Thermo Fisher Scientific AAV-MAX User Manual

AAV-MAX Helper-Free Production System, Prototype

USER GUIDE

For animal origin free, chemically deﬁned adeno-associated viral (AAV) vector production in mammalian suspension cells

for use with: Gibco™ Viral Production Cells 2.0, Prototype , Gibco™ Viral Production Medium,

Prototype, Gibco™ AAV-MAX Transfection Kit, Prototype , Gibco™ Viral-Plex Complexation Buer, Prototype , Gibco™ AAV-MAX Lysis Buer, Prototype

Publication Number MAN0019921

Revision A.0

For Research Use Only. Not for use in diagnostic procedures. Product is a prototype and performance characteristics of this product have not been established.

Manufacturer:

Life Technologies Corporation | 5781 Van Allen Way | Carlsbad, California 92008 USA

Manufacturer:

Life Technologies Corporation | 3175 Staley Road | Grand Island, New York 14072 USA

Products:

Viral Production Cells 2.0, Prototype AAV-MAX Transfection Kit, Prototype AAV-MAX Lysis Buer, Prototype

Products:

Viral Production Medium, Prototype Viral‑Plex™ Complexation buer, Prototype

For descriptions of symbols on product labels or product documents, go to thermoﬁsher.com/symbols-deﬁnition.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history: Pub. No. MAN0019921

Revision Date Description

A.0 10 February 2021 New document.

Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all applicable Limited Use Label Licenses.

TRADEMARKS: All trademarks are the property of Thermo Fisher Scientiﬁc and its subsidiaries unless otherwise speciﬁed.

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CHAPTER 1 Product information .................................................. 5

Product description ............................................................. 5

Contents and storage ............................................................ 5

System components ............................................................. 6

Viral Production Cells 2.0 .................................................... 6

Viral Production Medium ..................................................... 6

AAV‑MAX Transfection Reagent .............................................. 6

AAV‑MAX Transfection Booster ............................................... 6

AAV‑MAX Enhancer ......................................................... 7

Viral‑Plex™ Complexation Buer .............................................. 7

AAV‑MAX Lysis Buer ....................................................... 7

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CHAPTER 2 Culture Viral Production Cells 2.0 ................................... 8

Thaw and establish Viral Production Cells 2.0 ...................................... 8

Guidelines for handling cells ................................................. 8

Required materials not supplied .............................................. 8

Thaw Viral Production Cells 2.0 ................................................... 8

Subculture Viral Production Cells 2.0 .............................................. 9

Cryopreserve Viral Production Cells 2.0 .......................................... 11

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CHAPTER 3 Transfect Viral Production Cells 2.0 ............................... 12

Transfection guidelines ......................................................... 12

Equipment guidelines ........................................................... 12

Required materials ............................................................. 12

Optimized transfection conditions ................................................ 13

Transfection procedure ......................................................... 14

Day 0: Prepare and transfect cells ........................................... 14

Harvest AAV particles ........................................................... 16

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APPENDIX A Additional guidelines .............................................. 17

Plasmids ratio optimization ...................................................... 17

Crude AAV particle quantitation by qPCR (vg/mL) .................................. 18

AAV-MAX Helper-Free Production System, Prototype, User Guide

Contents

■

APPENDIX B Safety ............................................................... 19

Chemical safety ................................................................ 20

Biological hazard safety ......................................................... 21

Documentation and support ....................................................... 22

Customer and technical support ................................................. 22

Limited product warranty ........................................................ 22

AAV-MAX Helper-Free Production System, Prototype, User Guide

IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix

in this document.

Product description

The Gibco™ AAV‑MAX Helper-Free AAV Production System, Prototype is a high-yield Adeno-Associated Virus (AAV) production system based on a clonal HEK293F-derived cell line adapted to the chemically deﬁned Viral Production Medium in suspension format. The AAV‑MAX Helper-Free AAV Production System, Prototype includes cells, medium, transfection reagent, transfection booster, production enhancer, and lysis buer to produce scalable, high titer adeno-associated viral (AAV) vectors.

Contents and storage

Product information

The workﬂows described in this document are for use with the components in the following table.

Table 1 AAV‑MAX System, Prototype Components

Contents

Gibco™ Viral Production Cells 2.0 Prototype

Gibco™ Viral Production Medium, Prototype A49842DK 1 L 2°C to 8°C

Gibco™ AAV‑MAX Transfection Kit, Prototype:

•

Gibco™ AAV‑MAX Transfection Reagent, Prototype

•

Gibco™ AAV‑MAX Transfection Booster, Prototype

•

Gibco™ AAV‑MAX Enhancer, Prototype

Gibco™ Viral‑Plex™ Complexation Buer, Prototype

[1]

Cat. No. Amount Storage

A50232 1 mL

(1 × 107 cells/mL)

A49842BA 10 L (bag)

A50518 for 1 L culture

A50519 for 10 L culture

A49846DG 100 mL

Liquid nitrogen

Protect from light

[2]

AAV-MAX Helper-Free Production System, Prototype, User Guide

Chapter 1

System components

Table 1 AAV‑MAX System, Prototype Components (continued)

Gibco™ AAV‑MAX Lysis Buer, Prototype A50521 100 mL 2°C to 8°C

Gibco™ GlutaMAX™ Supplement 35050061 100 ML Room Temperature

[1]

[2]

Product information

Contents Cat. No. Amount Storage

Cells are cryopreserved in 90% Viral Production Medium and 10% DMSO. Store the frozen cells in liquid nitrogen immediately upon receipt and until ready to use. Do not store the cells at −80°C.

