GM AAV packaging services
- Efficient and Productive/ Unmatched Quality and Viability/ Diverse AAV Vector Grades/ Rapid Turnaround, High Purity, Enhanced Activity, and Superior Safety

Quick Quote AAV GOI Production Guide

GeneMedi excels in AAV packaging production, employing a meticulous AAV packaging protocol that integrates cutting-edge aav packaging plasmids and leverages the aav production process for maximum efficiency. Our state-of-the-art aav packaging mechanism ensures the delivery of AAV vectors with optimal packaging capacity, utilizing specialized aav packaging cell lines and aav production cell lines renowned for their reliability, such as the HEK293 cells. This choice underscores our commitment to aav packaging efficiency and the successful implementation of aav production in HEK293 cells, a method that has significantly advanced the field of gene therapy.

Our services are designed to navigate the complexities of the AAV packaging limit, facilitating the production of a wide range of AAV serotypes, including 4 aav, aav6, and aav9, each known for its distinct packaging capacity and applicability in various therapeutic contexts. The versatility of our aav manufacturing process, encompassing both raav production and scaav packaging capacity, allows us to meet the diverse needs of our clients, from early-stage research to clinical trials.

The core of our AAV vector production is the aav production workflow, which incorporates the highly efficient aav production triple transfection method. This advanced technique is a cornerstone of our ability to produce high-quality AAV vectors at scale, ensuring rapid turnaround times and adherence to the stringent quality standards required for clinical applications. Our GMP-grade AAV production protocol is meticulously designed to meet regulatory requirements, including the setup of GMP production lines and the preparation of IND pharmacological documentation, ensuring our products are ready for clinical trial applications.

At GeneMedi, we understand the critical role of AAV vector quality in the success of gene therapy projects. Our comprehensive range of AAV packaging services, from pilot to GMP grade, is tailored to support our clients throughout their research and development journey, ensuring the delivery of high-quality outcomes that cater to the specific requirements of each project. With a focus on customization, our skilled technical team works closely with each client, aligning our services with their unique needs and providing not just products, but solutions that contribute significantly to the success of their research and development efforts.

Advantages

GeneMedi is at the forefront of adeno-associated virus (AAV) vector production, offering a comprehensive array of AAV vector grades tailored to meet the diverse needs of both research and clinical endeavors. Our commitment to leveraging advanced proprietary technologies, optimized processes, and rigorous quality control measures positions us as a leader in the field, ensuring that our AAV vectors set the standard for efficiency, productivity, quality, and viability.

Efficient and Productive AAV Vector Production

Utilizing state-of-the-art technology and refined processes, GeneMedi achieves an unparalleled increase in AAV yield—enhancing it more than tenfold in HEK293 serum-free cell suspension systems. This remarkable efficiency not only accelerates production cycles but also meets the demands of urgent and large-scale projects, significantly reducing time from research to clinical application. Our enhanced production process boosts suspensive cell production by 10-25 fold, ensuring scalable operations that maintain a consistent supply of AAV vectors for expansive research initiatives and clinical trials.

Unmatched Quality and Viability

GeneMedi prioritizes the purity and viability of our AAV vectors. Through meticulous process development, we substantially minimize key impurities, such as host cell DNA (HCD) and endotoxins, ensuring each AAV vector batch meets high purity standards. This focus on quality reduces potential safety concerns and boosts therapeutic efficacy. Furthermore, our technological innovations lower the rate of empty capsids and increase the infection titer, enhancing the activity levels of our AAV vectors. This ensures high transduction efficiency and safety, crucial for the success of gene therapy treatments.

Diverse AAV Vector Grades for Varied Applications

GeneMedi offers an extensive range of AAV vector grades, including Pilot Grade for initial research phases, Research Grade for advanced studies, GMP-like Grade for preclinical trials, and GMP Grade for clinical applications. This versatility allows researchers and clinicians to select the most appropriate vector quality for their project's specific phase, ensuring compliance with regulatory standards when necessary.

Rapid Turnaround, High Purity, Enhanced Activity, and Superior Safety

Our streamlined production process, combined with cutting-edge technology, facilitates rapid turnaround times, critical for maintaining project schedules and achieving faster bench-to-bedside transitions. GeneMedi's AAV vectors are characterized by their high purity, achieved through advanced purification processes, and their enhanced activity, ensured by production techniques that increase the infectious titer. Together with a focus on minimizing the rate of empty capsids and reducing unwanted impurities, our vectors exhibit a superior safety profile, laying a reliable foundation for therapeutic applications.

GeneMedi's dedication to providing AAV vectors with rapid production cycles, high purity, enhanced activity, and elevated safety standards reflects our commitment to advancing gene therapy research and applications. Our ability to offer a wide range of vector grades, combined with our emphasis on efficiency, productivity, quality, and viability, establishes GeneMedi as a trusted partner in the scientific and medical communities, supporting the success of research projects and clinical trials globally. Our approach not only meets but exceeds client expectations, driving forward the potential of gene therapy with high-quality, reliable vector solutions.

