A non-affinity purification process for GMP production of prefusion-closed HIV-1 envelope trimers from clades A and C for clinical evaluation

Gulla, Krishana; Cibelli, Nicole; Cooper, Jonathan W.; Fuller, Haley; Schneiderman, Zachary J.; Witter, Sara; Zhang, Yaqiu; Changela, Anita; Geng, Hui; Hatcher, Christian; Narpala, Sandeep; Tsybovsky, Yaroslav; Zhang, Baoshan; Program, Vrc Production; McDermott, Adrian B.; Kwong, Peter D.; Gowetski, Daniel B.

doi:10.1016/j.vaccine.2021.04.063

Cited by 13 publications

(10 citation statements)

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“…Neutralization assessment indicated VRC40.01 to neutralize most of the glycan276-bearing strains of the 208-isolate panel; to visualize how VRC40.01 interacts with glycan276, we determined a cryo-EM structure of Env trimer BG505 SOSIPv5.2 ( Havenar-Daughton et al, 2016 ) in complex with the antigen-binding fragment (Fab) of VRC40.01 and an Env base-binding RM19R Fab ( Martin et al, 2020 ) at 3.3 Å ( Figures 2 , S3 , and S4 ; Table S3 ). We also determined a cryo-EM structure of BG505 DS-SOSIP ( Gulla et al, 2021 ) in complex with VRC40.01 Fab at 3.7 Å ( Figures 2 , S5 , and S6 ; Table S3 ). Modeling of the VRC40.01 Fab in the cryo-EM density was facilitated by a 2.83-Å crystal structure of VRC40.01 Fab ( Table S4 ).…”

Section: Resultsmentioning

confidence: 99%

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Structural basis of glycan276-dependent recognition by HIV-1 broadly neutralizing antibodies

et al. 2021

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Section: Resultsmentioning

confidence: 99%

“…We next obtained a cryo-EM structure of BG505 DS-SOSIP ( Gulla et al, 2021 ) in complex with VRC33.01 Fab at 3.7 Å resolution ( Figures 3 , S7 , and S8 ; Table S3 ). Modeling of the VRC33.01 Fab in the cryo-EM density was facilitated by a crystal structure of unliganded VRC33.01 Fab, which we determined at 1.54 Å resolution ( Table S4 ).…”

Section: Resultsmentioning

confidence: 99%

Structural basis of glycan276-dependent recognition by HIV-1 broadly neutralizing antibodies

et al. 2021

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“…Protein purity was analyzed by SDS-PAGE and concentrated where possible to ~10 mg/mL. BG505-SOSIP was requested from the Vaccine Research Center at the National Institute of Health ( 47 ).…”

Section: Methodsmentioning

confidence: 99%

Structural Basis of Antibody Conformation and Stability Modulation by Framework Somatic Hypermutation

et al. 2022

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Accumulation of somatic hypermutation (SHM) is the primary mechanism to enhance the binding affinity of antibodies to antigens in vivo. However, the structural basis of the effects of many SHMs remains elusive. Here, we integrated atomistic molecular dynamics (MD) simulation and data mining to build a high-throughput structural bioinformatics pipeline to study the effects of individual and combination SHMs on antibody conformation, flexibility, stability, and affinity. By applying this pipeline, we characterized a common mechanism of modulation of heavy-light pairing orientation by frequent SHMs at framework positions 39H, 91H, 38L, and 87L through disruption of a conserved hydrogen-bond network. Q39LH alone and in combination with light chain framework 4 (FWR4L) insertions further modulated the elbow angle between variable and constant domains of many antibodies, resulting in improved binding affinity for a subset of anti-HIV-1 antibodies. Q39LH also alleviated aggregation induced by FWR4L insertion, suggesting remote epistasis between these SHMs. Altogether, this study provides tools and insights for understanding antibody affinity maturation and for engineering functionally improved antibodies.

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“…An expression vector encoding the gene for S_dF_2P or HexaPro along with a DHFR selection marker was transfected into CHO-DG44 cells by electroporation using the MaxCyte STX® scalable transfection system (MaxCyte, Gaithersburg, MD) as previously described 28 . Transfected cells were cultivated in an Multitron shaker (Infors HT, Switzerland) set to 37 °C, 5% CO 2 , and 80% relative humidity with a shaking speed of 130 rpm (orbital throw of 1 inch) in CDM4CHO medium with 6 mM L-glutamine.…”

Section: Methodsmentioning

confidence: 99%

“…High-throughput chromatography resin screens using Tecan robotic liquid handlers and Repligen Robocolumns containing 0.1 mL of each respective resin were performed as previously described 26 – 28 to select lead candidates for process development. Additionally, to ensure a safety profile meeting regulatory agency guidance, viral clearance methods including low pH treatment and nanofiltration were screened for compatibility with the molecule and purification process 29 – 31 .…”

Section: Introductionmentioning

confidence: 99%

Advances in purification of SARS-CoV-2 spike ectodomain protein using high-throughput screening and non-affinity methods

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Arias

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et al. 2022

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The spike (S) glycoprotein of the pandemic virus, SARS-CoV-2, is a critically important target of vaccine design and therapeutic development. A high-yield, scalable, cGMP-compliant downstream process for the stabilized, soluble, native-like S protein ectodomain is necessary to meet the extensive material requirements for ongoing research and development. As of June 2021, S proteins have exclusively been purified using difficult-to-scale, low-yield methodologies such as affinity and size-exclusion chromatography. Herein we present the first known non-affinity purification method for two S constructs, S_dF_2P and HexaPro, expressed in the mammalian cell line, CHO-DG44. A high-throughput resin screen on the Tecan Freedom EVO200 automated bioprocess workstation led to identification of ion exchange resins as viable purification steps. The chromatographic unit operations along with industry-standard methodologies for viral clearances, low pH treatment and 20 nm filtration, were assessed for feasibility. The developed process was applied to purify HexaPro from a CHO-DG44 stable pool harvest and yielded the highest yet reported amount of pure S protein. Our results demonstrate that commercially available chromatography resins are suitable for cGMP manufacturing of SARS-CoV-2 Spike protein constructs. We anticipate our results will provide a blueprint for worldwide biopharmaceutical production laboratories, as well as a starting point for process intensification.

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A non-affinity purification process for GMP production of prefusion-closed HIV-1 envelope trimers from clades A and C for clinical evaluation

Cited by 13 publications

References 50 publications

Structural basis of glycan276-dependent recognition by HIV-1 broadly neutralizing antibodies

Structural basis of glycan276-dependent recognition by HIV-1 broadly neutralizing antibodies

Structural Basis of Antibody Conformation and Stability Modulation by Framework Somatic Hypermutation

Advances in purification of SARS-CoV-2 spike ectodomain protein using high-throughput screening and non-affinity methods

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