Monoclonal Antibody Purification

Quick Purification Strategy

Resolving Monoclonal Antibody
Charge Variants
Feed

CAPTURE

INTERMEDIATEGoals■■ Remove >90% hostcell proteins (HCP),DNA, and viruses■■ Concentrate target 		
monoclonal antibody 		(mAb)

Pure
Product

POLISHGoals■■ Separate closely
related species■■ Remove remnant
impurities

Goals■■ Minimize mAb
aggregate andvariant content■■ Clean up leached
Protein A

Cation Exchange

Ion Exchange/Mixed Mode

Recommended Resins

Affinity/Ion Exchange

Nuvia™ HR-SLoad

Bind/elute mode

Bind/elute mode

Bind/elute mode

Protein deamidation at neutral or alkaline pH is of significant concern to manufacturersof biotherapeutic proteins.
Deamidation at asparagine and glutamine residues:
Caused byResults in■■Enzymatic or alkaline hydrolysis

Affects■■

Immunogenicity, stability, and activity 		of proteins

Refer to experimental details on page 2.

Minimal changes in protein structureand charge■■ Downstream purification challenges due
to deaminated species■■ Molecular heterogeneity■■

Monoclonal Antibody Purification Strategy Details
Intermediate purification to resolve charge variants with Nuvia HR-S
Step

BufferEquilibration

Sample Loading

CaptureAffinity/ion exchange
chromatography

Column Volume

Flow Rate, cm/hr

10 mM sodium acetate, pH 4.5 (buffer A)

15300

Eluate from the capture step
diluted 1:4 in buffer A–300

Wash 1

Buffer A

15300

Wash 210 mM sodium citrate, 10 mM sodiumphosphate, pH 5.0 (buffer B)

15300

ElutionGradient of buffer B to 10 mM sodiumcitrate, 10 mM sodium phosphate, pH 8.0

15300

PolishIon exchange/
mixed-mode
chromatography

8.0
7.5
7.0pH6.5VDNALQSGNSQESVTEQDSK

6.0

Shoulder fraction protein sequence. Examination of the shoulder fraction protein
sequence confirms deamidation events on a single mAb peptide.

5.5
5.0
4.5

Resources
Visit bio-rad.com/web/NuviaHRS to get more information.

Shoulder

4.0

Purification of the mAb on Nuvia HR-S. The distinct shoulder on the front sideof the main peak is a modified mAb fragment. A280 (—); conductivity (—); pH (—).

Visit bio-rad.com/web/ProcessApplications to see otherapplications in which these resins can be used.

300

2,138.646Intensity, amu

200

Visit bio-rad.com/web/ProcessResins to get technical details
about Nuvia HR-S Resin.

10001502,136.142

100
5002,125

2,130

2,135

2,140

2,145m/z

2,150

2,155

2,160

2,165

Mass spectrometry (MS) analysis of the mAb. MS analyses of the shoulderedpeak (top spectrum) and the main peak (lower spectrum) show a mass shift of about
2.5 atomic mass units between the two peaks, which is consistent with twoor three deamidation events on a single peptide.

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