Quality Levels of AAV

			Pilot Grade	Research Grade	GMP-like Grade	GMP Grade
US upstream
	Packaging cell type		HEK293 three-plasmid transfection system or baculovirus/insect system	HEK293 three-plasmid transfection system or baculovirus/insect system	HEK293 three-plasmid transfection system or baculovirus/insect system	HEK293 three-plasmid transfection system or baculovirus/insect system
	Wall-mounted or suspended		Wall-mounted or suspended	Wall-mounted or suspended	Wall-mounted or suspended	Wall-mounted or suspended
	Cell bank		——	——	——	TBD
	Plasmid bank		——	——	——	TBD
	Minimum amount		——	——	TBD	TBD
DS downstream	QC project	method
Purification method			Crude purification	Iodixanol ultracentrifugation or Affinity chromatography	Tangential Flow Filtration+Affinity chromatography + anion chromatography	Tangential Flow Filtration+Affinity chromatography + anion chromatography
General inspection (physical inspection)	Appearance, clarity, visible foreign matter, insoluble particles, pH value, osmotic pressure, loading volume	Appearance-Light Inspection Visible foreign matter - light inspection Insoluble particles-resistance method pH-potentiometric method Osmolarity - Freezing point method Loading volume-capacity method	Appearance： Visual inspection"Clear solution, free of particulates"	Appearance： Visual inspection"Clear solution, free of particulates"	Appearance： Visual inspection"Clear solution, free of particulates" Visible particulates： Lamp inspection Subvisible particulates： Light blockage method pH： pH meter Product specific Osmolality： Cryoscopic method Excipient specific Filled volume： Volumetric Method-Meets USP/ChP Requirements	Appearance： Visual inspection"Clear solution, free of particulates" Visible particulates： Lamp inspection Subvisible particulates： Light blockage method pH： pH meter Product specific Osmolality： Cryoscopic method Excipient specific Filled volume： Volumetric Method-Meets USP/ChP Requirements
Identify	AAV genome sequence	Restriction enzyme map analysis	——	——	The result is subject to no mismatch	The result is subject to no mismatch
		Polymerase chain reaction (PCR)	——	——
		Reverse transcription-polymerase chain reaction (RT-PCR)	——	——
		Nucleic acid sequence determination	——	——
	Capsid protein identification	SDS-PAGE combined with Western blot to identify capsid protein VP1/2/3	——	VP1：VP2：VP3=1：1：10	VP1：VP2：VP3=1：1：10	VP1：VP2：VP3=1：1：10
	Identification of intact virus particles	Structural analysis, particle size distribution, refractive index analysis under electron microscope	——	——	Aggregates-TEM (negative stain)≤10%	Aggregates-TEM (negative stain)≤10%
Content and potency	Virus particle titer (V.P.)	ELISA	——	——	Report result/~1E14 VP/ml	Report result/~1E14 VP/ml
	Genomic Titer (V.G.)	ddPCR OR QPCR		The titer detected by Q-PCR is ≥ the protocol amount	Reported result/~1-5E13 VG/mL	Reported result/~1-5E13 VG/mL
	Total protein	BCA	——	——	Reported result	Reported result
	Protein expression (Cell)	ELISA/FIX	——	——	TBD	TBD
	Infectious titer (I.P.)	TCID50	——	——	Report result/1-5E11 TCID50/ml	Report result/1-5E11 TCID50/ml
	Specific activity	Specific activity = genome titer/infectious titer	——	——	<100	<100
	Potency - Genomic Expression	Detection of in vitro mRNA expression or protein expression of cells based on qPCR, ELISA, and Western Blot	——	——	Potency Content UV 0.8-1.2 mg/mL	Potency Content UV 0.8-1.2 mg/mL
	potency-activity	Bio-functional assay	——	——	In Vitro Potency- (between 1-15) vg/cell	In Vitro Potency- (between 1-15) vg/cell
Purity	Capsid protein purity test	SDS-PAGE electrophoresis combined with silver staining	——	SDS-PAGE ≥95%, VP1: VP2: VP3=1:1:10, no obvious contaminants were detected.	SDS-PAGE ≥95%, VP1: VP2: VP3=1:1:10, no obvious contaminants were detected.	SDS-PAGE ≥95%, VP1: VP2: VP3=1:1:10, no obvious contaminants were detected.
	Empty shell rate detection	ddPCR/ ELISA	——	——	Report result	Report result
		Transmission electron microscope (TEM)	——	——	Report result	Report result
		Analytical ultracentrifugation (AUC)	——	——	Report result	Report result
	Genomic aggregation detection	Size exclusion high performance liquid chromatography (SEC-HPLC method)	——	——	Report result	Report result
Security check	Replication-Competent Adeno-associated virus, or rcAAV for short, is an important evaluation indicator of product safety.	DNA immunoblotting (Southern blot) and qPCR	——	——	qPCR Negative	qPCR Negative
	Sterility test	membrane filtration	——	No growth	No growth	No growth
	Bacterial endotoxin test	Limulus reagent	——	TBD	LAL assay<10 EU/ml	LAL assay<10 EU/ml
	Mycoplasma	Culture method and PCR method	——	TBD	Negative	Negative
	exogenous factors	Culture	——	——	Negative	Negative
	bioburden	Culture	——	——	Report result	Report result
	Abnormal toxicity check	Animal experiment	——	——	Meets ChP Requirement	Meets ChP Requirement
Impurities	help DNA plasmid residue	ddPCR/qPCR	——	——	≤50ng/ml	≤50ng/ml
	Host cell DNA residue	ddPCR/qPCR	——	——	≤50 ng/ml	≤50 ng/ml
	Residual E1A/E1B DNA(copies/dose)	ddPCR/qPCR	——	——	≤(5-10) x 10^4 copies/mL E1A; (5-10) x 10^4 copies/mLE1B	≤(5-10) x 10^4 copies/mL E1A; (5-10) x 10^4 copies/mLE1B
	host cell protein residue	ELISA	——	——	≤0.5ug/10E^12/ml	≤0.5ug/10E^12/ml
	Bovine serum albumin residue	ELISA	——	——	≤50ng/ml	≤50ng/ml
	Residual Benzonase	ELISA	——	——	≤10 ng/ml	≤10 ng/ml
	Residues related to specific processes (such as iodixanol, transfection reagent, affinity ligand, Tween 20, detergent TritonX100, etc.)	HPLC	——	——	Report result	Report result

