skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: IgG-BSA separation and purification by internally staged ultrafiltration
Purification of IgG from residual host cell proteins (HCPs) in post-Protein A chromatography is important since some HCPs bind with Protein A and elute with the monoclonal antibody (mAb); removal of HCPs from CHO cell lines is essential. To that end, an advanced separation and purification technique in biopharmaceutical manufacturing, namely, internally staged ultrafiltration (ISUF), is investigated here. Choosing BSA as a model for HCPs in post-protein A eluate, separation of a binary mixture of IgG and BSA containing 1.0 mg/ml IgG and 0.1 mg/ml BSA is successfully demonstrated here using a modified ISUF technique: two Omega 100 kDa membranes on top followed by one Omega 70 kDa membrane at the bottom. This modified configuration demonstrated exceptional performance with almost complete rejection, 99 % purity, and 99.5 % retention of IgG, along with 96.5 % recovery of BSA over 10 diavolumes. This modified membrane stacking resulted from strategic considerations of membrane stacking and careful selection of molecular weight cutoffs and materials, and performance analysis of different membranes and stacking configurations using rejection behaviors, purity levels, and recovery rates under varying diavolume and pressure differential. The approach adopted here enhances flexibility in membrane choices in ISUF and provides valuable insights for optimizing membrane-based biopharmaceutical separation techniques.  more » « less
Award ID(s):
2310866
PAR ID:
10574556
Author(s) / Creator(s):
; ; ;
Publisher / Repository:
Elsevier
Date Published:
Journal Name:
Separation and Purification Technology
Volume:
354
Issue:
P7
ISSN:
1383-5866
Page Range / eLocation ID:
129245
Subject(s) / Keyword(s):
Modified internally staged ultrafiltration High recovery and purification of IgG from HCPs BSA as model HCP 100 kDa and 70kDa Omega ultrafiltration membrane Enhancement of high-performance tangential flow filtration
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; (, Journal of Physics: Energy)
    Abstract Membranes serve as important components for modern manufacturing and purification processes but are conventionally associated with excessive solvent usage. Here, for the first time, a procedure for fabricating large area polysulfone membranes is demonstrated via the combination of direct ink writing (DIW) with non-solvent induced phase inversion (NIPS). The superior control and precision of this process allows for complete utilization of the polymer dope solution during membrane fabrication, thus enabling a significant reduction in material usage. Compared to doctor blade fabrication, a 63% reduction in dope solution volume was achieved using the DIW technique for fabricating similarly sized membranes. Cross flow filtration analysis revealed that, independent of the manufacturing method (DIWvs.doctor blade), the membranes exhibited near identical separation properties. The separation properties were assessed in terms of bovine serum albumin (BSA) rejection and permeances (pressure normalized flux) of pure water and BSA solution. This new manufacturing strategy allows for the reduction of material and solvent usage while providing a large toolkit of tunable parameters which can aid in advancing membrane technology. 
    more » « less
  2. ; ; (, Current applications of cell culture engineering)
    Lee, G.M.; Kildegaard, H. Faustrup; Lee, S.Y. (Ed.)
    Host cell protein (HCP) impurities, endogenous proteins expressed from host cells, can challenge biopharmaceutical manufacturing. Certain HCPs can persist even after downstream purification, leading to adverse impacts on drug stability and potentially, patient safety. Thus, the quantification and control of HCPs is critical. Although many improvements have been made in HCP quantification and control methods, HCP-associated risks cannot be completely eliminated. A better biophysical understanding of Chinese hamster ovary (CHO) HCPs and advancement of monitoring assays will lead to better controlled biopharmaceutical manufacturing. This chapter will discuss (i) current HCP removal processes for various product types, (ii) the impact of residual HCPs on drug efficacy and safety, (iii) HCP quantification and monitoring methods such as proteomics approaches and enzyme-linked immunosorbent assays (ELISA) using anti-HCP antiserum, (iv) HCP control approaches in both upstream and downstream processes, and (v) future directions for effective HCP risk management strategies. 
    more » « less
  3. ; ; ; ; ; ; ; ; (, Polymers)
    null (Ed.)
