Product Code: LH0100001C
IMPORTANT NOTES
• Read this technical user guide before using the capsule.
• The LentiHERO® chromatography capsule contains a storage buffer of 20% ethanol. For long term storage, the capsule should be refrigerated between 2 ºC (36 ºF) and 8 ºC (46 ºF) and kept away from direct sunlight. For short term storage, the capsule can be kept at room temperature. Keep caps on the capsule until use. Do not freeze.
• This product is provided as sanitized, unless specified.
• This product is intended for single batch use. A clean-in-place method (CIP) is provided. As feedstocks and processes vary, it is recommended that the performance of this product is validated after CIP
• This technical information can change without notice. For the latest information, please refer to the Astrea Bioseparations website (https://www.astreabioseparations.com/) for the most current version of this document.
INTRODUCTION
LentiHERO® is a radial pre-packed chromatography capsule designed for downstream processing of lentiviral vectors from cell culture feedstock.
LentiHERO® consists of a proprietary high-flow and high-capacity composite nanofiber, AstreAdept®, housed in a protective shell specifically designed for fast flow rates
The combined characteristic of a large flowpath diameter, weak anion exchange chemistry, and mild elution conditions enable high recovery of purified lentiviral vectors.
TECHNICAL DATA
SPECIFICATIONS
LIGAND: Weak anion exchange
TYPICAL DYNAMIC BINDING CAPACITY:
Bovine serum albumin at 1 mg/mL in 10 mM at Tris pH 8 > 60 mg/mL
MAXIMUM PRESSURE AT 20 ºC:
6 Bar | 0.6 MPa | 87 psi
BED VOLUME (BV): 100 mL
NOMINAL VOID VOLUME: 300 mL
RECOMMENDED OPERATIONAL FLOW RATE (mL/min) [minimum to maximum range]: 500 [50 mL/min–1000 mL/min]
RECOMMENDED PRE-CAPSULE PRESSURE:
CHEMICAL STABILITY:
3 Bar | 0.3 MPa | 43 psi
Compatible with buffers commonly used in bioprocessing, including 20% EtOH, 1 M acetic acid, 1 M NaOH, and solutions containing up to 10 mM EDTA
CONNECTIONS: Sanitary flange (12.7 mm)
BLEED VALVE: Screw cap
REPEATED USE: Single batch use
MATERIALS OF CONSTRUCTION:
HOUSING: polypropylene, silicone
FUNCTIONAL MEMBRANE: Modified cellulose acetate
LentiHERO® 100
OPERATION
WARNING
Do not use high concentrations of organic solvents, for example, greater than 20% EtOH or 30% isopropyl alcohol.
Wear appropriate personal protective equipment during operation.
Deviation from recommended guidelines could result in personal harm or damage to the product or material.
Lentiviral vector feedstocks vary considerably in production method, lentiviral vector titer, genetic payload, and contamination profile. All these factors can influence the performance of a chromatography step. Optimization for use with any feedstock may be required.
Preparation of lentiviral vector feed
A general process is described below as an example of a purification process for lentiviral vectors
All lentiviral vector feeds should be treated with endonuclease before clarification (e.g., 50 µ/mL for 1 hour).
Lentiviral expression system
Adherent/ Suspension
Centrifugation or DEPTH filtration terminating in 1 µM followed by 0.45 µM filtration
Screening of clarification processes can be performed with Nereus LentiHERO® spin columns (Product code: NL100100).
Buffers
Processing VSV-G pseudotyped lentiviral vectors with buffers containing arginine has been demonstrated to give significant improvements in recovery (e.g., ~ 80% recovery from a load of 2.3E+9 TU/mL). Therefore, we recommend the VSV-G Buffers for use with VSV-G pseudotyped lentiviral vectors For non-VSV-G pseudotyped lentiviral vectors we recommend starting with the Universal buffers, and screening for optimization. VSV-G Buffer
A SANITIZATION: 0.5 M NaOH
B HIGH CONDUCTIVITY/STRIP: 20 mM Tris, 2 M NaCl, pH 7.2
C EQUILIBRATION:
20 mM Tris, 20 mM MgCl2, pH 7.2
D ELUTION: 20 mM Tris, 20 mM MgCl2, 0.8 M arginine, pH 7.2 Universal Buffer
A SANITIZATION: 0.5 M NaOH
B HIGH CONDUCTIVITY/STRIP: 20 mM Tris, 2 M NaCl, pH 7-8
C EQUILIBRATION:
20 mM Tris, 20 mM MgCl2, pH 7-8
D ELUTION: 20 mM Tris, 20 mM MgCl2, 0.6 M NaCl, pH 7-8
Installation
1. Attach the LentiHERO® 100 device to the liquid chromatography system with 12.7 mm TC connectors and appropriate size tubing (for example, PTFE/silicone tubing 9.5 mm ID).