Note：

Virus particle titer (V.P.): Enzyme-linked immunosorbent assay (ELISA), transmission electron microscopy (TEM), high-performance liquid chromatography (HPLC), analytical ultracentrifugation (AUC), etc. are common measurement methods. Among them, TEM and AUC are generally only used to detect empty shell AAV. Ratio of particle titer to solid AAV particle titer.

Genomic Titer (V.G.): qPCR and ddPCR are used to measure the genome titer. During the measurement, the DNA outside the AAV is first degraded by enzymes, and only the DNA packaged inside the virus particles is measured.

Infectious titer (I.P.): TCID50 (tissue culture half-infectious dose) is a commonly used indicator for virus titer evaluation.
Replication-Competent Adeno-associated virus, or rcAAV for short, is an important evaluation indicator of product safety.: Sensitive cells are used for multiple rounds of infection and amplification in the presence of helper virus. Genome extraction is performed after cell lysis. Finally, DNA immunoblotting (Southern blot) and qPCR are used to determine rep (regulatory genes) or cap (structural genes).

Host cell DNA residue: For ddPCR/qPCR, AAV, adenovirus and plasmid DNA residues produced by the 293 system are required to be checked, while the Bac system does not have this requirement. The detection items for the Bac system are SF9 residues and BEV-related baculovirus and rhabdovirus residues.

potency-activity： Detect virus activity based on cell-based in vitro biological activity and animal

Production steps of AAV

Upstream

1. Cell recovery:
It involves extracting cultured cells from a bioreactor to ensure their viability and readiness for subsequent production steps.

2. Cell inheritance:
It is a critical process for maintaining a healthy and sustainable cell population that involves transferring cells to fresh culture medium and allowing them to proliferate, ensuring a consistent and viable cell line for subsequent production steps.

3. Reactor culture:
It is the core step of AAV production. Genetically modified cells are cultured in bioreactors to maximize the production of target AAV vectors by optimizing growth parameters.

4. Plasmid transfection:
Transfecting cultured cells with viral vector DNA to initiate the synthesis of AAV particles and encourage the expression of the necessary genetic material is known as plasmid transfection.

5. Culture feed:
It is adding nutrients and growth factors to the culture medium to maintain optimal conditions for AAV vector production and ensure the continued health and efficient production of cells during the culture process.

6. Virus harvest:
It is the final step upstream and involves collecting AAV particles from cultured cells. Typically involving cell lysis and subsequent purification steps, a crude AAV vector product is obtained.

Downstream

1. Ultrafiltration and concentration:
It is a downstream processing step designed to concentrate harvested AAV particles. Through this technology, excess impurities are effectively removed and the volume of the material is reduced to prepare for further purification steps.

2. Crude chromatography:
It is a purification step in which concentrated AAV vectors are processed through chromatographic separation. This process helps separate the carrier from residual impurities, resulting in a partially purified product.

3. Refined chromatography:
It is a subsequent purification step aimed at obtaining a higher purity vector. Through additional chromatography technology, residual impurities are removed and a highly purified AAV vector product is obtained.

4. Solution replacement:
It involves changing the buffer composition of the AAV vector solution to optimize its compatibility with the selected purification technology and provide appropriate conditions for subsequent downstream steps.

5. Sterilizing filtration:
It is a critical step to ensure the elimination of potential contaminants. AAV vector solutions are filtered to remove bacteria or other unwanted particles, maintaining product integrity.

6. Canned finished product:
It is the final step in which the purified and sterilized AAV vector is carefully packed into appropriate containers. This step prepares the product for storage, distribution, and eventual use in therapeutic applications.