variability independent in the removal of the LPS performance content (assessed (Figure using the 3a). Nevertheless, standard error ofthethevariability mean, sem) in the wasremoval larger for performance (assessed the non-specific usingthan particles the standard error of the mean, sem) was larger for the non‑specific for the anti-Lipid A particles, which indicates that the reproducibility of the LPS removal particles than for the anti‑ Lipid system Abased particles, which indicates on electrostatic that the was interactions reproducibility lower compared of thewith LPS removal system the specific based antibody– on electrostatic antigen interactions interaction was lower under these compared conditions. with the specific Furthermore, antibody–antigen we noticed that LPS removal inter‑ action under was performance theselower conditions. for samplesFurthermore, containingwe noticedEU/mL. < 10,000 that LPS Weremoval performance rationalized that this was lower is likely for samples related to the containing < 10,000has fact that albumin EU/mL. been We rationalized described that this to strongly is likely interact withrelated LPS to the fact that albumin has been described to strongly interact via hydrophobic interactions between the acyl chains of Lipid A with one or more with LPS via hydrophobic interactions hydrophobicbetween bindingthe acyl sites in chains albumin of Lipid A with oneLPS [29]. Therefore, or more hydrophobic molecules binding can be partially sites in albumin [29]. Therefore, LPS molecules can be blocked by BSA, decreasing the interaction of LPS and the anti-Lipid A Ab, and, partially blocked by BSA, decreas‑ ing the interaction consequently, LPS ofis LPS not and the anti‑Lipid properly removed.AAAb, and, consequently, similar result was recentlyLPS isdescribed not properlyby removed. A similar result was recently described by Chen et Chen et al., who observed lower LPS removal as the ratio of human serum albumin to LPS al., who observed lower LPS removal increasedas[17].the Moreover, ratio of human givenserum albumin the strong to LPS positive netincreased charge of[17]. Moreover, non-specific given the particles, it strong positive net charge of non‑specific particles, it is also likely is also likely that BSA, which has a pI ≈ 4.7, adsorbs to the surface, forming a protein that BSA, which has a pI ≈ 4.7,which corona adsorbs to theLPS hinders surface, binding forming a protein and, thus, reducescoronaLPSwhich removal hinders [14]. LPS binding and, thus, reduces LPS removal [14]. Figure 3. Comparison between anti‑Lipid A particles and commercial non‑specific particles in aque‑ ous BSA solutions regarding (a) endotoxin removal and (b) protein recovery. All samples contain 1 mg/mL of BSA in water with different spiked amounts of LPS from E. coli O111:B4. Data shown are mean ± sem, n ≥ 3, and analyzed via t‑test (ns = not significant, ** p < 0.01, *** p < 0.001). Figure 3. Comparison between anti-Lipid A particles and commercial non-specific particles in aqueous BSA solutions regarding (a) endotoxin removal and (b) protein recovery. All samples contain 1 mg/mL of BSA in water with different spiked amounts of LPS from E. coli O111:B4. Data shown are mean ± sem, n ≥ 3, and analyzed via t-test (ns = Int. J. Mol. Sci. 2023, 24, 13971 not significant, ** p < 0.01, *** p < 0.001). 7 of 15 As previously mentioned, protein recovery is a crucial index to the selection of an endotoxin removal system, where the unwanted non-specific removal of protein As previously components should mentioned, be minimizedprotein recoveryavoided. or, ideally, is a crucial Whenindex to the selection comparing of an the protein endotoxin removal system, where the unwanted non‑specific removal of protein recovery performance of both particle types, a statistically significantly higher protein compo‑ nents should recovery yieldbewas minimized observedor,upon ideally, useavoided. When of anti-Lipid Acomparing the protein particles (Figure recovery per‑ 3b). Notably, for formance of both particle types, a statistically significantly higher protein recovery these particles, the protein recovery slightly increased with higher LPS concentrations yield was observed (93.2%, upon97.8% 96.8% and use offor anti‑Lipid 100, 1000Aand particles 10,000(Figure EU/mL, 3b). Notably, forwhile respectively), these the particles, non- the protein recovery slightly increased with higher LPS concentrations (93.2%, 96.8% specific particles did not follow this trend (84.0%, 88.1% and 87.3% for 100, 1000 and 10,000 and 97.8% for 100, 1000 and 10,000 EU/mL, respectively), while the non‑specific particles did EU/mL, respectively). The results obtained here highlighted the role of the specificity layer not follow this trend (84.0%, 88.1% and 87.3% for 100, 1000 and 10,000 EU/mL, respec‑ in reducing the non-specific removal of off-target molecules, allowing for higher protein tively). The results obtained here highlighted the role of the specificity layer in reducing recovery. the non‑specific removal of off‑target molecules, allowing for higher protein recovery. 2.3.2. 