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  • 1
    In: Biotechnology Progress, Wiley, Vol. 35, No. 6 ( 2019-11)
    Abstract: In this work, two phage biopanning strategies were developed to identify affinity peptides for a single Fab and multiple kappa Fabs. For the biopanning rounds, protein L beads were employed to bind Fab targets in a fixed orientation, and NHS functionalized magnetic beads were used to facilitate evaluation of low pH elution conditions. The resulting peptide sequences were synthesized and the binding to different Fabs was evaluated using fluorescence polarization. The first biopanning approach yielded a peptide with similar affinities for two forms of the Fab (recombinantly expressed and post papain‐digestion) as well as the intact antibody. While moderate affinity was observed toward a murine variant of the Fab with the same complementarity determining regions (CDR) region but different framework, minimal binding occurred to a Fab with high sequence homology but containing different CDR loops. The second biopanning strategy yielded a peptide with affinity for all three kappa Fabs indicating that it may be a good lead for the development of more general affinity reagents for recombinant kappa Fabs. Finally, an affinity peptide column was developed, and its efficacy was demonstrated for Fab purification from a complex cell culture fluid mixture. The results presented in this article demonstrate that different peptide‐based phage biopanning strategies can be effectively employed to identify affinity peptide leads for specific Fab and more general kappa Fab purifications.
    Type of Medium: Online Resource
    ISSN: 8756-7938 , 1520-6033
    Language: English
    Publisher: Wiley
    Publication Date: 2019
    detail.hit.zdb_id: 2003526-3
    SSG: 12
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  • 2
    In: Journal of Biotechnology, Elsevier BV, Vol. 338 ( 2021-09), p. 1-4
    Type of Medium: Online Resource
    ISSN: 0168-1656
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2021
    detail.hit.zdb_id: 2016476-2
    SSG: 12
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  • 3
    Online Resource
    Online Resource
    Elsevier BV ; 2020
    In:  Journal of Biotechnology Vol. 309 ( 2020-02), p. 59-67
    In: Journal of Biotechnology, Elsevier BV, Vol. 309 ( 2020-02), p. 59-67
    Type of Medium: Online Resource
    ISSN: 0168-1656
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2020
    detail.hit.zdb_id: 2016476-2
    SSG: 12
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  • 4
    In: Journal of Chemical Technology & Biotechnology, Wiley, Vol. 94, No. 7 ( 2019-07), p. 2345-2354
    Abstract: In this study, we have demonstrated the design, screening and selection of peptide ligands for the affinity capture of human growth hormone (hGH) from yeast cell cultures. Results Ligand design was carried out using multiple approaches based on primary sequence and structures of natural binding partners of hGH. Screening of potential affinity peptides was conducted using high throughput microarray platforms followed by assessment of in‐solution binding to hGH using fluorescence polarization. Peptide leads were subsequently immobilized on chromatographic resins and the binding and desorption behavior was examined using batch adsorption studies. A lead candidate was examined in further details in column chromatography studies which indicated that while high purity was attained, further refinement was necessary for improved product recovery. Histidine scanning was employed to successfully improve the recovery of hGH from cell culture fluid while still maintaining high purity. Finally, proof‐of‐concept was demonstrated in the column format using complex feed stock where a product purity of 95% was attained at 80% yield. Conclusion The approaches presented here can be translated to other biologics of interest for the rapid development of affinity based purification processes. © 2019 Society of Chemical Industry
    Type of Medium: Online Resource
    ISSN: 0268-2575 , 1097-4660
    URL: Issue
    Language: English
    Publisher: Wiley
    Publication Date: 2019
    detail.hit.zdb_id: 1479465-2
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  • 5
    In: Microbiology Spectrum, American Society for Microbiology, Vol. 9, No. 2 ( 2021-10-31)
