Process intensification in fed-batch production bioreactors using non-perfusion seed cultures

Area of Science:

• Biotechnology
• Biopharmaceutical Manufacturing
• Cell Culture Technology

Background:

• The biopharmaceutical industry predominantly uses fed-batch processes for monoclonal antibody (mAb) production.
• Conventional fed-batch methods often start with low inoculation viable cell densities (VCDs).
• Process intensification, including higher inoculation VCDs, aims to increase manufacturing output and shorten production timelines.

Purpose of the Study:

• To investigate the impact of increased inoculation VCD on mAb titer and product quality in fed-batch production.
• To evaluate non-perfusion methods for achieving high VCD in seed trains (N-1 step).
• To compare the efficacy of non-perfusion N-1 seeds with perfusion N-1 seeds in scaled-up bioreactors.

Main Methods:

• Utilized Chinese Hamster Ovary (CHO) GS cell lines to produce three different mAbs.
• Implemented intensified fed-batch and batch modes for the N-1 seed train with enriched media.
• Inoculated production bioreactors (N stage) at elevated VCDs (3-6 × 10^6 cells/mL).
• Conducted studies in 5-L bioreactors and scaled up to 500-L and 1000-L N-stage bioreactors.

Main Results:

• Increased inoculation VCD significantly improved final mAb titer within a 14-day fed-batch duration.
• Non-perfusion N-1 methods achieved high VCDs (22-34 × 10^6 cells/mL) using enriched media.
• Non-perfusion N-1 seeds yielded comparable titer and product quality to perfusion N-1 seeds across scales.
• Enrichment of basal medium was critical for batch mode N-1 operations and subsequent intensified fed-batch steps.

Conclusions:

• Non-perfusion N-1 seed strategies provide a simpler, effective alternative to perfusion for intensifying fed-batch mAb production.
• These methods shorten seed train and production durations or improve titer.
• The principles are applicable to various protein production systems and scales in biologics manufacturing.