N-GLYcanyzer monitoring of a microperfusion process reveals critical parameters' impact on Trastuzumab biosimilar N-glycoform time evolution
- 1Rutgers, The State University of New Jersey
Abstract
Controlling monoclonal antibody (mAb) N-glycosylation as a critical quality attribute (CQA) remains challenging in continuous and intensified biomanufacturing, where conventional end-point analyses obscure dynamic pathway responses. Here, we deployed the N-GLYcanyzer process analytical technology (PAT) for automated monitoring of a microperfusion process that reveals critical parameters' impact on Trastuzumab biosimilar N-glycoform time evolution across a 20-day culture duration of Chinese Hamster Ovary (CHO) cells in a ~2 mL scale-down microbioreactor perfusion platform. Using offline PAT assays combined with our integrated at-line PAT workflow, we systematically screened six perfusion process parameters: carbon source, viable cell density (VCD), perfusion rate, temperature, and pH. While nutrient limitation and thermal/acidic stress significantly disrupted N-glycans maturation, notably triggering high-mannose (Man5) accumulation and severe productivity penalties under pH 6.7 and 39 {degrees}C operation, targeted feed supplementation with galactose and MnCl2 consistently shifted glycoform profiles away from G0F toward galactosylated species (G1F, G1F', G2F). Crucially, coupling this combined feed strategy with an intensified perfusion rate (1.2 VVD) achieved a sustained, desired galactosylation shift without a productivity cost. These findings demonstrate that nutrient feed composition and perfusion rate are primary levers for steering continuous intensified microperfusion processes for biologics production with desired N-glycoform states and highlight the utility of automated at-line PAT like N-GLYcanyzer for enabling real-time attribute-based control in continuous biomanufacturing.
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BibTeX
@misc{narvekar2026nglycanyzer,
title = {{N-GLYcanyzer monitoring of a microperfusion process reveals critical parameters' impact on Trastuzumab biosimilar N-glycoform time evolution}},
author = {A. Narvekar and M. Harrison and R. Thakur and P. Khot and P. Semira and K. Lee and S. P. S. Chundawat},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.31.763318},
url = {https://www.biorxiv.org/content/10.64898/2026.10.31.763318v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - N-GLYcanyzer monitoring of a microperfusion process reveals critical parameters' impact on Trastuzumab biosimilar N-glycoform time evolution
AU - A. Narvekar
AU - M. Harrison
AU - R. Thakur
AU - P. Khot
AU - P. Semira
AU - K. Lee
AU - S. P. S. Chundawat
PY - 2026
DA - 2026/10/31
PB - bioRxiv
DO - 10.64898/2026.10.31.763318
UR - https://www.biorxiv.org/content/10.64898/2026.10.31.763318v1
AB - Controlling monoclonal antibody (mAb) N-glycosylation as a critical quality attribute (CQA) remains challenging in continuous and intensified biomanufacturing, where conventional end-point analyses obscure dynamic pathway responses. Here, we deployed the N-GLYcanyzer process analytical technology (PAT) for automated monitoring of a microperfusion process that reveals critical parameters' impact on Trastuzumab biosimilar N-glycoform time evolution across a 20-day culture duration of Chinese Hamster Ovary (CHO) cells in a ~2 mL scale-down microbioreactor perfusion platform. Using offline PAT assays combined with our integrated at-line PAT workflow, we systematically screened six perfusion process parameters: carbon source, viable cell density (VCD), perfusion rate, temperature, and pH. While nutrient limitation and thermal/acidic stress significantly disrupted N-glycans maturation, notably triggering high-mannose (Man5) accumulation and severe productivity penalties under pH 6.7 and 39 {degrees}C operation, targeted feed supplementation with galactose and MnCl2 consistently shifted glycoform profiles away from G0F toward galactosylated species (G1F, G1F', G2F). Crucially, coupling this combined feed strategy with an intensified perfusion rate (1.2 VVD) achieved a sustained, desired galactosylation shift without a productivity cost. These findings demonstrate that nutrient feed composition and perfusion rate are primary levers for steering continuous intensified microperfusion processes for biologics production with desired N-glycoform states and highlight the utility of automated at-line PAT like N-GLYcanyzer for enabling real-time attribute-based control in continuous biomanufacturing.
ER -