Home › Immunology › Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System
Immunology JoVE (Open Access) Citable · DOI

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

DOI: 10.3791/58947-v
What you'll learn
  • ✓ Purify monoclonal antibodies from CHO cell culture using automated microbioreactor systems
  • ✓ Quantify purified antibody concentration via protein assay
  • ✓ Characterize N-glycan profiles using LC-MS analytics
  • ✓ Assess charge variants to determine critical quality attributes
Protocol

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Difficulty
advanced
Total time
~3–5 days (culture to final analytics)
Model organism
Chinese Hamster Ovary (CHO) cells
Biosafety
BSL-1

Steps

1
Purify monoclonal antibody from harvested cell culture fluid

Apply automated microbioreactor-derived cell culture fluid to chromatography for antibody isolation and purification. This step removes contaminants and concentrates the target monoclonal antibody for downstream analysis.

▶ 00:39
2
Determine purified antibody concentration

Measure total protein concentration of the purified antibody sample using a quantitative protein assay. This establishes the yield and enables normalization for subsequent analytics.

▶ 01:49
3
Label and isolate N-glycans from antibody

Enzymatically release N-linked glycans from the purified antibody and apply fluorescent labeling to enable mass spectrometry detection. Isolate labeled glycans prior to LC-MS analysis.

▶ 02:33
4
Analyze labeled N-glycans by LC-MS

Separate and characterize the labeled N-glycan structures using liquid chromatography coupled to mass spectrometry to determine glycosylation profiles and quality attributes.

▶ 05:00
5
Prepare charge variant sample for analysis

Format and condition purified antibody sample to assess post-translational modifications that alter charge, a key quality attribute reflecting product heterogeneity.

▶ 06:17
6
Prepare microfluidic chip for charge variant separation

Load and condition a capillary electrophoresis chip with appropriate buffer and sample to perform charge variant profiling and separation.

▶ 07:38
7
Interpret representative purified antibody analysis results

Review and interpret representative data from purification and analytics workflows, including concentration, glycosylation, and charge variant profiles from CHO microbioreactor cultures.

▶ 08:31
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