2.3.2.LPS LPSRemoval Removaland andProtein ProteinRecovery Recoveryin in11mg/mL mg/mLInsulin Insulinin inHEPES HEPES25 25mMmM With Withthe theadvent adventof ofrecombinant recombinantDNA DNAtechnology, technology,insulin insulinstarted startedto tobebeproduced producedin in bacteria, more specifically in E.coli, and was the first FDA-approved bacteria, more specifically in E. coli, and was the first FDA‑approved recombinant recombinant drug [30,31]. To ensure drug [30,31]. safetysafety To ensure for humans, for humans, the the endotoxin endotoxin level in in level insulin insulinneeds needs totobebewell- well‑ controlled, controlled, with the threshold of a specific insulin formulation being dependent onon with the threshold of a specific insulin formulation being dependent its its po‑ potency tency andand the the recommended recommended dose dose [7,32]. [7,32]. We We therefore therefore chose chose to to investigate investigate whether whether anti-Lipid anti‑Lipid AA particles particles could could bebe used used for for LPS LPS removal removal inin the the presence presence of of insulin insulin inin buffer. buffer. Similarly Similarlytotothetheprevious previous experiment experiment performed performedwithwithBSABSAin inwater, water,different differentamounts amountsof ofLPS LPSfrom fromE.E.coli coliO111:B4 O111:B4were werespiked spiked with insulin (final insulin concentration was 1 mg/mL) and the SNs with insulin (final insulin concentration was 1 mg/mL) and the SNs were evaluated for LPS were evaluated for LPS and protein content. No significant difference was and protein content. No significant difference was observed between observed between the two different two different particle particle types regarding regardingLPS LPSremoval removal (Figure (Figure 4a). 4a). Here, Here, differently differently from previous from previous experi‑ experiments ments with BSA,withthe BSA, LPSthe LPS contamination contamination level did notlevelinfluence did notthe influence removalthe removal performance performance of particles,due of particles, presumably presumably due to lower to lower non‑specific non-specific interactions betweeninteractions insulin and between LPS as insulin comparedandwith LPS as compared with BSA-LPS. BSA‑LPS. Figure 4. Comparison between anti‑Lipid A particles and commercial non‑specific particles in in‑ sulin in HEPES buffer regarding (a) endotoxin removal and (b) protein recovery. All samples Figure contain4.1 Comparison between mg/mL of insulin anti-Lipid in HEPES 25 mMApH particles 7.1 withand commercial different non-specific spiked amounts of LPS from particles in insulin E. coli O111:B4. Datainshown HEPES mean ± arebuffer sem, n ≥ (a) regarding endotoxin 3, and analyzedremoval and with t‑test (ns(b) protein = not significant, recovery. * p < 0.05). All samples contain 1 mg/mL of insulin in HEPES 25 mM pH 7.1 with different The BCA assay showed a higher statistically significant protein (insulin) recovery when using anti‑Lipid A particles compared with non‑specific particles in two out of three conditions (Figure 4b). In fact, anti‑Lipid A particles achieved up to 100% protein recovery for 100 and 10,000 EU/mL, while non‑specific particles achieved a maximum recovery of 92.9%, for 100 EU/mL. Although not significant, the mean protein recovery for 1000 EU/mL was still higher for the specific anti‑Lipid A particles (99.6%) than for the non‑specific parti‑ when using anti-Lipid A particles compared with non-specific particles in two out of three conditions (Figure 4b). In fact, anti-Lipid A particles achieved up to 100% protein recovery for 100 and 10,000 EU/mL, while non-specific particles achieved a maximum recovery of 92.9%, for 100 EU/mL. Although not significant, the mean protein recovery for 1000 Int. J. Mol. Sci. 2023, 24, 13971 EU/mL was still higher for the specific anti-Lipid A particles (99.6%) than for the 8non-of 15 specific particles (90.6%), consistent with the other two samples and the original intention and rationale of our specific anti-Lipid A particle platform. cles (90.6%), consistent with the other two samples and the original intention and rationale 2.3.3. of ourLPS Removal specific and Protein anti‑Lipid Recovery A particle in Naturally LPS-Contaminated BPE in DPBS platform. Given the intrinsic complexity of LPS-contaminated protein solutions, endotoxin 2.3.3. LPSsystems removal Removal areand Protein mainly Recovery tested in Naturally in controlled LPS‑Contaminated samples where a known BPE in DPBS amount and Given serotype the is of LPS intrinsic spiked in complexity a sample. of ForLPS‑contaminated practical applications, protein theresolutions, is an interestendotoxin to test removal systems endotoxin removalare mainly from tested in controlled higher-complexity samples matrixes, such where as BPEs, a known amount and which represents a serotype of naturally LPS is spiked insample. LPS-contaminated a sample. BirchForpollen practical applications, is the main causethere is an rhinitis of allergic interest into test endotoxin northern removalEurope and central from higher‑complexity [33], containing matrixes, more thansuch 150asallergens BPEs, which represents including the a naturally protein Bet LPS‑contaminated sample. Birch v 1 [33,34]. Upon extraction pollen of birch is theBPE pollen, maincontains cause ofglycans allergicas rhinitis well as in northern and central Europe low-molecular-weight [33], containing compounds, includingmore LPS than 150 allergens originating includingbacterial from different the pro‑ tein Betencountered species v 1 [33,34]. Upon on theextraction birch pollen of birch surfacepollen, BPETo [35,36]. contains isolate glycans the immune as well as low‑ response molecular‑weight compounds, including LPS originating from from allergens and to help clarify their role, LPS should be removed from the extractsdifferent bacterial species encountered before on the exposing it birch pollen to cells. Assurface BPE is [35,36]. To isolate a genuine the immune solution, nature-derived response fromLPS allergens and to contamination help is of an clarify unknown their role, LPS source, adding should be removed another from the extracts level of complexity before to our study. exposing Initial LPSitcontaminations to cells. As BPEwere is a measured genuine nature‑derived to be ≈ 2.3 EU/mL. solution, Thus,LPS theycontamination were much lower is of an unknown source, adding another level of complexity to our than the spiked samples in previous experiments (the lowest tested condition was 100 study. Initial LPS contam‑ inations were measured to be ≈ 2.3 EU/mL. Thus, they were much lower than the spiked EU/mL). samples Here,inweprevious incubatedexperiments 1 mL of BPE (the lowest solution tested withcondition was 100 5 mg of either EU/mL). particles non-specific Here, we incubated 1 mL of BPE solution with 5 mg of either or anti-Lipid A particles. Due to interferences of BPE with the LAL assay, a cell-based non‑specific particles or anti‑Lipid A particles. Due to interferences of BPE with the assay was employed instead for the endotoxin quantification in the processed SN samples.LAL assay, a cell‑based assayHEK-Blue™-hTLR4 The was employed instead cells for werethe stimulated endotoxin quantification in the presence in oftheLPS, processed SN sam‑ triggering the ples. The HEK‑Blue™‑hTLR4 cells were stimulated in the presence inducible expression of secreted embryonic alkaline phosphatase (SEAP). SEAP was then of LPS, triggering the inducible expression of secreted embryonic alkaline phosphatase determined using HEK-Blue™ detection. Endotoxin removal was measurable after (SEAP). SEAP was then determined using HEK‑Blue™ detection. Endotoxin removal was measurable after incuba‑ incubation with both particle types, albeit in a lower range (ca. 20%), with no significant tion with both particle types, albeit in a lower range (ca. 20%), with no significant difference difference between the two systems (Figure 5a). Some environmental bacteria have the between the two systems (Figure 5a). Some environmental bacteria have the capability to capability to produce LPS with uncommon chemical structures, including modifications produce LPS with uncommon chemical structures, including modifications in the Lipid A in the Lipid A region [37]. Therefore, in addition to the fact that the BPE contains LPS from region [37]. Therefore, in addition to the fact that the BPE contains LPS from unknown unknown bacterial sources, which might not be recognized by our anti-Lipid A antibody, bacterial sources, which might not be recognized by our anti‑Lipid A antibody, further further non-specific interactions between BPE constituents and LPS that would, similarly non‑specific interactions between BPE constituents and LPS that would, similarly to BSA, to BSA, hinder the LPS removal cannot be excluded. hinder the LPS removal cannot be excluded. Figure 5. Comparison between anti‑Lipid A particles and commercial non‑specific particles in BPE in DPBS regarding (a) endotoxin removal and (b) protein recovery. Samples are solutions of BPE in DPBS with natural LPS contamination of unknown source. Data shown are mean ± sem, n ≥ 3, and analyzed with t‑test (ns = not significant, *** p < 0.001). However, when evaluating the protein recovery, the difference between using anti‑ Lipid A particles and non‑specific particles was striking. Anti‑Lipid A particles were able to recover up to 85% of the original proteins in the solution, whereas the non‑specific par‑ ticles only recovered around 50% (Figure 5b). A possible explanation for this observation could be the formation of a protein corona on the surface of charged non‑specific particles, which would also reduce the interaction with and, consequently, the removal of LPS. Int. J. Mol. Sci. 2023, 24, 13971 9 of 15 3. Materials and Methods 3.1. Materials Carboxy polystyrene particles of 3.00 µm (PC05003, Lot 10014) were acquired from Bangs Laboratories Inc. (Fishers, IN, USA). Phosphate‑buffered saline (PBS), succinimidyl‑ 4‑(N‑maleimidomethyl)cyclohexan‑1‑carboxylat (SMCC), aminodextran (AmDex), PBS‑5 mM EDTA (PE) and Endotoxin Removal Beads (130‑093‑657, here also referred to as ‘non‑ specific particles’) were produced by Miltenyi Biotec. Endotoxin‑free water (W50‑1000) was purchased from Lonza. LPS from E. coli O111:B4 (L2630‑25MG), insulin (I0516‑5ML), N‑Hydroxysuccinimide (NHS, 8045180025), tris (2‑carboxyethyl)phosphine hydrochloride (TCEP, 75259‑1G), N‑Ethylmaleimide (NEM, 04259‑5G), N‑(3‑Dimethylaminopropyl)‑N′ ‑ethylcarbodiimide hydrochloride (EDC, 03450‑ 5G), sodium azide (SIAL71289‑250G), dimethyl sulfoxide (DMSO, D2650‑5X10mL), HEPES (H0887‑20ML), Dulbecco’s