Browsing by Author "Patrick V. Gurgel, Committee Member"

Filter results by typing the first few letters
Now showing 1 - 2 of 2
  • No Thumbnail Available
    Fc-binding Hexamer Peptide Ligands for Immunoglobulin Purification
    (2008-08-18) Yang, Haiou; Carla Mattos, Committee Member; Patrick V. Gurgel, Committee Member; Jason M. Haugh, Committee Member; Ruben G. Carbonell, Committee Chair; Peter K. Kilpatrick, Committee Member
    Antibodies and their fragments have found a wide array of applications as pharmaceutical compounds, in addition to their common usage in the purification and localization of proteins. Antibody-based therapeutics accounts for roughly 20% of the therapeutic products in development in the USA, with the majority being of the immunoglobulin G (IgG) isotype. For IgG purification, affinity chromatography has been greatly used where Staphylococcus aureus Protein A and Streptococcus Protein G are the most common affinity ligands for IgG. However, the drawbacks associated with these two proteins have given rise to the searching for alternative affinity ligands for antibody purification. We therefore set our research goal as to find a small peptide that can bind hIgG through its Fc portion and can be used in purification of antibodies and Fc-fusion proteins. Small peptides are interesting for their advantages of being more stable, less immunogenic, less expensive and milder in elution than protein ligands. Peptides were searched by a radiolabeled-screening technique in a combinatorial linear hexamer peptide library built on solid phase Toyopearl AF-Amino resins. The screening identified a family of ligands with homogenous composition of His + aromatic group + positively charge group having the ability to match Protein A in binding human IgG (hIgG) through its Fc portion. The selectivity to the Fc portion is comparable to Protein A. The HWRGWV ligand of the Fc-binding peptide family has been investigated in many aspects and exhibits some interesting tributes. It has broad binding spectrum. It can bind all subclasses of hIgG, human IgD, IgE, IgM, and to a less extent human secretory IgA. It also displays the ability to retain chicken and several mammalian IgGs. Deglycosylation of hIgG has no influence on its binding to the HWRGWV ligand and the ligand does not compete with Protein A or Protein G in binding hIgG. It is suggested by the mass spectrometry data that HWRGWV binds to the pFc portion of hIgG and interacts with the amino acids SNGQPEN in the loop Ser383 — Asn389 by specific interactions. The selectivity of the HWRGWV resin to Fc over Fab is affected by its peptide density which also influences the equilibrium constant for hIgG. Increasing density increases both the association constant which is in the order of 105 M-1 and the binding of the Fab fragment. A ligand density of around 0.1 meq—g was determined to have both the specificity and appropriate affinity to Fc. HWRGWV demonstrates the ability to purify IgG. It can isolate hIgG from mammalian cell culture media containing 10% fetal calf serum (cMEM) with more than 95% of both purity and yield. The ligand was also used to isolate hIgG from Cohn II+III paste and an yield of 82% and purity of 73% were obtained in one step. The bound IgG can be recovered using phosphate buffer at pH 4 and its binding capacity for hIgG is 130 mg⁄g-dry-resin. Acetylation of the N-terminal amine does not pose any influence on either the static binding or the dynamic isolation of hIgG. Temperature has no significant influence on hIgG isolation from cMEM. Increasing peptide density improve the yield but with a compensation of the purity to a similar degree. Feed hIgG concentration in the range of 0.5 — 10 mg⁄mL affects the recovery yield where the yield is favored with higher IgG concentration. The separation of hIgG from cMEM by HWRGWV is comparable to Protein A at an initial hIgG concentration of 10 mg⁄mL and to A2P agarose gel at both 10 and 0.5 mg⁄mL, but using a milder pH 4 elution condition. HWRGWV was immobilized on Sepharose CL-4B. HWRGWV-modified Sepharose CL-4B can isolate hIgG from cMEM with similar purity to and a lower yield than on Toyopearl AF-Amino under the conditions optimized on the latter matrix. Our experiments also show that 2% AcOH is not strong enough to completely remove the bound proteins on HWRGWV. A better column regeneration and sanitation procedure is needed for longer column lifetime. This work demonstrates the possibility to use a peptide as short as six amino acids to mimic Protein A in IgG isolation by binding through the Fc portion.
  • No Thumbnail Available
    Hybrid Particle-Nonwoven Membrane Materials for Bioseparations
    (2009-07-07) Herigstad, Matthew Omon; Ruben G. Carbonell, Committee Chair; George W. Roberts, Committee Member; Jason M. Haugh, Committee Member; Behnam Pourdeyhimi, Committee Member; Patrick V. Gurgel, Committee Member
    Adsorption separations performed in feed streams containing large particulates pose interesting problems, the solution of which would aid in many fields of bioseapartions. Production of biologically derived protein products is one of the most rapidly expanding sectors in the global economy. The capture and purification of these products has, of late, become the bottleneck of the industry and can account for approximately 50-80% of the production costs. The biopharmaceutical industry has begun to focus on improving overall economics by merging two or more separation schemes into one. The majority of the emphasis has been on combining the initial protein capture and host cell clearance steps; however, many of the currently available methods have shown little efficacy at large-scale. Additionally, interest in the clearance of pathogenic activity, most importantly infectious prions, from blood and blood derived products has grown over the past decade with the increased threat of blood-transfusion of variant Creutzfeldt-Jakob disease. This work characterizes the transport and binding properties of a novel hybrid particle-nonwoven membrane medium in which a polymeric chromatographic resin is entrapped between layers of a nonwoven polypropylene membrane (a particle-impregnated membrane or PIM). This membrane-supported resin construct offers the advantage of increased interstitial pore diameter to allow passage of cells and other debris in the feed, while providing sufficiently high surface area for product capture within the resin particles. Columns packed with stacked disks of PIM displayed excellent flow distribution, and had an interstitial porosity of εb = 0.48 ± 0.01, a 25-60% increase over those typically observed in a packed bed. These columns were able to pass over 95% of E. coli cells and human red blood cell concentrate (RBCC) in 30 column volumes, while maintaining a pressure drop significantly lower than that of a packed bed. The dynamic binding capacity of the chromatographic resin entrapped in the PIM packed column for bovine serum albumin (BSA) was essentially the same as that observed with the same volume of resin in a packed bed. Additionally, the binding of prion was characterized to PIM constructs containing an affinity ligand for PrPSc, in saline, RBCC, and human IgG solutions. The General Rate (GR) model of chromatography was used to analyze experiments indicating that the breakthrough and elution behaviors of the PIM column are predictable, and very similar to those of a normal packed bed. These results indicate that PIM constructs can be designed to process viscous mobile phases containing particulates while retaining the desirable binding characteristics of the embedded chromatographic resin. The PIM systems could find uses in adsorption separation processes from complex feed streams such as whole blood, cell culture, and food processing and could offer a process alternative to expanded beds.