Outlook on Next Generation Technologies and Strategy Considerations for ADC Process Development and Manufacturing

Marcq, Olivier

doi:10.1007/978-3-319-78154-9_6

Cited by 3 publications

(1 citation statement)

References 121 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Especially the conjugation step is considered to demand adequate mixing and careful considerations of the payload addition method or rate. In addition, single-use reactors are becoming more commonly used in ADC manufacturing ( Marcq and Damelin, 2018 ), ( Schmidhalter et al, 2019 ). However, their reactor design and mixing geometry is different from conventional stirred vessels which might add further possibilities to affect the product.…”

Section: Introductionmentioning

confidence: 99%

Kinetic studies and CFD-based reaction modeling for insights into the scalability of ADC conjugation reactions

Weggen

Seidel

Bean³

et al. 2023

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

The manufacturing of antibody-drug conjugates (ADCs) involves the addition of a cytotoxic small-molecule linker-drug (= payload) to a solution of functionalized antibodies. For the development of robust conjugation processes, initially small-scale reaction tubes are used which requires a lot of manual handling. Scale-up to larger reaction vessels is often knowledge-driven and scale-comparability is solely assessed based on final product quality which does not account for the dynamics of the reaction. In addition, information about the influence of process parameters, such as stirrer speed, temperature, or payload addition rates, is limited due to high material costs. Given these limitations, there is a need for a modeling-based approach to investigate conjugation scale-up. In this work, both experimental kinetic studies and computational fluid dynamics (CFD) conjugation simulations were performed to understand the influence of scale and mixing parameters. In the experimental part, conjugation kinetics in small-scale reaction tubes with different mixing types were investigated for two ADC systems and compared to larger bench-scale reactions. It was demonstrated that more robust kinetics can be achieved through internal stirrer mixing instead of external mixing devices, such as orbital shakers. In the simulation part, 3D-reactor models were created by coupling CFD-models for three large-scale reaction vessels with a kinetic model for a site-specific conjugation reaction. This enabled to study the kinetics in different vessels, as well as the effect of process parameter variations in silico. Overall, it was found that for this conjugation type sufficient mixing can be achieved at all scales and the studied parameters cause only deviations during the payload addition period. An additional time-scale analysis demonstrated to aid the assessment of mixing effects during ADC process scale-up when mixing times and kinetic rates are known. In summary, this work highlights the benefit of kinetic models for enhanced conjugation process understanding without the need for large-scale experiments.

show abstract

Section: Introductionmentioning

confidence: 99%

Kinetic studies and CFD-based reaction modeling for insights into the scalability of ADC conjugation reactions

Weggen

Seidel

Bean³

et al. 2023

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

show abstract

Engineering designer beta cells with a CRISPR-Cas9 conjugation platform

et al. 2020

View full text Add to dashboard Cite

Genetically fusing protein domains to Cas9 has yielded several transformative technologies; however, the genetic modifications are limited to natural polypeptide chains at the Cas9 termini, which excludes a diverse array of molecules useful for gene editing. Here, we report chemical modifications that allow site-specific and multiple-site conjugation of a wide assortment of molecules on both the termini and internal sites of Cas9, creating a platform for endowing Cas9 with diverse functions. Using this platform, Cas9 can be modified to more precisely incorporate exogenously supplied single-stranded oligonucleotide donor (ssODN) at the DNA break site. We demonstrate that the multiple-site conjugation of ssODN to Cas9 significantly increases the efficiency of precision genome editing, and such a platform is compatible with ssODNs of diverse lengths. By leveraging the conjugation platform, we successfully engineer INS-1E, a β-cell line, to repurpose the insulin secretion machinery, which enables the glucose-dependent secretion of protective immunomodulatory factor interleukin-10.

show abstract

A conjugation platform for CRISPR-Cas9 allows efficient β-cell engineering

Lim

Sreekanth

Cox

et al. 2019

Preprint

View full text Add to dashboard Cite

Genetically fusing protein domains to Cas9 has yielded several transformative technologies; however, these fusions are polypeptidic, limited to the Cas9 termini and lack multivalent display, and exclude diverse array of molecules. Here, we report a platform for the site-specific and multivalent display of a wide assortment of molecules on both the termini and internal sites on Cas9. Using this platform, we endow Cas9 with the functionality to effect precision genome edits, which involves efficient incorporation of exogenously supplied single-stranded oligonucleotide donor (ssODN) at the break site. We demonstrate that the multivalent display of ssODN on Cas9 significantly increased precision genome edits over those of Cas9 bearing one or no ssODN, and such display platform is compatible with large oligonucleotides and rapid screening of ssODNs. By hijacking the insulin secretion machinery and leveraging the ssODN display platform, we successfully engineer pancreatic β cells to secrete protective immunomodulatory factor interleukin-10. TOC GRAPHIC MAINCRISPR-Cas9 is a DNA endonuclease that can be targeted to a genomic site using a guide RNA (gRNA) bearing sequence complementarity to the target site. 1 Genetic fusion of Cas9 with effector domains (e.g., a transcription activator) has yielded transformative technologies; 2,3 however, this approach is limited to fusions that are generally linear, polypeptidic, and located on the termini of Cas9. A conjugation platform that allows the creation of fusions that are non-polypeptidic (e.g., nucleic acids, small molecules, polyethylene glycol [PEG] chains), unnatural peptides/proteins, internally located on Cas9, and branched with a multivalent display, would provide a greater diversity of technologies and applications. For example, precise sequence alteration at the Cas9 cleavage site requires the efficient incorporation of exogenously supplied single-stranded oligonucleotide donor DNA (ssODN) 4 via the homology-directed repair (HDR) pathway. 5-7 However, most cells instead employ the nonhomologous end-joining (NHEJ) repair, which results in unpredictable insertions and deletions of bases at the cleavage site, some of which are large enough to have pathogenic consequences. 3,8,9 Displaying ssODNs on Cas9 can increase their local concentration around the DNA strand break site to allow enhanced incorporation of the desired sequence. In another application, appending PEG chains to Cas9 may reduce the immunogenicity of this bacterial protein. 10 Multivalent display of DNA repair pathway inhibitors (e.g., NHEJ or p53 pathway inhibitors) or cell-specific ligands could also enhance precision and efficacious genome editing. 11,12 An ideal conjugation platform for Cas9 should have several characteristics. First, the platform should be compatible with a diverse set of cargoes (e.g., small molecules, nucleic acids, nanoparticles, antibodies, PEG chains) and allow their multivalent display. Second, the platform should be robust and implementable by nonspecialists, given the diverse u...

show abstract

Outlook on Next Generation Technologies and Strategy Considerations for ADC Process Development and Manufacturing

Cited by 3 publications

References 121 publications

Kinetic studies and CFD-based reaction modeling for insights into the scalability of ADC conjugation reactions

Kinetic studies and CFD-based reaction modeling for insights into the scalability of ADC conjugation reactions

Engineering designer beta cells with a CRISPR-Cas9 conjugation platform

A conjugation platform for CRISPR-Cas9 allows efficient β-cell engineering

Contact Info

Product

Resources

About