Phosphate‑Buffered Saline (DPBS, D8537‑500ML) and Dulbe‑ cco’s Modified Eagle’s Medium—high glucose (DMEM, D5671‑500ML) were acquired from Sigma‑Aldrich. Endpoint chromogenic LAL test (LAL, A39553), polyclonal Goat anti‑Lipid A antibody (anti‑Lipid A, PA1‑73178), Pierce™ BCA Protein Assay (23227), Pierce™ BCA Protein Assay Kit—Reducing Agent Compatible (23250), Coomassie Protein Assay Reagent (1856209) and Betula pendula (batch No 012519101) were purchased from Thermo Fisher Scientific. Donkey anti‑goat‑phycoerythrin antibody (anti‑Goat‑PE, 705‑115‑147) and goat anti‑human‑phycoerythrin (anti‑Human‑PE, 109‑116‑098) were purchased from Jackson Immuno Research. Bovine serum albumin (BSA, P6154‑500GR) was purchased from BioW‑ est. Pluronic F‑68 (A1288,0100) was purchased from PanReac AppliChem. β ‑mercaptoeth‑ anol (β‑ME, 444203) was acquired from Calbiochem. Copper grid (01801) was acquired from Ted‑pella. Normocin (ant‑nr‑1), HEK‑Blue™ Selection (hb‑sel), HEK Blue™hTLR4 cells (hkb‑htlr4) and HEK Blue™detection (hb‑det2) were obtained from Invivogen. All plasticware used was sterile and either endotoxin‑free certified or tested for the experiments. The following equipment was used during this study: Synergy H1 microplate reader from Biotek (Winooski, VT, USA) using software Gen5 v3.04; Countess II FL from Invitro‑ gen (Waltham, MA, USA); NTA LM10 and its software NanoSight v3.2.16 from Malvern instruments Ltd. (Malvern, UK); MACSQuant 10 and its software MACSQuantify v2.13; and FEI Magellan 400L XHR SEM. 3.2. Methods When not described differently, washing and centrifugation steps refer to 3000× g, 5 min, RT. An amount of 0.1 ng/mL of LPS from E. coli is considered to present a potency of 1 EU/mL [6]. Therefore, 1000 ng/mL LPS from E. coli O111:B4 is considered the same as 10,000 EU/mL in this study. 3.2.1. Anti‑Lipid A Particles’ Conjugation The 10 mg carboxy PS particles were initially washed once with ethanol 70% to remove potential contamination and a second time with PBS‑0.03% Pluronic F‑68. Particles were then centrifuged, and the pellet resuspended in 0.5 mL EDC (20 mg/mL) and 0.5 mL NHS (12 mg/mL) and incubated for 15 min. The sample was then centrifuged, resuspended in an aqueous aminodextran solution (33 mg/mL) and incubated in an overhead mixer. After 2 h, particles were washed and resuspended in 1 mL PBS‑0.03% Pluronic F‑68 containing 30 µL of SMCC in DMSO and incubated for 1 h. Then, coupling of the anti‑Lipid A antibody was performed via Michael addition, as previously described [38–40]. Here, the anti‑Lipid A Ab was reduced for one hour with 1.5 mM TCEP prior to the addition to SMCC‑activated particles. After the lapse of 2 h, the reaction was stopped via addition of 50 mM β‑ME followed by 40 mM NEM. The particles were then washed 3 times and the first SN was saved for protein quantification. The final pellet was resuspended in PBS‑0.03% Pluronic F‑68 and sodium azide was added to a final concentration of 0.05%. Particles were stored at 2–8 ◦ C until further use. Int. J. Mol. Sci. 2023, 24, 13971 10 of 15 3.2.2. Anti‑Lipid A Particles’ Characterization After the conjugation, the SN containing the unconjugated Ab was quantified via reducing‑agent‑compatible BCA, using anti‑Lipid A Ab for the calibration curve. The amount of conjugated Ab was calculated by subtracting the total amount used for con‑ jugation from the amount calculated with the BCA test. Additionally, anti‑Lipid A particles were stained with secondary antibodies for 20 min in the dark and washed with PE buffer to remove the unbound secondary Ab. As the anti‑ Lipid A Ab is a goat‑raised Ab, the anti‑Goat‑PE should recognize its epitope and increase the MFI in the B2/PE channel (filter 585/40 nm) in MACSQuant 10. On the other hand, the anti‑Human‑PE should not bind to the anti‑Lipid Ab conjugated on the particles and, therefore, not show any signal in the B2/PE channel. Flow cytometry data were analyzed in MACSQuantify. To evaluate the aggregation state of the conjugates, particles were optically imaged in solution in Countess Cell Counter (dilution 1:200 in PBS‑0.03% Pluronic F‑68). Moreover, in addition to determining the percentage of single particles in flow cytometry through FSC‑A x FSC‑H [41], we additionally gated the main population of single particles on FSC‑ A x SSC‑A plots, with FSC/SSC set to log scale (Figure S4). Morphological characterization of microparticles was performed on an FEI Magellan 400L XHR SEM operating at 20 kV. Samples (10 µL) were deposited on an ultrathin formvar‑ coated 200‑mesh copper grid. More than 100 particles were analyzed from STEM images with ImageJ to obtain the particle size distribution profile. 