2. Attach the column outlet from the chromatography system to the inlet of the device (center connector), and the column inlet from the chromatography to the outlet of the device (outer connector) and loosen the stopper on the bleed valve.
3. If required, run sanitization buffer A at 1 bed volume (BV)/minute for 30 minutes.
4. Once liquid is visibly exiting the bleed valve, close with the stopper. Continue running until the pH of the chromatography system outlet is stable and at the same pH as sanitization buffer A.
5. Equilibrate the unit with equilibration buffer C at 1 BV/min, until pH and conductivity are stable. THIS BUFFER MUST NOT CONTAIN MAGNESIUM SULPHATE DUE TO THE POSSIBILITY OF PRECIPITATION AT HIGH pH.
6. Run 20 BV of high conductivity buffer B through the LentiHERO® to charge the device.
7. Equilibrate with 10 BV of low conductivity equilibration buffer C at 5 BV/min, until pH and conductivity are stable.
8. The system is now ready to load clarified feed that has been treated with endonuclease.
Chromatography method
All steps (except where noted) are run at 5 bed volumes (BV)/min.
1. Load clarified lentiviral vector feed to approximately 4E+9 TU of lentiviral vectors/mL of adsorbent.
2. Wash with 30 BV of equilibration buffer C.
3. Elute with 20 BV of elution buffer D collecting the elution peak by UV absorbance. Peak volume will be determined by hold up in the system and tubing.
a. When using VSV-G Buffers, arginine will act as the as the eluting salt. In this case it is not necessary to dilute the elution pool.
b. When using the Universal Buffers, the elution peak should be diluted immediately 1:1 with the equilibration buffer or an appropriate media to lower overall conductivity to approximately 30 mS/cm. For example, the elution peak can be collected into an equal volume of equilibrium buffer, including any appropriate stabilizing additives.
c. Non-VSV-G pseudotyped lentiviral vectors may require optimisation of the elution conditions, a gradient of 1 M NaCl in 20 mM Tris, pH 7.2, 20 mM MgCl2 is recommended.
4. NOTE: WASH WITH 30 BV OF HIGH CONDUCTIVITY BUFFER B TO REMOVE THE MGCL2 PRIOR TO ANY CIP OR SANITIZATION STEP AFTER USE, IF RE-USING.
5. Sanitize with 10 BV of sanitization buffer C 0.5 M NaOH at 1 BV/min.
The LentiHERO® can now be disposed of safely or re-charged and re-equilibrated for use with the same target (see installation instructions).
TROUBLESHOOTING
Problem
High back pressure during loading
Lentiviral vectors fail to bind to the LentiHERO®
Probable cause
Incomplete clarification
Early breakthrough of lentiviral vectors
Conductivity of load too high
1. Higher TU titer than expected
Or 2. Competitive binding with other impurities
Reduced recovery of lentiviral vectors
Instability of lentiviral vectors
Action
1. Follow recommended procedure for clarification. Use a larger surface area of filter train or DEPTH filters between 1 µM to 0.45 µM.
2. Diafilter with 100 kDa molecular weight cut off (MWCO) membrane in equilibration buffer at 100 mM salt.
Dilute load with equilibration buffer until conductivity is approximately 15 mS/cm
1. Check TU of load.
2. Check dsDNA contamination in feed. Optimize removal of charged contaminants using diafiltration (e.g., buffer exchange with 100 kDa molecular weight membrane in equilibration buffer at 100 mM salt)
Screen additional elution salts, for example NaCl, and additives to stabilize the lentiviral vectors and add these to the elution and the diluent if using NaCl in the elution, for example 10% sucrose, or trehalose. The optimal amounts will be dependent on vector and GoI
QUALITY ASSURANCE
The product meets the standards as described below.
COMPLIANCE OF MATERIALS:
DECLARATIONS OF SUITABILITY:
QUALITY:
COMPLIANCE OF PERFORMANCE AND IDENTIFICATION:
All wetted parts have been assessed for low toxicity compliance and biocompatibility against either USP VI <88>, ISO 10993, 21CFR Part 177, or by risk assessment
TSE/BSE free, non-animal derived, nitrosamine free statements, GMO and Country of Origin statements are available within the associated Process-ready Regulatory Support File
Manufactured within an ISO 9001:2015 Quality Management System, the product is controlled and traceable. Manufactured within an ISO 7 environment before being released by the Quality Team.
Flow test, visual inspection, bioburden, and endotoxin
For further details, refer to the associated Process-ready Regulatory Support File.
