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
Loading characteristics of streptavidin on polypropylene capillary channeled polymer fibers and capture performance towards biotinylated proteins
continuing emphasis. Polypropylene (PP) capillary-channeled polymer (C-CP) fiber columns are modified with the biotin- binding protein streptavidin (SAV) to capture biotinylated proteins. The loading characteristics of SAV on fiber supports were determined using breakthrough curves and frontal analysis. Based on adsorption data, a 3-min on-column loading at a flow rate of 0.5 mL min−1 (295.2 cm h−1) with a SAV feed concentration of 0.5 mg mL−1 produces a SAV loading capacity of 1.4 mg g−1 fiber. SAV has an incredibly high affinity for the small-molecule biotin (10−14 M), such that this binding relationship can be exploited by labeling a target protein with biotin via an Avi-tag. To evaluate the capture of the biotinylated proteins on the modified PP surface, the biotinylated versions of bovine serum albumin (b-BSA) and green fluorescent protein (b-GFP) were utilized as probe species. The loading buffer composition and flow rate were optimized towards protein capture. The non-ionic detergent Tween-20 was added to the deposition solutions to minimize non-specific binding. Values of 0.25–0.50% (v/v) Tween-20 in PBS exhibited better capture efficiency, while minimizing the non-specific binding for b-BSA and b-GFP, respectively. The C-CP fiber platform has the potential to provide a fast and low-cost method to capture targeted proteins for applications including protein purification or pull-down assays.
more »
« less
- Award ID(s):
- 2107882
- PAR ID:
- 10535285
- Publisher / Repository:
- Springer
- Date Published:
- Journal Name:
- Analytical and Bioanalytical Chemistry
- Volume:
- 415
- Issue:
- 27
- ISSN:
- 1618-2642
- Page Range / eLocation ID:
- 6711 to 6721
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Protein A (ProA) chromatography is a mainstay in the analytical and preparative scale isolation/purification of monoclonal antibodies (mAbs). One area of interest is continuous processing or continuous chromatography, where ProA chromatography is used in the large-scale purification of mAbs. However, filtration is required prior to all ProA isolations to remove large particulates in cell culture supernatant, consisting of a mixture of cell debris, host cell contaminants, media components, etc. Currently, in-line filters are used to remove particles in the supernatant, requiring replacement over time due to fouling; regardless of the scale. Here we demonstrate the ProA isolation of unfiltered Chinese hamster ovary (CHO) cell media using capillary-channel polymer (C-CP) fiber stationary phases modified with S. aureus Protein A (rSPA). The base polymer of the analytical scale C-CP columns costs ~$5 per 30 cm column, and when modified with ProA, the base cost is ~$25 per 30 cm column, a cost-effective option in comparison to analytical-scale commercial columns. To directly sample unfiltered media, a 5 cm gap was created at the head of the C-CP column, where the large particulates are trapped, while molecular solutes flow through the capillary channels without sacrifice in analytical performance, mAb loading capacity, or backpressure increases. The binding capacity of the gap ProA C-CP column was ~ 2 mg mL− 1 of IgG per bed volume. The same analytical column could be operated after processing a total of ~ 56 column bed volumes of supernatant (>25 analytical cycles) without the need for caustic clean-in-place processing.more » « less
-
Biotin-labeled proteins are widely used as tools to study protein–protein interactions and proximity in living cells. Proteomic methods broadly employ proximity-labeling technologies based on protein biotinylation in order to investigate the transient encounters of biomolecules in subcellular compartments. Biotinylation is a post-translation modification in which the biotin molecule is attached to lysine or tyrosine residues. So far, biotin-based technologies proved to be effective instruments as affinity and proximity tags. However, the influence of biotinylation on aspects such as folding, binding, mobility, thermodynamic stability, and kinetics needs to be investigated. Here, we selected two proteins [biotin carboxyl carrier protein (BCCP) and FKBP3] to test the influence of biotinylation on thermodynamic and kinetic properties. Apo (without biotin) and holo (biotinylated) protein structures were used separately to generate all-atom structure-based model simulations in a wide range of temperatures. Holo BCCP contains one biotinylation site, and FKBP3 was modeled with up to 23 biotinylated lysines. The two proteins had their estimated thermodynamic stability changed by altering their energy landscape. In all cases, after comparison between the apo and holo simulations, differences were observed on the free-energy profiles and folding routes. Energetic barriers were altered with the density of states clearly showing changes in the transition state. This study suggests that analysis of large-scale datasets of biotinylation-based proximity experiments might consider possible alterations in thermostability and folding mechanisms imposed by the attached biotins.more » « less
-
Protein self-assembly plays a vital role in a myriad of biological functions and in the construction of biomaterials. Although the physical association underlying these assemblies offers high specificity, the advantage often compromises the overall durability of protein complexes. To address this challenge, we propose a novel strategy that reinforces the molecular self-assembly of protein complexes mediated by their ligand. Known for their robust noncovalent interactions with biotin, streptavidin (SAv) tetramers are examined to understand how the ligand influences the mechanical strength of protein complexes at the nanoscale and macroscale, employing atomic force microscopy-based single-molecule force spectroscopy, rheology, and bioerosion analysis. Our study reveals that biotin binding enhances the mechanical strength of individual SAv tetramers at the nanoscale. This enhancement translates into improved shear elasticity and reduced bioerosion rates when SAv tetramers are utilized as cross-linking junctions within hydrogel. This approach, which enhances the mechanical strength of protein-based materials without compromising specificity, is expected to open new avenues for advanced biotechnological applications, including self-assembled, robust biomimetic scaffolds and soft robotics.more » « less
-
Background: DJ-1 is a protein whose mutation causes rare heritable forms of Parkinson’s disease (PD) and is of interest as a target for treating PD and other disorders. This work used high performance affinity microcolumns to screen and examine the binding of small molecules to DJ-1, as could be used to develop new therapeutics or to study the role of DJ-1 in PD. Non-covalent entrapment was used to place microgram quantities of DJ-1 in an unmodified form within microcolumns, which were then used in multiple studies to analyze binding by model compounds and possible drug candidates to DJ-1. Results: Several factors were examined in optimizing the entrapment method, including the addition of a reducing agent to maintain a reduced active site cysteine residue in DJ-1, the concentration of DJ-1 employed, and the entrapment times. Isatin was used as a known binding agent (dissociation constant, ~2.0 µM) and probe for DJ-1 activity. This compound gave good retention on 2.0 cm × 2.1 mm inner diameter DJ-1 microcolumns made under the final entrapment conditions, with a typical retention factor of 14 and elution in ~8 min at 0.50 mL/min. These DJ-1 microcolumns were used to evaluate the binding of small molecules that were selected in silico to bind or not to bind DJ-1. A compound predicted to have good binding with DJ-1 gave a retention factor of 122, an elution time of ~15 min at 0.50 mL/min, and an estimated dissociation constant for this protein of 0.5 µM. Significance: These chromatographic tools can be used in future work to screen additional possible binding agents for DJ-1 or adapted for examining drug candidates for other proteins. This work represents the first time protein entrapment has been deployed with DJ-1, and it is the first experimental confirmation of binding to DJ-1 by a small lead compound selected in silico.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1007/s00216-023-04948-5