3.2.3. LPS Removal Capacity of Anti‑Lipid A Particles To assess the ability of anti‑Lipid A particles to remove LPS, a saturation curve was generated. In short, a 1000 ng/mL ≈ 10,000 EU/mL solution of LPS from E. coli O111:B4 was prepared in endotoxin‑free water via serial dilution of 5 mg/mL to 100 µg/mL, then to 1000 ng/mL. Between each dilution step, LPS was left 15 min in an overhead mixing at RT for homogenization and subsequently vortexed for 2 min. A total of 5 mg of anti‑Lipid A particles were washed twice in water and then resuspended in 1 mL of LPS solution. The vial was incubated for 20 min using an overhead mixer. After 20 min, the sample was cen‑ trifuged, and the SN was saved. The pellet was resuspended again in 1 mL of LPS solution, and the procedure was repeated 12 times. For LPS quantification on the SN, the traditional endpoint chromogenic LAL test was employed following the manufacturer’s instructions, always using a freshly prepared calibration curve (Figure S5a) and diluting samples as nec‑ essary. All SNs were previously filtered with a 0.65 µm sterile centrifugal filter to remove leftover particles and prevent interferences with the LAL. After that, the amount of LPS calculated in the SN (Figure S5b) was compared with the initially added (10,000 EU/mL) and the endotoxin removal percentage was plotted (Equations (S1) and (S2)). Complemen‑ tarily, due to the amphiphilic nature of LPS and its tendency to form supramolecular struc‑ tures, these SNs were also characterized using NTA. The NanoSight software automatically reports the number of particles/frame for each sample. It is important to note that the set‑ tings for processing (screen gain and threshold) were adjusted for the control samples and kept constant for further measurements. The LPS removal percentage was calculated con‑ sidering the number of particles/frame in each SN and the number of particles/frame in the reference solution (Figure S8 and Equations (S2) and (S3)). Here, 0% removal is associated with SN with the number of particles/frame equal to the number of particles/frame in the 10,000 EU/mL control. 3.2.4. Longitudinal Stability To evaluate the Ab‑conjugated particles’ stability over time, anti‑Lipid A particles were stored at 2–8 ◦ C at a concentration of 20 mg/mL in PBS‑Pluronic 0.03% with 0.05% sodium azide. At different time points (i.e., days 8, 14, 25, 34, 40, 41, 60, 77, 156, 161 and 251), particles were centrifuged and incubated with LPS from E. coli O111:B4 in water. In short, a solution of 1000 ng/mL of LPS was prepared as previously described. Then, 1 mL of Int. J. Mol. Sci. 2023, 24, 13971 11 of 15 the LPS solution was added to 5 mg of anti‑Lipid A particles and incubated in an overhead mixer for 20 min at RT. The sample was then centrifuged, and the SN was collected and filtered with a 0.65 µm centrifugal filter before endotoxin quantification using LAL test. 3.2.5. LPS Removal and Protein Recovery in 1 mg/mL BSA in Water Different concentrations of LPS from E. coli O111:B4 were prepared (1000 ng/mL ≈ 10,000 EU/mL; 100 ng/mL ≈ 1000 EU/mL; 10 ng/mL ≈ 100 EU/mL). Dried BSA was weig‑ hted and used without previous LPS detoxification and resuspended in either LPS‑free water or directly using the different LPS solutions to achieve a consistent final BSA concen‑ tration of 1 mg/mL in all cases. The intrinsic LPS content in 1 mg/mL BSA in pure water was determined to be 14 EU/mL. All samples were mixed in an overhead mixer for at least 15 min at RT before usage. A total of 5 mg of anti‑Lipid A particles and 5 mg of non‑specific particles were washed twice with water and finally resuspended in 1 mL of the BSA‑LPS solutions. Vials were incubated for 20 min in an overhead mixer at RT. After 20 min, sam‑ ples were centrifuged, and the SNs were filtered through a 0.65 µm sterile centrifugal filter. After that, each SN was subjected to LAL and BCA assays to evaluate its endotoxin content and protein concentration, respectively. For the LAL, samples were diluted as necessary to ensure a concentration < 50 µg/mL of BSA in order to avoid interferences [42]. The LPS removal performance was calculated considering the initial endotoxin contamination of the sample (10,000 EU/mL, 1000 EU/mL or 100 EU/mL) and the resulting concentration determined in the acquired SN via the LAL test. The protein recovery was calculated com‑ paring the initial protein content to the final concentration after the removal process, both assessed via BCA. 3.2.6. LPS Removal and Protein Recovery in 1 mg/mL Insulin Solution in HEPES 25 mM Insulin stock solution was used without detoxification, with an intrinsic LPS concen‑ tration of less than 0.1 EU/mL at a final concentration of 1 mg/mL in HEPES buffer 25 mM, pH 7.1. Insulin was diluted with LPS from E. coli O111:B4 to achieve a final concentration of 1 mg/mL insulin containing 1000 ng/mL ≈ 10,000 EU/mL, 100 ng/mL ≈ 1000 EU/mL or 10 ng/mL ≈ 100 EU/mL in HEPES 25 mM, pH 7.1. Solutions were kept in an overhead mixer for at least 15 min at RT before usage. A total of 5 mg of anti‑Lipid A particles and 5 mg of non‑specific particles were washed twice with HEPES 25 mM pH 7.1 and resuspended in 1 mL of the insulin‑LPS solutions. Vials were incubated for 20 min in an overhead mixer at RT. After 20 min, all samples were centrifuged, and SNs were further filtered with a 0.65 µm sterile centrifugal filter. After that, SN were evaluated regarding endotoxin and protein content as described in Section 3.2.5. 