    In the textile industry, a high-efficiency dye removal and low-retention of salt is demanded for recycling wastewater. In this study, polyvinylidene fluoride (PVDF) ultrafiltration membrane was transformed to a negatively charged loose nanofiltration (NF) membrane through UV-grafting of acrylic acid. At the optimal exposure of PVDF membrane in UV light for 5 min, the membrane had a high dye recovery above 99% (Congo red and Eriochrome® Black T) and a low sodium chloride (NaCl) rejection of less than 15% along with pure water flux of 26 L∙m−2∙h−1∙bar−1. Its antifouling and oleophobicity surface properties were verified using fluorescent- bovine serum albumin (BSA) and underwater mineral oil contact angle, respectively. According to the fluorescent microscopic images, the modified membrane had ten times lower adhesion of protein on the surface than the unmodified membrane. The underwater oil contact angle was raised from 110° to 155°. Moreover, the salt rejection followed this sequence: Na2SO4 > MgSO4 > NaCl > MgCl2, which agreed with the typical negatively charged NF membrane. In addition, the physicochemical characterization of membranes was further investigated to understand and link to the membrane performance, such as surface functional group, surface elements analysis, surface roughness/morphology, and surface hydrophilicity. 
    more » « less
  4. ; ; ; ; (, International Journal of Molecular Sciences)
    null (Ed.)
    While antibodies remain established therapeutic and diagnostic tools, other protein scaffolds are emerging as effective and safer alternatives. Affibodies in particular are a new class of small proteins marketed as bio-analytic reagents. They feature tailorable binding affinity, low immunogenicity, high tissue permeation, and high expression titer in bacterial hosts. This work presents the development of affibody-binding peptides to be utilized as ligands for their purification from bacterial lysates. Affibody-binding candidates were identified by screening a peptide library simultaneously against two model affibodies (anti-immunoglobulin G (IgG) and anti-albumin) with the aim of selecting peptides targeting the conserved domain of affibodies. An ensemble of homologous sequences identified from screening was synthesized on Toyopearl® resin and evaluated via binding studies to select sequences that afford high product binding and recovery. The affibody–peptide interaction was also evaluated by in silico docking, which corroborated the targeting of the conserved domain. Ligand IGKQRI was validated through purification of an anti-ErbB2 affibody from an Escherichia coli lysate. The values of binding capacity (~5 mg affibody per mL of resin), affinity (KD ~1 μM), recovery and purity (64–71% and 86–91%), and resin lifetime (100 cycles) demonstrate that IGKQRI can be employed as ligand in affibody purification processes. 
    more » « less
  5. ; ; ; ; (, J. Mater. Chem. A)
    We demonstrated for the first time that inkjet printing can be a low-cost, easy, fast, and scalable method for depositing ultrathin (7.5–60 nm) uniform graphene oxide (GO) nanofiltration membranes on polymeric supports for highly effective water purification. A large area (15 × 15 cm 2 ) GO nanofiltration membrane was printed successfully on a modified polyacrylonitrile (M-PAN) support. Water permeance and rejection of small organic molecules (<1 nm, charged and uncharged) of printed GO membranes can be adjusted by controlling the GO “ink” concentration and/or printing time. Compared with commercial polymeric nanofiltration membranes, printed GO membranes, after optimization, showed approximately one order of magnitude higher water permeance and much higher rejection (>95%) of small organic molecules. Printed GO membranes also showed excellent performance in removing pharmaceutical contaminants, with ∼95% rejection and <10% water permeance decline over extended-period permeation testing. We believe that inkjet printing could be an effective method for preparing ultrathin GO membranes for effective water nanofiltration purification. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email