ee20d87156d3502de9350eb9b404e433 pmc_10531372.pdf 8d76eede0829cb02cc2baf81e586c28148fd1dab pmc_10531372.pdf 5833c0c0bc14ec742e9f8daf9c4e88856d294b4ce0aa61c222615a4b849570d5 pmc_10531372.pdf Title: Recovering What Matters: High Protein Recovery after Endotoxin Removal from LPS-Contaminated Formulations Using Novel Anti-Lipid A Antibody Microparticle Conjugates Subject: 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 symptoms. Therefore, regulatory limits for endotoxin content apply to injectable pharmaceuticals, and excess LPS must be removed before commercialization. The majority of available endotoxin removal systems are based on the non-specific adsorption of LPS to charged and/or hydrophobic surfaces. Albeit effective to remove endotoxins, the lack of specificity can result in the unwanted loss of essential proteins from the pharmaceutical formulation. In this work, we developed microparticles conjugated to anti-Lipid A antibodies for selective endotoxin removal. Anti-Lipid A particles were characterized using flow cytometry and microscopy techniques. These particles exhibited a depletion capacity > 6 103 endotoxin units/mg particles from water, as determined with two independent methods (Limulus Amebocyte Lysate test and nanoparticle tracking analysis). Additionally, we compared these particles with a non-specific endotoxin removal system in a series of formulations of increasing complexity: bovine serum albumin in water < insulin in buffer < birch pollen extracts. We demonstrated that the specific anti-Lipid A particles show a higher protein recovery without compromising their endotoxin removal capacity. Consequently, we believe that the specificity layer integrated by the anti-Lipid A antibody could be advantageous to enhance product yield. Keywords: LPS contamination; polystyrene particles; bioconjugation; supramolecular structures; LAL; NTA Author: Cristiane Casonato Melo, Alexandra C. Fux, Martin Himly, Neus G. Bastús, Laura Schlahsa, Christiane Siewert, Victor Puntes, Albert Duschl, Isabel Gessner and Jonathan A. Fauerbach Creator: LaTeX with hyperref Producer: xdvipdfmx (20200315) CreationDate: Tue Sep 12 06:22:01 2023 CEST ModDate: Tue Sep 12 06:25:35 2023 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 15 Encrypted: no Page size: 595.28 x 841.89 pts (A4) Page rot: 0 File size: 2299178 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- UWWHSC+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 9 0 TZDFGO+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 16 0 YWSPUO+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 23 0 MRLLGP+VNR8 Type 1C Builtin yes yes yes 30 0 RQRWVU+CMSY10 Type 1C Builtin yes yes yes 35 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 56 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 59 0 HHATMK+EURM10 Type 1C Builtin yes yes yes 85 0 PMCBCD+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 101 0 PMCBCE+PalatinoLinotype CID TrueType Identity-H yes yes yes 104 0 PMCBCF+PalatinoLinotype TrueType WinAnsi yes yes no 110 0 PMCBCH+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 113 0 PMCBNE+PalatinoLinotype TrueType MacRoman yes yes no 116 0 PMCBCD+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 130 0 PMCBCE+PalatinoLinotype CID TrueType Identity-H yes yes yes 133 0 PMCBCF+PalatinoLinotype TrueType WinAnsi yes yes no 139 0 PMCBCH+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 142 0 PMCBNE+PalatinoLinotype TrueType MacRoman yes yes no 145 0 PMCBCD+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 161 0 PMCBCE+PalatinoLinotype CID TrueType Identity-H yes yes yes 164 0 PMCBCF+PalatinoLinotype TrueType WinAnsi yes yes no 170 0 PMCBCH+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 173 0 PMCBNE+PalatinoLinotype TrueType MacRoman yes yes no 176 0 PMCBCD+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 189 0 PMCBCE+PalatinoLinotype CID TrueType Identity-H yes yes yes 192 0 PMCBCF+PalatinoLinotype TrueType WinAnsi yes yes no 198 0 PMCBCH+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 201 0 PMCBNE+PalatinoLinotype TrueType MacRoman yes yes no 204 0 PMCBCD+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 217 0 PMCBCE+PalatinoLinotype CID TrueType Identity-H yes yes yes 220 0 PMCBCF+PalatinoLinotype TrueType WinAnsi yes yes no 226 0 PMCBCH+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 229 0 PMCBNE+PalatinoLinotype TrueType MacRoman yes yes no 232 0 OBNUUE+VNR9 Type 1C Builtin yes yes yes 252 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-02-02 02:32:52 CET RepresentationInformation: pmc_10531372.