3.2.7. LPS Removal and Protein Recovery in Naturally LPS‑Contaminated BPE in DPBS Birch pollen extract was prepared based on the protocol in Johnson et al. with slight modifications [43]. In total, 100 mg of Betula pendula pollen was dispersed in 1 mL of DPBS. This dispersion was incubated at 4 ◦ C in an overhead mixer for 18 h to allow for the ex‑ traction of a plethora of substances, including LPS. After that, the vial was centrifuged at 4 ◦ C, 14,000 rpm for 30 min. The SN was filtered with a 0.22 µm filter and frozen at −20 ◦ C until use. For LPS removal, a 1:10 dilution of the above‑mentioned BPE solution was prepared in DPBS. Briefly, 5 mg of anti‑Lipid A particles and 5 mg of non‑specific particles were washed twice with DPBS and resuspended with 1 mL of BPE. The vial was incubated for 20 min in an overhead mixer at RT. After 20 min, samples were centrifuged, and the SN was further filtered with a 0.65 µm sterile centrifugal filter to remove potential remaining particles. Due to interferences of BPE with the LAL assay, a cell‑based assay was employed for the endotoxin quantification in the obtained SN. HEK‑Blue™‑hTLR4 cells were used for endotoxin quantification in BPE, based on a protocol provided by Johnson et al. [43] with slight modifications. The cells were cultured in DMEM supplemented with penicillin‑ streptomycin, glutamine, normocin, inactivated FBS and HEK‑Blue™ Selection. Upon Int. J. Mol. Sci. 2023, 24, 13971 12 of 15 stimulation of cells with LPS, an inducible expression of SEAP was triggered and deter‑ mined using HEK‑Blue™ detection. In short, 20 µL of blank (water), negative control (DPBS), positive control (10 EU/mL), standards (calibration curve), sample control (BPE) and the SN after the removal process were added to a 96‑well plate and incubated for 10 min at 37 ◦ C, 5% CO2 . When that time elapsed, 180 µL of 2.5 × 105 cells/mL in sup‑ plemented DMEM without HEK‑Blue™ Selection were added to each well. The plate was then incubated for 22 h at 37 ◦ C, 5% CO2 . On the following day, 20 µL of the SN were transferred to a clean 96‑well plate and 180 µL of HEK Blue™ detection was added to each well. The plate was further incubated for 6 h at 37 ◦ C, 5% CO2 . After that time, the ab‑ sorbance at 650 nm was measured in a plate reader. The endotoxin quantified on samples was compared with the initial endotoxin content and the removal percentage was calcu‑ lated (Equations (S2) and (S4)). In a similar manner, BPE interferes in the BCA assay for protein quantification; therefore, the Bradford assay was applied for this purpose. Here, 40 µL of blank (water), negative control (DPBS), standards (calibration curve), sample con‑ trol (BPE) and the SN after the removal process were added to a 96‑well plate and 160 µL of Coomassie was added to each well. The absorbance at 595 nm was measured and the protein concentrations were calculated based on a BSA calibration curve. The protein re‑ covery was calculated comparing the initial protein content to the final concentration after the removal process, both assessed via Bradford assay. 4. Conclusions Endotoxin removal from sensible samples is a challenging process due to the wide range of possible interactions that these amphiphilic molecules can have with sample con‑ stituents. Non‑specific endotoxin removal methods can be quite effective, resulting in sam‑ ples with acceptable endotoxin contents. However, the loss of non‑target active compo‑ nents can be a limitation for some applications. In this work, we showed how the conjugation of polystyrene microparticles to a poly‑ clonal anti‑Lipid A antibody can help to avoid the non‑specific depletion of other active molecules while achieving a similarly high LPS removal in comparison with non‑specific removal particles. This was tested by measuring the endotoxin removal and protein re‑ covery performance over a series of solutions with increasing matrix complexity from water < BSA in water < insulin in HEPES 25 mM < BPE in DPBS, where the latter was a naturally LPS‑contaminated sample. In addition to the well‑established LAL assay, we, to the best of our knowledge, were the first to use NTA as an alternative method to charac‑ terize LPS, with results corresponding to those obtained with the LAL assay. It should be noted that in our study NTA was suitable for the detection of LPS supramolecular struc‑ tures only if no other light‑scattering molecules were present (i.e., LPS formulations in pure water). The potential use of NTA in complex LPS samples is still to be explored and developed. Overall, we observed that, independent of the buffer and sample con‑ stituents, anti‑Lipid A particles enhanced the protein recovery for all conditions compared with non‑specific particles. In fact, the advantage of the antibody–antigen interactions was most prominent in samples containing BPE, where the protein recovery was 35% higher for anti‑Lipid A particles. On the other hand, while one would assume that high LPS con‑ centrations are the weak point of particle‑based removal systems given their surface satura‑ tion, we observed that LPS removal was challenging at LPS concentrations ≤ 100 EU/mL, where LPS interactions and competition on the binding sites by other molecules play a crucial role [14]. Overall, we present a new system that allows for efficient and specific LPS removal within 20 min, while maintaining protein recoveries > 85%, even in complex solutions such as BPE. Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms241813971/s1. Int. J. Mol. Sci. 2023, 24, 13971 13 of 15 Author Contributions: Conceptualization, C.C.M., M.H., I.G. and J.A.F.; Data curation, C.C.M.; For‑ mal analysis, C.C.M., A.C.F., N.G.B., I.G. and J.A.F.; Funding acquisition, N.G.B., C.S., V.P., A.D. and J.A.F.; Investigation, C.C.M. and A.C.F.; Methodology, C.C.M., M.H., I.G. and J.A.F.; Project admin‑ istration, I.G. and J.A.F.; Resources, M.H., N.G.B., V.P. and J.A.F.; Supervision, A.D., I.G. and J.A.F.; Validation, C.C.M. and J.A.F.; Visualization, C.C.M., I.G. and J.A.F.; Writing—original draft, C.C.M., I.G. and J.A.F.; Writing—review and editing, C.C.M., A.C.F., M.H., N.G.B., L.S., C.S., V.P., A.D., I.G. and J.A.F. All authors have read and agreed to the published version of the manuscript. Funding: The project leading to this application has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska‑Curie grant agree‑ ment No 812661, H2020‑MSCA‑ITN‑2018‑812661 (ENDONANO). Institutional Review Board Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: We thank Litty Johnson, Benjamin Punz, Christian Langer, Nadine Borgelt and Ramona Siemer for sharing the protocols that were used as starting points for this study. We thank Paola Italiani for coordinating the ENDONANO project. Figures 1a and 2a were created with BioRen‑ der.com accessed on 19 August 2023. Conflicts of Interest: C.C.M., A.C.F., L.S., C.S., I.G. and J.A.F. are employees of Miltenyi Biotec B.V. & Co. KG. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References 1. Silhavy, T.J.; Kahne, D.; Walker, S. The Bacterial Cell Envelope. Cold Spring Harb. Perspect. Biol. 2010, 2, a000414. [CrossRef] 2. Stromberg, L.R.; Mendez, H.M.; Mukundan, H. Detection Methods for Lipopolysaccharides: Past and Present. In Escherichia coli—Recent Advances on Physiology, Pathogenesis and Biotechnological Applications; Samie, A., Ed.; IntechOpen: London, UK, 2017. [CrossRef] 3. Sampath, V. Bacterial endotoxin‑lipopolysaccharide; structure, function and its role in immunity in vertebrates and invertebrates. Agric. Nat. Resour. 2018, 52, 115–120. [CrossRef] 4. Williams, K.L. Historical and Emerging Themes. In Endotoxins: Pyrogens, LAL Testing and Depyrogenation; Williams, K.L., Ed.; Informa Healthcare Inc.: Richmond, VA, USA, 2007. 5. Fux, A.C.; Melo, C.C.; Michelini, S.; Swartzwelter, B.J.; Neusch, A.; Italiani, P.; Himly, M. Heterogeneity of Lipopolysaccharide as Source of Variability in Bioassays and LPS‑Binding Proteins as Remedy. Int. J. Mol. Sci. 2023, 24, 8395. [CrossRef] [PubMed] 6. Schneier, M.; Razdan, S.; Miller, A.M.; Briceno, M.E.; Barua, S. Current technologies to endotoxin detection and removal for biopharmaceutical purification. Biotechnol. Bioeng. 2020, 117, 2588–2609. [CrossRef] 7. U.S. Food & Drug Administration. Bacterial Endotoxins/Pyrogens. Available online: https://www.fda.gov/inspections‑comp liance‑enforcement‑and‑criminal‑investigations/inspection‑technical‑guides/bacterial‑endotoxinspyrogens (accessed on 17 De‑ cember 2020). 8. Dawson, M. Endotoxin Limits for Parenteral Drug Products; BET White Paper; Associates of Cape Cod, Inc.: East Falmouth, MA, USA, 2017; Volume 1. 9. Sandle, T. A Comparative Study of Different Methods for Endotoxin Destruction. Am. Pharm. Rev. 2013, 16, 15–17. 10. Bononi, I.; Balatti, V.; Gaeta, S.; Tognon, M. Gram‑Negative Bacterial Lipopolysaccharide Retention by a Positively Charged New‑Generation Filter. Appl. Environ. Microbiol. 2008, 74, 6470–6472. [CrossRef] 11. Sandle, T. A Practical Approach to Depyrogenation Studies Using Bacterial Endotoxin. J. GXP Compliance 2011, 15, 90–96. 12. de Oliveira Magalhães, P.O.; Lopes, A.M.; Mazzola, P.G.; Rangel‑Yagui, C.; Penna, T.C.V.; Pessoa Júnior, A. Methods of endotoxin removal from biological preparations: A review. J. Pharm. Pharm. Sci. 2007, 10, 388–404. 13. Razdan, S.; Wang, J.‑C.; Barua, S. PolyBall: A new adsorbent for the efficient removal of endotoxin from biopharmaceuticals. Sci. Rep. 2019, 9, 8867. [CrossRef] 14. Anspach, F.B.; Petsch, D. Membrane adsorbers for selective endotoxin removal from protein solutions. Process. Biochem. 2000, 35, 1005–1012. [CrossRef] 15. Vagenende, V.; Ching, T.‑J.; Chua, R.‑J.; Thirumoorthi, N.; Gagnon, P. Amide‑Mediated Hydrogen Bonding at Organic Crys‑ tal/Water Interfaces Enables Selective Endotoxin Binding with Picomolar Affinity. ACS Appl. Mater. Interfaces 2013, 5, 4472–4478. [CrossRef] [PubMed] Int. J. Mol. Sci. 2023, 24, 13971 14 of 15 16. Li, J.; Shang, G.; You, M.; Peng, S.; Wang, Z.; Wu, H.; Chen, G.‑Q. Endotoxin Removing Method Based on Lipopolysaccharide Binding Protein and Polyhydroxyalkanoate Binding Protein PhaP. Biomacromolecules 2011, 12, 602–608. [CrossRef] 17. Chen, J.; Shi, Z.; Yang, X.; Zhang, X.; Wang, D.; Qian, S.; Sun, W.; Wang, C.; Li, Q.; Wang, Z.; et al. Broad‑Spectrum Clearance of Lipopolysaccharides from Blood Based on a Hemocompatible Dihistidine Polymer. ACS Appl. Mater. Interfaces 2023, 15, 32251–32261. [CrossRef] 18. Shi, Z.; Zhang, X.; Yang, X.; Zhang, X.; Ma, F.; Gan, H.; Chen, J.; Wang, D.; Sun, W.; Wang, J.; et al. Specific Clearance of Lipopolysaccharide from Blood Based on Peptide Bottlebrush Polymer for Sepsis Therapy. Adv. Mater. 