GENE THERAPY

AAV Purification: Separate Empty and Full Capsids with MCSGP

Continuous AIEX polishing for full capsids.

Improve AAV purification by recovering the full capsids that batch AIEX discards in overlapping side fractions. Published on AAV2 by two independent research groups: 5× Higher full capsid yield | 2.5× Productivity | 3× Lower process mass intensity. Develop on the Contichrom® CUBE and transfer it to the TWIN LPLC for manufacturing.

Müller et al., Biotechnol. Bioeng., 2025; Grob et al., J. Chromatogr. A, 2025 (independent AAV2 AEX studies)

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Read the Latest AAV2 Paper

AAV viral vector purification — illustration

Talk to Our Gene Therapy Specialists

Read the Latest AAV2 Paper

How AAV Purification Works: From Capture to Polishing

A chromatography-based AAV purification process typically includes harvest clarification, concentration and buffer exchange, affinity capture, and polishing. Affinity chromatography concentrates the viral vector and removes many process-related impurities, while AEX polishing separates genome-containing full capsids from empty capsids.

ChromaCon’s MCSGP technology addresses the high-resolution polishing stage by continuously recycling overlapping empty/full capsid fractions. CaptureSMB is also being developed for continuous AAV affinity capture, allowing capture and polishing methods to be evaluated on the same Contichrom® platform.

The AAV Downstream Bottleneck: Empty Capsids

Adeno-associated virus (AAV) is the leading vector platform in gene therapy — with multiple approved products (Luxturna®, Zolgensma®, Hemgenix®, Roctavian®, Elevidys®) and over 130 clinical trials underway. But manufacturing recombinant AAV at the yields and quality needed for clinical and commercial supply remains a defining challenge.

A critical problem is the high proportion of empty capsids in every AAV harvest. Empty capsids — identical in size and shape to full, genome-containing capsids but lacking therapeutic DNA — routinely comprise 50–95% of total viral particles in crude material. They are therapeutically inactive and at high levels may trigger immune responses and increase patient dosing burden.

Empty and full AAV capsids are near-identical in size and surface chemistry, differing only slightly in isoelectric point — meaning they co-elute across a narrow, overlapping zone in anion exchange chromatography. Full capsid yield using Batch AEX chromatography can be very low, leaving much room for optimization of the polishing step.

The Solution: MCSGP with AutoPeak® for AAV Full Capsid Enrichment

MCSGP (Multi-column Countercurrent Solvent Gradient Purification) resolves the yield-purity trade-off that makes batch AEX polishing of AAV so wasteful. Two identical columns operate in a continuous countercurrent mode — recycling the overlapping empty/full capsid fractions that batch chromatography discards. The process uses the same AEX resins, buffers, and gradient conditions as your existing batch AAV method — these conditions are the starting point for MCSGP process development.

AutoPeak® dynamic process control monitors the AAV elution profile in real-time via the UV260/280 absorbance ratio — a reliable online indicator of the empty/full capsid transition zone — automatically adjusting the recycling and collection windows to maintain robust, unattended full-capsid enrichment.

Same resin. Same buffers. Superior performance. MCSGP works with the AEX resins and gradient methods already validated for your AAV serotype — Capto Q, POROS HQ, Fractogel TMAE, or others. The online UV260/280 ratio provides a reagent-free PAT signal for the empty/full transition, enabling AutoPeak® to deliver consistent full-capsid enrichment run-to-run, even as upstream empty/full ratios fluctuate.

Learn About MCSGP + AutoPeak®

Continuous chromatography - MCSGP — illustration

AAV Purification Results: MCSGP vs. Batch AEX

5×

Higher Full Capsid Yield

2.5×

Higher Productivity

3×

Lower Process Mass Intensity (PMI)

30% → 68%

Full Capsid Enrichment

Case Study 1: Proof-of-Concept MCSGP Enrichment of Full AAV2 Capsids

Setting: AAV2 purification from HEK293 cell lysate via anion exchange chromatography. Starting material with a challenging empty/full capsid ratio.

Batch AEX starting point:

Full capsid content: ~30% (ddPCR) / ~27% (cryo-TEM)
Product loss in side-fractions: significant — full capsids recovered at low yield

MCSGP result (6 cycles, same AEX conditions):

Full capsid content: 68% (ddPCR) / 61% (cryo-TEM) — more than doubling full capsid purity
Full capsid recovery: substantially higher than batch — yield-purity trade-off overcome
Online UV260/280 ratio monitoring: successfully identified the empty/full transition zone in real time; confirmed by offline multi-angle light scattering (MALS) and cryo-TEM

Key finding: MCSGP process development starts from your validated batch AEX method — resin, buffers, and gradient conditions are preserved. The online UV260/280 ratio provides a practical, reagent-free PAT signal suitable for AutoPeak® integration. Both analytical techniques (ddPCR and cryo-TEM) confirmed the enrichment — validating the UV ratio as an accessible process monitoring tool for full/empty capsid content.