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-01-10 02:27:49 CET Size: 2299178 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 504 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: 0 Item: Title: Results and Discussion Destination: 1 Children: Item: Title: Anti-Lipid A Particles: Conjugation and Characterization Destination: 2 Item: Title: Anti-Lipid A Particles’ Functionality and Stability Destination: 3 Children: Item: Title: LPS Removal Capacity of Anti-Lipid A Particles Destination: 4 Item: Title: Longitudinal Stability Destination: 5 Item: Title: LPS Removal and Protein Recovery Destination: 6 Children: Item: Title: LPS Removal and Protein Recovery in 1 mg/mL BSA in Water Destination: 7 Item: Title: LPS Removal and Protein Recovery in 1 mg/mL Insulin in HEPES 25 mM Destination: 8 Item: Title: LPS Removal and Protein Recovery in Naturally LPS-Contaminated BPE in DPBS Destination: 9 Item: Title: Materials and Methods Destination: a Children: Item: Title: Materials Destination: b Item: Title: Methods Destination: c Children: Item: Title: Anti-Lipid A Particles’ Conjugation Destination: d Item: Title: Anti-Lipid A Particles’ Characterization Destination: e Item: Title: LPS Removal Capacity of Anti-Lipid A Particles Destination: f Item: Title: Longitudinal Stability Destination: 10 Item: Title: LPS Removal and Protein Recovery in 1 mg/mL BSA in Water Destination: 11 Item: Title: LPS Removal and Protein Recovery in 1 mg/mL Insulin Solution in HEPES 25 mM Destination: 12 Item: Title: LPS Removal and Protein Recovery in Naturally LPS-Contaminated BPE in DPBS Destination: 13 Item: Title: Conclusions Destination: 14 Item: Title: References Destination: 15 Info: Title: Recovering What Matters: High Protein Recovery after Endotoxin Removal from LPS-Contaminated Formulations Using Novel Anti-Lipid A Antibody Microparticle Conjugates Author: Cristiane Casonato Melo, Alexandra C. Fux, Martin Himly, Neus G. Bastús, Laura Schlahsa, Christiane Siewert, Victor Puntes, Albert Duschl, Isabel Gessner and Jonathan A. Fauerbach Subject: 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 symptoms. Therefore, regulatory limits for endotoxin content apply to injectable pharmaceuticals, and excess LPS must be removed before commercialization. The majority of available endotoxin removal systems are based on the non-specific adsorption of LPS to charged and/or hydrophobic surfaces. Albeit effective to remove endotoxins, the lack of specificity can result in the unwanted loss of essential proteins from the pharmaceutical formulation. In this work, we developed microparticles conjugated to anti-Lipid A antibodies for selective endotoxin removal. Anti-Lipid A particles were characterized using flow cytometry and microscopy techniques. These particles exhibited a depletion capacity > 6 103 endotoxin units/mg particles from water, as determined with two independent methods (Limulus Amebocyte Lysate test and nanoparticle tracking analysis). Additionally, we compared these particles with a non-specific endotoxin removal system in a series of formulations of increasing complexity: bovine serum albumin in water < insulin in buffer < birch pollen extracts. We demonstrated that the specific anti-Lipid A particles show a higher protein recovery without compromising their endotoxin removal capacity. Consequently, we believe that the specificity layer integrated by the anti-Lipid A antibody could be advantageous to enhance product yield. 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