System components

The following section provides descriptions of components in the AAV‑MAX Production System, Prototype.

Viral Production Cells 2.0

Protect from light

Gibco™ Viral Production Cells 2.0 (VPCs 2.0), Prototype are a clonal derivative of the HEK293F cell line and have been adapted to suspension, high-density culture in Gibco™ Viral Production Medium. These cells can be thawed directly into Gibco™ Viral Production Medium.

Cell line characteristics:

•

Transformed via culture with sheared human adenovirus 5 DNA

•

Does not contain the SV40 large T antigen

•

Cell doubling time of <24 hours

•

Achieves maximum cell densities of >1.2 × 107 cells/mL in shaker ﬂask cultures

•

Maximal AAV production can be achieved between cell passages 4 to 25

Viral Production Medium

Gibco™ Viral Production Medium, Prototype is a chemically deﬁned, serum-free, protein-free, animal origin-free medium developed for growth and transfection of VPCs 2.0. Prior to use, the medium requires supplementation with 4 mM GlutaMAX™ Supplement.

AAV‑MAX Transfection Reagent

Gibco™ AAV‑MAX Transfection Reagent, Prototype is a chemically deﬁned, cationic lipid-based reagent that has been uniquely designed for high eciency co-transfection of multiple plasmids DNA into high-density VPCs 2.0 with low toxicity.

AAV

‑MAX Transfection Booster

Gibco™ AAV‑MAX Transfection Booster, Prototype is a chemically deﬁned reagent that has been uniquely designed to boost eciency of co-transfection of multiple plasmids DNA into high density VPCs 2.0. AAV‑MAX Transfection Booster works together with AAV‑MAX Transfection Reagent to deliver superior transfection eciency for high-titer AAV vector production.

AAV-MAX Helper-Free Production System, Prototype, User Guide

AAV‑MAX Enhancer

Gibco™ AAV‑MAX Enhancer, Prototype is a chemically deﬁned, serum-free, protein-free formulation that is designed to enhance AAV vector production in VPCs 2.0.

Viral‑Plex™ Complexation Buer

Gibco™ Viral‑Plex™ Complexation Buer, Prototype is a chemically deﬁned, serum-free, protein-free, phenol-red free formulation that is designed to facilitate DNA complexation with transfection reagent during DNA transfection.

AAV‑MAX Lysis Buer

Gibco™ AAV‑MAX Lysis buer, Prototype is a ready-to-use, chemically deﬁned, Tween®-based cell lysis reagent for the extraction of AAV particles from producer HEK293 cells. The buer is supplied as a 10X solution that can be directly added to HEK293 AAV production cultures to induce cell lysis.

Chapter 1 Product information

System components

AAV-MAX Helper-Free Production System, Prototype, User Guide

Culture Viral Production Cells 2.0

Thaw and establish Viral Production Cells 2.0

Guidelines for handling cells

•

IMPORTANT! Store the frozen cells in liquid nitrogen until ready to use. Do not store the cells at

–80°C.

•

Avoid subjecting cells to short-term, extreme temperature changes.

•

After storing cells in liquid nitrogen following receipt on dry ice, allow the cells to remain in liquid nitrogen for 3 to 4 days before thawing.

•

For all cell manipulations, mix cells by gentle swirling and avoid vigorous shaking/pipetting.

•

For routine cell culture maintenance, subculture cells every 3 to 4 days when they reach 4 to 6 × 106 cells/mL. Do not subculture cells that have not reached early log phase growth of ≥4 × 106 cells/mL.

Required materials not supplied

•

125‑mL PETG Erlenmeyer Flasks (e.g., Nalgene™ Single-Use PETG Erlenmeyer Flasks with Plain Bottom: Sterile, Cat. No. 4115-0125)

•

Orbital shaker (e.g., MaxQ™ HP™ Tabletop Orbital Shaker, Cat. No. SHKE416HP)

•

Temperature and CO2 controlled incubator (e.g., Large-Capacity Reach-In CO2 Incubator, Cat. No.

3950)

•

Reagents and equipment to determine cell viability (e.g., hemocytometer with trypan blue or cell counter)

Thaw Viral Production Cells 2.0

Prepare 30 mL of pre-warmed Viral Production Medium supplemented with 4 mM GlutaMAX Supplement into a 125‑mL PETG Erlenmeyer shaker ﬂask.

Remove a vial of VPCs 2.0 from liquid nitrogen and swirl in a 37°C water bath for 1 to 2 minutes to thaw the cells rapidly until only a small amount of ice remains.

Note: Do not submerge the vial in the water.

Just before the cells are completely thawed, decontaminate the vial with 70% ethanol before opening it in a laminar ﬂow hood.

™

AAV-MAX Helper-Free Production System, Prototype, User Guide

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