Müller, J. et al. Biotechnol. Bioeng. (2025). PubMed: 40468522

Case Study 2: Model-Based Design and Implementation of MCSGP for AAV2 Empty/Full Separation

Setting: AAV2 purification via anion exchange chromatography, Purity specification (Pspec) = 70.0% full capsids.

Batch AEX baseline:

Yield: ~10–11% full capsids at 70% purity
Productivity: 3.85×10¹³ VP/(L_resin·h) with 20 CV gradient
Shallow gradients (30 CV) improve yield slightly (to 11.3%) but dramatically reduce productivity — a hard trade-off

MCSGP result (same AEX resin, same Pspec):

Yield: 57.8% — approximately 5× higher than batch
Productivity: 8.65×10¹³ VP/(L_resin·h) — approximately 2.5× higher than the productivity-optimized batch case
Process mass intensity (PMI): reduced to 1/3 of batch — 3-fold reduction in buffer consumption
Both high yield and high productivity achieved simultaneously — the trade-off eliminated

Key finding: MCSGP is the only technique that simultaneously improves yield, productivity, and sustainability metrics for the AAV empty/full separation. The study validated MCSGP performance through model-based process design, substantially reducing experimental effort for method transfer.

J. Chromatogr. A (2025). DOI: 10.1016/j.chroma.2025.465001

CaptureSMB for Continuous AAV Affinity Capture (Coming Soon)

For AAV programs using commercial affinity resins (POROS CaptureSelect AAVX, AAV8, AAV9; Capto AVB; AVIPure®), CaptureSMB extends twin-column periodic countercurrent chromatography to the capture step — the same principle that delivers 2.5× Protein A productivity for mAbs.

In CaptureSMB mode, two affinity columns operate in a coordinated interconnected and parallel cycle. The lead column loads beyond its single-pass breakthrough point; any breakthrough is captured on the second column in series. This overcomes a key challenge of AAV affinity capture: low dynamic binding capacity relative to the large capsid size (~25 nm) limits resin utilization in conventional batch loading.

Potential benefits for AAV affinity capture:

Higher resin utilization — load beyond the breakthrough point without product loss
Higher productivity per column volume — relevant given the high cost of specialty AAV affinity resins
Reduced buffer consumption — fewer equilibration cycles per gram captured
Smaller column footprint — important in scale-limited GMP environments

CaptureSMB for AAV affinity capture is in active development. Contact our application specialists for a feasibility assessment. The CUBE supports both CaptureSMB and MCSGP, making it the ideal platform for developing and comparing both steps on the same instrument.

AAV Purification: Batch AEX vs. MCSGP

Parameter	Batch AEX	MCSGP (Contichrom)
Full capsid yield (at 70% purity spec)	~10–11%	~58% (+5×)
Productivity	3.85×10¹³ VP/(L_resin·h)	8.65×10¹³ VP/(L_resin·h) (+2.5×)
Process mass intensity (buffer)	Baseline	3× lower
Full capsid enrichment	Limited by center-cut	30% → 68% (ddPCR)
Yield-purity trade-off	Hard constraint — cannot improve both	Eliminated
Process continuity	Batch cycles, idle time	Continuous, 24/7
Resin / method change	Current validated method	Same resin, same gradient
Online monitoring (PAT)	Offline analytics only	UV260/280 ratio — real time
Process variability compensation	Manual re-optimization	AutoPeak® automatic adjustment

💡 How MCSGP Improves AAV Purification Yield and Productivity

In batch AEX, lengthening the gradient to recover more full capsids proportionally reduces productivity — the two objectives are fundamentally opposed. MCSGP resolves this by recycling the overlapping fractions, allowing both to be maximized independently. This is particularly critical for AAV, where upstream empty capsid content is inherently variable and often unpredictable — a challenge that AutoPeak® continuously adapts to in real time.

AAV Purification Systems from Development to GMP Manufacturing

All systems run MCSGP and CaptureSMB with UV260/280 ratio monitoring for empty/full capsid detection. Methods scale predictably from CUBE → TWIN LPLC.

Systems Overview

$Contichrom CUBE multi-column FPLC chromatography system with UV detectors and FOXY fraction collector$

Contichrom® CUBE

The essential platform for chromatography process development. This versatile 100-bar benchtop FPLC system runs batch, integrated batch, and continuous chromatography — including MCSGP and CaptureSMB — out of the box. Develop purification processes for mAbs, peptides, oligonucleotides, ADCs, and viral vectors, with verified scale-up at over 100× to pilot and production systems.

Contichrom TWIN LPLC continuous chromatography system for capture and polishing

Contichrom® TWIN LPLC – Capture

Accelerate biopharmaceutical capture chromatography at production scale. This sanitary GMP system uses CaptureSMB® with AutomAb® to maximize resin utilization — achieving up to 3× faster processing and 50% less Protein A resin and buffer consumption compared to batch. CIP-ready design for mAbs, viral vectors, ADCs, and recombinant proteins.

Contichrom® TWIN LPLC – Polishing

Bring continuous polishing to biopharmaceutical manufacturing at production scale. This sanitary GMP system uses MCSGP with AutoPeak® to eliminate the yield-purity trade-off achieving +10–50% absolute yield increase at target purity while reducing buffer consumption compared to batch. CIP-ready design for mAbs, viral vectors, ADCs, and recombinant proteins.

Systems Overview

MCSGP Applications Across AAV Purification

Use Case 1 — AAV2 Purification by Polishing (AEX)

The most thoroughly published serotype for MCSGP-based empty/full separation. Two independent 2025 publications demonstrated 5× full-capsid yield improvement and 2.5× productivity gain vs. batch AEX. Directly applicable to any AAV2 program currently using AEX polishing.

Use Case 2 — MCSGP Development for AAV5, AAV8, AAV9 and Other Serotypes

MCSGP applies to any serotype for which an AEX gradient method achieves at least partial empty/full resolution. The CUBE development platform enables rapid method screening and adaptation for new serotypes without published separation data.

Use Case 3 — AAV Purification for Variable Upstream Feeds

AAV empty/full ratios vary batch-to-batch based on cell system, transfection efficiency, and serotype. AutoPeak® compensates in real time using the UV260/280 ratio — maintaining consistent full-capsid output without manual re-optimisation between runs.

Use Case 4 — Integrated AAV Affinity Capture + AEX Polishing

For programs using pan-AAV affinity resins, MCSGP replaces or augments batch AEX polishing. A complete two-step continuous chromatography solution applicable to the broadest range of AAV programs.

Getting Started with MCSGP for AAV Gene Therapy

See the Contichrom® platform before you commit. Computationally assess MCSGP suitability for your AAV AEX separation using your existing batch data and UV260/280 fraction analysis. Develop your continuous method on a Contichrom CUBE with integrated empty/full capsid discrimination, then scale to the TWIN LPLC for GMP production.

See It First — Free Demo or Webinar

Not yet familiar with the Contichrom® platform? Start here. YMC ChromaCon offers no-charge demonstrations and introductory webinars tailored to your team — no starting material, no commitment required.

On-site in Zurich (half day, 2–4 hours): see the CUBE in person, run ChromIQ® live, discuss your AAV capsid separation challenge with YMC ChromaCon application scientists
Remote webinar (1–3 hours): interactive live session covering MCSGP, CaptureSMB, N-Rich, ChromIQ® software, and the full Contichrom® ecosystem
Sessions can focus on MCSGP for AEX-based empty/full capsid separation and the AutoPeak® UV260/280 PAT control concept

→ Request a free demo or introductory webinar

Modeling Assessment for AAV

Predict MCSGP performance on your AAV AEX separation before running any new experiments. YMC ChromaCon evaluates applicability and estimates expected capsid yield improvement from your existing batch data.

Submit your single-column AEX batch chromatogram, gradient, resin, and column dimensions — along with UV260/280 absorbance data from your batch fractions
YMC ChromaCon confirms MCSGP applicability for your serotype, estimates the expected improvement in empty capsid depletion, and identifies key process parameters for your purity specification
Data can come from any HPLC or FPLC system — no Contichrom® required
Typical timeline: 3–5 weeks from receipt of data; no material shipment required

→ MCSGP Process Modeling Service

Develop at Lab Scale — Rent or Purchase a CUBE

MCSGP method development is performed on the Contichrom CUBE using your existing AEX resin and buffer system. Available for rental or purchase.

