Optimization Strategy for Downstream Process of Lentivirus

Lentivirus vector (LV) is an enveloped virus of the Retroviridae family, which can stably integrate and express transgenes in dividing and non-dividing cells. It has the advantages of large packaging capacity, low cytotoxicity and immunogenicity, wide infection spectrum, and stable expression, making it one of the widely used viral vectors for gene therapy and cell therapy.

Lentiviral vectors are enveloped RNA viruses with a diameter of 80-120 nm, exhibiting icosahedral symmetry and spherical shape, with a packaging capacity of 4.5-5 kb. The activity of lentiviruses is easily affected by stress, shear forces, temperature, salt concentration, pH, and other factors during production processes.

Next, we will introduce the downstream process development strategy of lentivirus from several core steps.

The downstream purification process of lentiviral vectors basically uses chromatography separation technology and hollow fiber membrane technology. Generally, virus vector purification chromatography involves various chromatography methods such as molecular sieve, complex mode, and ion exchange chromatography; hollow fiber technology includes clarification filtration and tangential flow filtration.

The basic process of lentivirus purification and optimization points of core process steps are as follows：

01 Clarification harvest

Clarification and filtration mainly remove cells and cell debris as well as some HCD and HCP in the resin, and reduce viscosity. Common methods include deep filtration, centrifugation, dead-end filtration, or a combination of these two methods. However, for the clarification of lentiviral vectors with a scale of more than 50L, deep filtration or 0.2-0.5μm hollow fiber is preferred.

02 Ultrafiltration concentration 1

Using a 300-750KDa hollow fiber column to concentrate and exchange the lentiviral vector solution for clarified and filtered solution can remove some protein, DNA fragments and other impurity molecules. During the concentration process, the shear force is generally controlled at 2000-4000/s-1, TMP is 3-7psi, and the flux is >50LMH, with a 5-10-fold concentration.

03 Nuclease incubation

Add Benzonase to reduce the size and viscosity of DNA molecules. The general temperature for nuclease incubation is 25-37°C, with a treatment time of 30-60 minutes; or the temperature is controlled at 2-8°C, with overnight treatment, and the concentration of nuclease is 20-50 U/mL, with a Mg2+ ion concentration of 1-10 mM.

04 Chromatography purification

The requirement is not to change the paragraph format: Chromatography purification plays an important role in lentiviral vectors. Generally, the combination of MaXtar Q and two-step chromatography is selected to remove impurities such as HCP, DNA fragments, and plasmid residues. MaXtar COLL 700 (molecular sieve or multimodal chromatography) chromatography is used to separate HCP, HCD, and other impurities with a molecular weight less than 700KD from the inner pores of the chromatography resin baseball, which are retained by hydrophobic interactions through ionic interactions. Lentiviruses with a molecular weight greater than 700KD do not enter the inner pores and flow through, thus achieving separation. MaXtar Q is combined with lentiviral vectors through ionic interactions and retained on the MaXtar Q chromatography column. Through a salt gradient, process impurities are removed and high-activity lentiviral vectors are harvested.

Due to the influence of stress, shear force, temperature, salt concentration, pH, and other factors, lentiviral vectors are prone to aggregation and inactivation. The general process parameters in the purification process are optimized under chromatography conditions at 15-25°C: MaXtar COLL 700/400 loading capacity: 3-15CV, flow rate: 150-300cm/h; MaXtar Q loading capacity: 5-50CV, elution salt concentration: 0.5-0.8M NaCl, elution solution should be diluted in time to reduce salt concentration.

Case sharing of MaXtar COLL 700 from BioLink in the use of lentivirus purification

Case 1: Application of COLL 700 in lentivirus purification

Case 2: Application of COLL 700 on lentivirus

Conclusion: The recovery rate and impurity removal effect have reached the expected level.

05 Ultrafiltration concentration 2

Use a 300-750KDa hollow fiber column to concentrate and exchange the lentiviral vector solution for clarified and filtered solution. During the concentration process, the shear force is generally controlled at 2000-4000/s-1, and the TMP is controlled at 3-7psi. Concentrate the lentiviral vector to the appropriate titer and exchange it for the buffer required for the formulation.

For different lentiviral vectors, the following products from BioLink can be selected for purification and separation:

The characteristics of the above several BioLink chromatography resin products can be summarized as follows: