AEX new angle: AEX resins for high productivity in viral vector purification

May 28,2025

bestchrom

As gene therapy and vaccine R&D rapidly rise and develops, this field has attracted extensive attention from both academia and industry. As the core delivery system of gene therapy and vaccine development, the optimization of viral vector purification is plays keep role in the Critical Quality Attributes, CQAs) of final product.

Among various purification technologies, AEX (Anion Exchange Chromatography) has gradually become a vital part of mainstream viral vector purification process due to its advantages including good selectivity, easy scale-up and controllable operation.

Currently, viral vectors mainly consist of lentivirus (LV), retrovirus (RV), adenovirus (ADV) and adeno-associated virus (AAV). The following are information for relevant viral vectors, which can be used for the recommendation of suitable purification protocols.

A summary of viral vector information

Most widely used viral vectors:

The purification of viral vectors usually include the following steps:

Cell capture：Ultracentrifugation（100,000×g/2h），Deep filtration（0.45μm+0.22μm connection）.

Nucleic acid treatment：Benzonase（50U/mL，37℃/1h）

UF/DF：750KDa/300kDa/100kDa concentration & filtration

Chromatographic purification: AEX is the key step

1. Retrovirus（RV）/ Lentivirus (LV) purification

Retrovirus is a kind of enveloped single-stranded RNA virus. Being an important member of retrovirus family, Lentiviral vector is a gene delivery system based on human immunodeficiency virus type 1 (HIV-1). Because retrovirus vector and lentivirus vector are negatively charged, AEX chromatography serves as main purification protocol in the conventional capture of lentivirus.

Classic chromatography protocol:

• Resin：Diamond Q or Diamond Q Mustang

• Equilibration buffer：20mM Tris+150mM NaCl，pH7.4

• Elution buffer：20mM Tris+0.3~0.6M NaCl gradient elution，pH7.4

Recommended new protocol:

Choose Mix-mode resins Diamond Layer 400/Layer 700, which enjoy excellent performance in viral purity and recovery.

Charge disparity on the surface of lentivirus limits the selective capture when using traditional AEX resins. To solve this challenge, choosing mix-mode chromatography resins such as Diamond Layer 400/Layer 700 to achieve efficient purification.

Examples of Chromatography protocol ：

• Resin：Diamond Layer 400/Layer 700

• Equilibration：20mM Tris+150mM NaCl，pH7.4

• Load sample：Conductance <5mS/cm，residence time :6~10min

• Elution： Collect flow-through peak

Experimental result：

2. The purification of Adenovirus (ADV)

ADV is non-enveloped double stranded DNA virus with relatively large particle size (~90nm). Purification method of ADV is very similar to that used in the purification of retrovirus (RV) and lentivirus (LV) vectors. Both anion exchange (AEX)chromatography and mixed-mode chromatography resins can be used.

3. The purification of adeno-associated virus(AAV)

Diamond Q Mustang/Q Bestarose HP。

AAV is a small-sized, non-enveloped single-stranded DNA virus, belonging to the dependent virus genus of Parvoviridae. With a size of 25nm, decision to use AEX or CEX resin is mainly dependent on the charges on the surface of molecules. Capsid protein of AAV has a pI of 5.9-6.3. Meanwhile, pI of empty shell protein impurity is different from that of capsid protein. The removal of empty shell protein is mainly achieved by AEX resin with high resolution. Try Diamond Q Mustang/Q Bestarose HP provided by Bestchrom.  

Purification case using Bestchrom resin

• Resin：Diamond Q Mustang/Q Bestarose HP

• Equilibration buffer：20mM PB pH7.4

• Elution buffer：20mM PB+50mM~500mM NaCl  gradient eluiton，pH7.4

Conclusion: AEX chromatography, a highly effective tool for viral vector purification

AEX chromatography resin is now a key link in the processing of viral vector purification, thanks to its advantages including high resolution, stable operation and easy scale-up. IEX is playing an indispensable role in vaccine production and preparation of gene therapy. As chromatography technology and resin performance continuously updates, AEX will continue to be a crucial part in the R&D of viral vectors production process.