ChromIQ® MCSGP Wizard builds the initial MCSGP operating point from your batch AEX chromatogram and UV260/280 fraction data
AutoPeak® is configured using the online UV260/280 absorbance ratio as the PAT signal for the empty/full capsid transition zone — enabling real-time, automated empty capsid discrimination without offline analytics between runs
Development typically completed in a few days of experimental work; no iterative batch screening required
Upstream affinity capture can also be developed as CaptureSMB on the same CUBE, enabling an end-to-end continuous downstream workflow

→ Contichrom CUBE Rental Program

→ Purchase a Contichrom CUBE

Scale to GMP Production — Contichrom TWIN LPLC

The MCSGP method developed on the CUBE transfers directly to the TWIN LPLC for manufacturing-scale AAV production. ChromaCon provides scale-up consulting and GMP documentation support.

TWIN LPLC Polishing for continuous MCSGP-based empty/full capsid separation at production scale — AutoPeak® UV260/280 control logic is preserved from CUBE development
TWIN LPLC Capture for upstream affinity capture (if applicable), enabling an end-to-end continuous downstream process on the same platform family
Parameters scale predictably — column chemistry, gradient conditions, and AutoPeak® control logic are all maintained at scale
GMP documentation support (IQ/OQ/PQ) and scale-up consulting available for regulatory submissions

→ Scale-Up Consulting and Training

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Ready to Increase Full Capsid Yield in Your AAV Process?

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How does MCSGP fit into an AAV purification process?

MCSGP is primarily used during the AEX polishing stage of AAV purification. After affinity capture has concentrated the vector and removed many process-related impurities, MCSGP separates overlapping empty and full capsid populations while recycling fractions that conventional batch AEX would discard.

Can MCSGP separate empty and full AAV capsids from serotypes other than AAV2?

Yes — MCSGP applies to any serotype where AEX gradient chromatography achieves at least partial empty/full resolution. Published proof-of-concept is on AAV2 (Müller et al. 2025; Grob et al. 2025). The Contichrom CUBE enables rapid method adaptation to AAV5, AAV8, AAV9, and engineered capsid variants.

Does MCSGP require a different resin or buffer system from my current AEX polishing method?

No. MCSGP uses the same anion exchange resin, buffers, and gradient conditions as your existing validated batch AEX method. Transitioning from batch to MCSGP requires only translation of the batch gradient into an MCSGP operating strategy — ChromaCon’s application specialists support this as part of the standard feasibility assessment.

How does AutoPeak® monitor the empty/full capsid transition during MCSGP?

AutoPeak® uses the real-time UV260/280 absorbance ratio as the primary PAT signal. Empty capsids have a lower UV260/280 ratio than full capsids. As elution transitions through the mixed zone, AutoPeak® detects the shift online and automatically defines recycling window boundaries — confirmed against cryo-TEM and multi-angle light scattering (Müller et al. 2025).

What full-capsid purity and yield can I expect from MCSGP?

At a 70% full-capsid purity specification, MCSGP achieved ~58% yield versus ~10–11% for batch — a ~5× improvement (Grob et al. 2025). Starting from 30% full capsids, Müller et al. (2025) achieved 68% full-capsid content. Expected outcomes for your program depend on starting material and target purity; ChromaCon’s feasibility assessment provides molecule-specific projections.

How many MCSGP cycles are needed, and how long does a run take?

In Müller et al. (2025), 6 MCSGP cycles achieved the reported enrichment. Cycle number and run time depend on feed volume, column size, gradient length, and target purity. Method development on the Contichrom CUBE typically requires one to a few days. Manufacturing-scale batch duration is modeled as part of a feasibility study.

Is there an empty/full ratio specification I need to meet for regulatory filing?

FDA guidance and ICH Q6B call for characterization of capsid content as a critical quality attribute. Many programs target ≥90% full capsids in the final drug substance. MCSGP with AutoPeak® enables reproducible full-capsid enrichment from variable upstream feeds — directly supporting lot-to-lot consistency and regulatory compliance.

Can the same Contichrom system run both MCSGP polishing and CaptureSMB capture?

Yes. Both the CUBE and TWIN LPLC support MCSGP and CaptureSMB on the same hardware, switchable via ChromIQ® software. A single instrument covers affinity capture development (CaptureSMB) and polishing development (MCSGP) — enabling a fully integrated continuous two-step purification strategy.

What are the scale-up options for clinical and commercial AAV manufacturing?

The Contichrom TWIN LPLC covers clinical through commercial scale (column volumes from 100 mL to 2 L). Scale-up from CUBE to TWIN LPLC is linear — column chemistry, gradient conditions, and AutoPeak® control logic are preserved. cGMP design, 21 CFR Part 11 compliance, and IQ/OQ/PQ documentation packages are available.