Image of an AAV particle

CTS AAV-MAX Helper Free AAV Production System

Accelerate the path to clinic with complementary research and cGMP-manufactured AAV-MAX solutions. One complete AAV production system for facilitating a seamless translation from research to clinical and commercial production.

Research & Development → Preclinical → Clinical and commercial production

Icons depicting the culture, transfection and production, and AAV extraction components of the AAV-MAX System
Figure 1. Components of the CTS AAV-MAX Helper Free AAV Production System.

Comparison of RUO and CTS AAV-MAX production systems

The AAV-MAX Helper-Free AAV Production System enables a smooth transition from discovery through research and commercial development manufacturing, offering one optimized, scalable platform with research-grade and cGMP-manufactured reagent options. Figure 2 shows the equivalence of the CTS and RUO systems with two serotypes.

Bar chart comparing titers obtained from CTS and RUO AAV-MAX systems

 

Figure 2. Comparison of AAV titers between research-grade (RUO) and CTS reagents. AAV serotypes AAV2 and AAV6 were produced at 30 mL production scale in 125 mL shake flasks. Performance of the RUO and CTS AAV-MAX system reagents was shown to be equivalent as measured by viral titers. Titers were measured by qPCR.

AttributesAAV-MAX (RUO)CTS AAV-MAX
Intended useFor research onlyFor research use or manufacturing of cell, gene, or tissue-based products
Catalog pack sizes1-10 L production scale1–100 L production scale
FormulationSame (medium requires GlutaMAX supplementation)Same (medium contains GlutaMAX)
PerformanceSameSame
Producer cell line
  • Research use only bank
  • Tested for mycoplasma, viability, and sterility
  • Product documentation includes COA
  • cGMP-banked
  • Tested for mycoplasma, viability, sterility, and human pathogens (adventitious agents)
  • Product documentation includes COA and COO
  • Cell line documentation package available
Regulatory support documentationNone
  • DMF
  • Regulatory Support File
GMP manufacturingManufactured at ISO 90001 or ISO 13485 certified sitesManufactured in conformance with GMP for medical devices, 21 CFR Part 820, USP<1043>, and Ph Eur 5.2.12. Manufactured at sites that are ISO 13485-certified

Components of the CTS AAV-MAX Helper Free AAV Production System

CTS Viral Production Cells (VPCs) 2.0

  • Clonal, 293F-derived, high-producing cell line
  • Optimized for high-density suspension culture (>12 x 106 cells/mL) in a chemically defined medium
  • No SV40 large T antigen or genetic engineering
  • Robust scalability and passage stability 
  • Documented, cGMP bank–manufactured as per 21 CFR 211 and EudraLex, Volume 4, and characterized as per ICH Q5A and ICH Q5D guidelines
Graph of the VPC 2.0 cell line growth curve
Figure 3. Viral Production Cells 2.0 maintain similar growth profiles over multiple passages. Cells were cultured in Viral Production Medium, and culture viability and VCD were measured from day 4 to day 7 post-seeding.

Medium

  • CTS Viral Production Medium
    • Animal origin–free
    • Chemically defined
    • Protein-free
    • Phenol-red free
    • Available off-the-shelf in pack sizes up to 100 L
    • Advanced Granulation Technology (AGT) formats
      • Granular dry media format
      • Animal origin–free
      • Chemically defined
      • Protein-free
      • Phenol-red free
      • Available off-the-shelf in pack sizes up to 100 L
      • AGT granules dissolve rapidly and are pre-adjusted for pH and osmolality
    Graph of the CTS AAV-MAX viable cell density and viability
    Figure 4. CTS Viral Production Medium supports the high density and robust growth of VPCs 2.0 for 7 days without additional nutrient support in multiple scales of cell culture. VPC2.0 were started in culture at 0.5 x 106 cells/mL in each size of culture and measure the growth for 7 days with Vi-Cell XR. All shake flasks used were Nalgene (Thermo Fisher Scientific) with the exception of the 5 L shake flask, which is a product of Corning.

    Transfection and production

    • CTS AAV-MAX Transfection Reagent and Transfection Booster (AOF, CD)
    • CTS AAV-MAX Enhancer (AOF, CD)
    • CTS Viral-Plex™ Complexation Buffer (AOF, CD, protein-free)

    Figure 5. Results of AAV production with and without added Enhancer. The Enhancer was added to prepared VPCs 2.0 with 3 x 106 cells/mL on the day of transfection (T=0). For transfection, a GFP-expressing transfer plasmid and helper plasmid were used with respective Rep/Cap plasmids. 72 hours post transfection, AAV-MAX Lysis buffer was added to the culture directly to harvest AAV for 2 hours at 37°C, followed by AAV titer measurement by qPCR. The data were normalized to the titer (vg/mL) of AAV production with Enhancer.

    Figure 6. Results of AAV production with and without added Booster during transfection. For transfection, a GFP-expressing transfer plasmid and a helper plasmid were used with respective Rep/Cap plasmids. 72 hours post transfection, AAV-MAX Lysis buffer was added to cultures directly to harvest AAV for 2 hours at 37C then followed by AAV titer measurement by qPCR. The data was normalized to the titer (vg/mL) of AAV production with Booster.

    Cells

    CTS Viral Production Cells (VPCs) 2.0

    • Clonal, 293F-derived, high-producing cell line
    • Optimized for high-density suspension culture (>12 x 106 cells/mL) in a chemically defined medium
    • No SV40 large T antigen or genetic engineering
    • Robust scalability and passage stability 
    • Documented, cGMP bank–manufactured as per 21 CFR 211 and EudraLex, Volume 4, and characterized as per ICH Q5A and ICH Q5D guidelines
    Graph of the VPC 2.0 cell line growth curve
    Figure 3. Viral Production Cells 2.0 maintain similar growth profiles over multiple passages. Cells were cultured in Viral Production Medium, and culture viability and VCD were measured from day 4 to day 7 post-seeding.
    Medium

    Medium

    • CTS Viral Production Medium
      • Animal origin–free
      • Chemically defined
      • Protein-free
      • Phenol-red free
      • Available off-the-shelf in pack sizes up to 100 L
      • Advanced Granulation Technology (AGT) formats
        • Granular dry media format
        • Animal origin–free
        • Chemically defined
        • Protein-free
        • Phenol-red free
        • Available off-the-shelf in pack sizes up to 100 L
        • AGT granules dissolve rapidly and are pre-adjusted for pH and osmolality
      Graph of the CTS AAV-MAX viable cell density and viability
      Figure 4. CTS Viral Production Medium supports the high density and robust growth of VPCs 2.0 for 7 days without additional nutrient support in multiple scales of cell culture. VPC2.0 were started in culture at 0.5 x 106 cells/mL in each size of culture and measure the growth for 7 days with Vi-Cell XR. All shake flasks used were Nalgene (Thermo Fisher Scientific) with the exception of the 5 L shake flask, which is a product of Corning.
      Transfection and production

      Transfection and production

      • CTS AAV-MAX Transfection Reagent and Transfection Booster (AOF, CD)
      • CTS AAV-MAX Enhancer (AOF, CD)
      • CTS Viral-Plex™ Complexation Buffer (AOF, CD, protein-free)

      Figure 5. Results of AAV production with and without added Enhancer. The Enhancer was added to prepared VPCs 2.0 with 3 x 106 cells/mL on the day of transfection (T=0). For transfection, a GFP-expressing transfer plasmid and helper plasmid were used with respective Rep/Cap plasmids. 72 hours post transfection, AAV-MAX Lysis buffer was added to the culture directly to harvest AAV for 2 hours at 37°C, followed by AAV titer measurement by qPCR. The data were normalized to the titer (vg/mL) of AAV production with Enhancer.

      Figure 6. Results of AAV production with and without added Booster during transfection. For transfection, a GFP-expressing transfer plasmid and a helper plasmid were used with respective Rep/Cap plasmids. 72 hours post transfection, AAV-MAX Lysis buffer was added to cultures directly to harvest AAV for 2 hours at 37C then followed by AAV titer measurement by qPCR. The data was normalized to the titer (vg/mL) of AAV production with Booster.

      Scaling the CTS AAV-MAX System

      Challenges associated with existing AAV production systems include low titers, high cost of cGMP plasmid DNA, poor scalability, and a lack of fit-for-purpose cGMP manufactured reagents. The CTS AAV-MAX system can help you overcome these challenges by allowing you to produce high AAV titers using preoptimized reagents backed by product-specific regulatory support and developed for AAV production in a scalable suspension platform that maintains volumetric viral titers as you scale up.

      Figure 7. AAV6 genome titers for AAV production in 250 mL shake flasks and 50 L bioreactors. The complete CTS AAV-MAX system was used to produce AAV6. Titers in crude samples were measured via ddPCR.

      Cell Therapy Systems (CTS) reagents for AAV-MAX production workflows

      Gibco Cell Therapy Systems (CTS) reagents are GMP-manufactured products designed for cell and gene therapy, so you can transition your therapy to the clinic with confidence.

      cGMP manufacturingTesting and documentationProven use
      Manufactured in conformance with GMP for medical devices, 21 CFR Part 820, USP<1043>, and Ph Eur 5.2.12.Manufacturing sites are FDA-registered, ISO 13485–certified, and regularly audited.

      Traceability documentation—including Drug Master Files (DMFs) and/or Regulatory Support Files (RSFs), Certificates of Origin (CoOs).

      Product safety testing—including sterility, endotoxin, and mycoplasmas on applicable products.

      Used in FDA-approved and EMA-approved CAR T therapies and the first FDA-approved therapeutic cancer vaccine. [1]

      Used in over 100 clinical trials.

      1. Madan RA and Gulley JL. (2011) Sipuleucel-T: harbinger of a new age of therapeutics for prostate cancer. Expert Rev Vaccines.

      Discover our research use AAV-MAX production system

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      Intended use of the products mentioned on this page vary. For specific intended use statements please refer to the product label.