2023, 35, 2302560. [CrossRef] 19. Ribeiro, L.N.d.M.; Couto, V.M.; Fraceto, L.F.; de Paula, E. Use of nanoparticle concentration as a tool to understand the structural properties of colloids. Sci. Rep. 2018, 8, 982. [CrossRef] 20. Filipe, V.; Hawe, A.; Jiskoot, W. Critical Evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the Measurement of Nanoparticles and Protein Aggregates. Pharm. Res. 2010, 27, 796–810. [CrossRef] 21. Comfort, N.; Cai, K.; Bloomquist, T.R.; Strait, M.D.; Ferrante, A.W.J.; Baccarelli, A.A. Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles. JoVE J. Vis. Exp. 2021, 28, e62447. 22. Williams, K.L. Endotoxin Relevance and Control Overview. In Endotoxins: Pyrogens, LAL Testing and Depyrogenation; Informa Healthcare Inc.: Richmond, VA, USA, 2007. 23. Kundu, N.; Banik, D.; Sarkar, N. Self‑Assembly of Amphiphiles into Vesicles and Fibrils: Investigation of Structure and Dynamics Using Spectroscopy and Microscopy Techniques. Langmuir 2018, 34, 11637–11654. [CrossRef] 24. Parikh, S.J.; Chorover, J. Infrared spectroscopy studies of cation effects on lipopolysaccharides in aqueous solution. Colloids Surf. B Biointerfaces 2007, 55, 241–250. [CrossRef] 25. Santos, N.C.; Silva, A.C.; Castanho, M.A.R.B.; Martins‑Silva, J.; Saldanha, C. Evaluation of Lipopolysaccharide Aggregation by Light Scattering Spectroscopy. ChemBioChem 2003, 4, 96–100. [CrossRef] [PubMed] 26. Miltenyi Biotec B.V. & Co. KG. Endotoxin Removal Beads. Available online: https://static.miltenyibiotec.com/asset/1506554056 41/document_505pfa7o0l78p0kmisccvm4f29?content‑disposition=inline (accessed on 26 January 2022). 27. Ionova, Y.; Wilson, L. Biologic excipients: Importance of clinical awareness of inactive ingredients. PLoS ONE 2020, 15, e0235076. [CrossRef] [PubMed] 28. ThermoFisher Scientific. Bovine Serum Albumin (BSA Protein). Available online: https://www.thermofisher.com/be/en/home/ life‑science/cell‑culture/mammalian‑cell‑culture/media‑supplements/bovine‑serum‑albumin.html (accessed on 17 May 2023). 29. Esparza, G.A.; Teghanemt, A.; Zhang, D.; Gioannini, T.L.; Weiss, J.P. Endotoxin·albumin complexes transfer endotoxin monomers to MD‑2 resulting in activation of TLR4. Innate Immun. 2012, 18, 478–491. [CrossRef] 30. Landgraf, W.; Sandow, J. Recombinant Human Insulins—Clinical Efficacy and Safety in Diabetes Therapy. Eur. Endocrinol. 2016, 12, 12–17. [CrossRef] 31. Gupta, V.; Sengupta, M.; Prakash, J.; Tripathy, B.C. Production of Recombinant Pharmaceutical Proteins. Basic Appl. Asp. Biotech‑ nol. 2016, 77–101. [CrossRef] 32. Donner, T.; Sarkar, S. Insulin—Pharmacology, Therapeutic Regimens, and Principles of Intensive Insulin Therapy; MDText.com, Inc.: South Dartmouth, MA, USA, 2000. 33. Biedermann, T.; Winther, L.; Till, S.J.; Panzner, P.; Knulst, A.; Valovirta, E. Birch pollen allergy in Europe. Allergy 2019, 74, 1237–1248. [CrossRef] 34. Pablos, I.; Wildner, S.; Asam, C.; Wallner, M.; Gadermaier, G. Pollen Allergens for Molecular Diagnosis. Curr. Allergy Asthma Rep. 2016, 16, 31. [CrossRef] [PubMed] 35. Pointner, L.; Bethanis, A.; Thaler, M.; Traidl‑Hoffmann, C.; Gilles, S.; Ferreira, F.; Aglas, L. Initiating Pollen Sensitization— Complex Source, Complex Mechanisms. Clin. Transl. Allergy 2020, 10, 36. [CrossRef] 36. Manirajan, B.A.; Hinrichs, A.‑K.; Ratering, S.; Rusch, V.; Schwiertz, A.; Geissler‑Plaum, R.; Eichner, G.; Cardinale, M.; Kuntz, S.; Schnell, S. Bacterial Species Associated with Highly Allergenic Plant Pollen Yield a High Level of Endotoxins and Induce Chemokine and Cytokine Release from Human A549 Cells. Inflammation 2022, 45, 2186–2201. [CrossRef] 37. Di Lorenzo, F.; Duda, K.A.; Lanzetta, R.; Silipo, A.; De Castro, C.; Molinaro, A. A Journey from Structure to Function of Bacterial Lipopolysaccharides. Chem. Rev. 2021, 122, 15767–15821. [CrossRef] [PubMed] 38. Jokerst, J.V.; Floriano, P.N.; Christodoulides, N.; Simmons, G.W.; McDevitt, J.T. Integration of semiconductor quantum dots into nano‑bio‑chip systems for enumeration of CD4+ T cell counts at the point‑of‑need. Lab A Chip 2008, 8, 2079–2090. [CrossRef] 39. Tiwari, D.K.; Tanaka, S.‑I.; Inouye, Y.; Yoshizawa, K.; Watanabe, T.M.; Jin, T. Synthesis and Characterization of Anti‑HER2 Antibody Conjugated CdSe/CdZnS Quantum Dots for Fluorescence Imaging of Breast Cancer Cells. Sensors 2009, 9, 9332–9354. [CrossRef] 40. Woythe, L.; Tholen, M.M.E.; Rosier, B.J.H.M.; Albertazzi, L. Single‑Particle Functionality Imaging of Antibody‑Conjugated Nanoparticles in Complex Media. ACS Appl. Bio Mater. 2023, 6, 171–181. [CrossRef] [PubMed] 41. Bio‑Rad Doublet Discrimination . Available online: https://www.bio‑rad‑antibodies.com/flow‑cytometry‑doublet‑discriminati on.html (accessed on 3 July 2023). Int. J. Mol. Sci. 2023, 24, 13971 15 of 15 42. Petsch, D.; Deckwer, W.‑D.; Anspach, F. Proteinase K Digestion of Proteins Improves Detection of Bacterial Endotoxins by the Limulus Amebocyte Lysate Assay: Application for Endotoxin Removal from Cationic Proteins. Anal. Biochem. 1998, 259, 42–47. [CrossRef] [PubMed] 43. Johnson, L.; Aglas, L.; Punz, B.; Dang, H.‑H.; Christ, C.; Pointner, L.; Wenger, M.; Hofstaetter, N.; Hofer, S.; Geppert, M.; et al. Mechanistic insights into silica nanoparticle–allergen interactions on antigen presenting cell function in the context of allergic reactions. Nanoscale 2023, 15, 2262–2275. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual au‑ thor(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.