Question 1

What is the difference between vg/mL and GC/mL for AAV titer?

Accepted Answer

vg/mL (viral genomes per mL) and GC/mL (genome copies per mL) measure the same thing — the number of vector DNA molecules per milliliter — and are numerically equivalent. vg/mL is the convention in process development and clinical dosing, while GC/mL is used in qPCR and ddPCR analytical reports. Both are determined by quantitative PCR targeting a sequence in the transgene cassette (typically ITR or transgene-specific primers).

Question 2

How do I convert AAV capsid titer to genome titer?

Accepted Answer

Genome titer (vg/mL) = capsid titer (capsids/mL) × fraction of full capsids. For example, if your ELISA reports 1 × 10^13 capsids/mL and AUC analysis shows 20% full capsids, the genome titer is 2 × 10^12 vg/mL. The full:empty ratio varies by serotype and production method — typically 10-30% full for classical triple-transfection AAV, and higher (30-50%) for optimised baculovirus or stable producer systems.

Question 3

What is the typical full:empty capsid ratio for AAV?

Accepted Answer

For standard HEK293 triple-transfection production, typical full:empty ratios are 10-30% full capsids depending on serotype, with AAV2 often at the higher end (20-30%) and AAV8/AAV9 sometimes lower (10-20%). After AEX polishing (e.g., CIMmultus QA or POROS CaptureSelect), ratios can be enriched to 50-80% full. AUC (analytical ultracentrifugation) and charge-detection mass spectrometry (CDMS) are the gold-standard methods for measuring full:empty ratio.

Question 4

How do I convert AAV titer to transducing units (TU/mL)?

Accepted Answer

Transducing units (TU/mL or IU/mL) measure functionally infectious particles, determined by transduction assays (e.g., GFP+ cells by flow cytometry). The vg:TU ratio (also called particle-to-infectivity ratio) is serotype- and cell-type-dependent: typically 100:1 to 1000:1 for AAV2 on HEK293, 1000:1 to 10,000:1 for AAV8/AAV9 on HEK293 (these serotypes transduce HEK293 poorly but work well in vivo). TU/mL = vg/mL ÷ vg:TU ratio.

Question 5

How do I calculate the dose volume for an AAV gene therapy?

Accepted Answer

Dose volume (mL) = (target dose in vg/kg × patient body weight in kg) ÷ product titer in vg/mL. For example, a 1 × 10^14 vg/kg dose for a 70 kg adult from a 2 × 10^13 vg/mL product requires (1 × 10^14 × 70) ÷ (2 × 10^13) = 350 mL. Clinical AAV doses range from ~10^11 vg/kg (intrathecal/intravitreal) to ~10^14 vg/kg (systemic IV for neuromuscular diseases like SMA).

Question 6

What AAV titer methods are used in GMP manufacturing?

Accepted Answer

GMP release testing typically includes: (1) qPCR or ddPCR for genome titer (vg/mL) — ddPCR is increasingly preferred for its absolute quantification without standard curves; (2) Capsid ELISA (e.g., Progen AAV titration kits) for total capsid concentration; (3) AUC or CDMS for full:empty ratio; (4) In vitro potency assay (cell-based transduction) for functional titer. The combination of genome titer and capsid ELISA gives the vg:capsid ratio, which is an orthogonal measure of full:empty fraction.

Serotype	Typical Full:Empty (%)	vg:TU Ratio (HEK293)	Primary Tropism	Clinical Dose Range
AAV2	15-30%	100-500:1	Broad (liver, muscle, CNS)	10¹¹-10¹³ vg/kg
AAV5	10-20%	1,000-5,000:1	Liver, CNS, lung	10¹³-10¹⁴ vg/kg
AAV8	10-20%	3,000-10,000:1	Liver, muscle	10¹³-10¹⁴ vg/kg
AAV9	8-18%	3,000-10,000:1	CNS, muscle, heart, liver	10¹³-10¹⁴ vg/kg
AAVrh10	10-20%	1,000-5,000:1	CNS, liver	10¹³-10¹⁴ vg/kg

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