    Abstract: Routine testing for SARS-CoV-2 is rare for institutes of higher education due to prohibitive costs and supply chain delays. During spring 2021, we routinely tested all residential students 1 to 2 times per week using pooled, RNA-extraction-free, reverse transcription quantitative PCR (RT-qPCR) testing of saliva at a cost of $0.43/sample with same-day results. The limit of detection was 500 copies/ml on individual samples, and analysis indicates 1,000 and 2,500 copies/ml in pools of 5 and 10, respectively, which is orders of magnitude more sensitive than rapid antigen tests. Importantly, saliva testing flagged 83% of semester positives (43,884 tests administered) and was 95.6% concordant with nasopharyngeal diagnostic results (69.0% concordant on the first test when the nucleocapsid gene (N1) cycle threshold ( C T ) value was  〉 30). Moreover, testing reduced weekly cases by 59.9% in the spring despite far looser restrictions, allowing for more normalcy while eliminating outbreaks. We also coupled our testing with a survey to clarify symptoms and transmissibility among college-age students. While only 8.5% remained asymptomatic throughout, symptoms were disparate and often cold-like (e.g., only 37.3% developed a fever), highlighting the difficulty with relying on symptom monitoring among this demographic. Based on reported symptom progression, we estimate that we removed 348 days of infectious individuals by routine testing. Interestingly, viral load ( C T value) at the time of testing did not affect transmissibility ( R 2 = 0.0085), though those experiencing noticeable symptoms at the time of testing were more likely to spread the virus to close contacts (31.6% versus 14.3%). Together, our findings support routine testing for reducing the spread of SARS-CoV-2. Implementation of cost- and resource-efficient approaches should receive strong consideration in communities that lack herd immunity. IMPORTANCE This study highlights the utility of routine testing for SARS-CoV-2 using pooled saliva while maintaining high sensitivity of detection (under 2,500 copies/ml) and rapid turnaround of high volume (up to 930 samples in 8 h by two technicians and one quantitative PCR [qPCR] machine). This pooled approach allowed us to test all residential students 1 to 2 times per week on our college campus during the spring of 2021 and flagged 83% of our semester positives. Most students were asymptomatic or presented with symptoms mirroring common colds at the time of testing, allowing for removal of infectious individuals before they otherwise would have sought testing. To our knowledge, the total per-sample consumable cost of $0.43 is the lowest to date. With many communities still lagging in vaccination rates, routine testing that is cost-efficient highlights the capacity of the laboratory’s role in controlling the spread of SARS-CoV-2.
    Type of Medium: Online Resource
    ISSN: 2165-0497
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2021
    detail.hit.zdb_id: 2807133-5
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  • 6
    In: Biotechnology and Bioengineering, Wiley, Vol. 117, No. 12 ( 2020-12), p. 3775-3784
    Abstract: Affinity precipitation using stimulus‐responsive biopolymers such as elastin‐like polypeptides (ELPs) have been successfully employed for the purification of monoclonal antibodies. In the current work, we extend these studies to the development of an ELP‐peptide fusion for the affinity precipitation of the therapeutically relevant small non‐mAb biologic, AdP. A 12‐mer affinity peptide ligand (P10) was identified by a primary phage biopanning followed by a secondary in‐solution fluorescence polarization screen. Peptide P10 and AdP interacted with a K D of 19.5 µM. A fusion of P10 with ELP was then shown to be successful in selectively capturing the biologic from a crude mixture. While pH shifts alone were not sufficient for product elution, the use of pH in concert with fluid‐phase modifiers such as NaCl, arginine, or ethylene glycol was effective. In particular, the use of pH 8.5 and an arginine concentration of 500 mM enabled 〉 80% product recovery. The overall process performance evaluated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and reversed‐phase ultra‐performance liquid chromatography analyses indicated successful single‐step purification of the biologic from an Escherichia coli lysate resulting in ∼90% purity and 〉 80% recovery. These results demonstrate that phage display can be readily employed to identify a peptide ligand capable of successfully carrying out the purification of a non‐antibody biological product using ELP‐based affinity precipitation.
    Type of Medium: Online Resource
    ISSN: 0006-3592 , 1097-0290
    URL: Issue
    Language: English
    Publisher: Wiley
    Publication Date: 2020
    detail.hit.zdb_id: 1480809-2
    detail.hit.zdb_id: 280318-5
    SSG: 12
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