Dr. Barrett's Bench to Batch Advice

Every AAV program comes with critical manufacturing decisions and a labyrinth of options. In this advice column, our Head of Technical Sales and Scientific Advisory, Dr. Brianna Barrett, Ph.D., answers real developer questions about program manufacturing strategy with practical guidance shaped by supporting over 60 gene therapy programs. Consider it direct access to a manufacturing strategist from early planning through delivery.

Got a question? Email Dr. Barrett!  

Dear Dr. Barrett,

We are interested in gaining real-time insights to empty vs. full AAV capsid content throughout our process. We've heard of mass photometry; is this commonly used at your site? How does this compare to the use of analytical ultracentrifugation?
— Real-Time Ratios

Dear Real-Time Ratios,

Mass photometry (MP) is gaining real traction for exactly this purpose, and I understand why teams are drawn to it. The technique measures individual particles as they land on a glass surface, converting light scattering into mass with single-molecule resolution. Because empty and full capsids differ in mass by roughly the weight of the packaged genome, you get a clean separation between populations in a matter of minutes, using only microliters of sample and no labeling.

That speed is the real value for in-process use. MP will not replace your release assay, but it does not need to. What it offers is a fast read at critical points in the process (post-transfection, after affinity capture, across AEX or CsCl fractions) so you can make real-time decisions about pooling, loading, or when a step has reached its endpoint, rather than waiting on a slower orthogonal method to confirm what already happened.

Analytical ultracentrifugation (AUC) remains the gold standard for release because it resolves subpopulations (partial genomes, aggregates, dimers) with a precision mass photometry cannot fully replicate, and it carries the validation history regulators expect on a CoA. The two methods work well together: AUC anchors your final specification, while MP gives you eyes on the process as it runs, becoming a crucial insight during development of a process.

With FULL confidence,

— Dr. Barrett

August 2026

Dear Dr. Barrett,

What manufacturing considerations are there when advancing a gene therapy program from Phase 1/2 clinical to Phase 3/pivotal trials?

—Pivoting to Pivotal

Dear Pivoting to Pivotal,

Great question, and one that catches a lot of programs off guard. Phase 1/2 manufacturing is built for speed and flexibility. Phase 3 manufacturing has to be built for consistency, and comparability, and that shift touches nearly every part of the process. A few areas to focus on:

Process lock and comparability. Incorporating process changes from early manufacturing learnings can result in substantial program gains. However, the key to late-phase is process lock of your commercial representative process. Strategically incorporating process changes early to reduce rework and additional regulatory paperwork can make the transition to late-phase smooth. All process changes should be assessed for potential impact to product comparability, such as potency, purity, and impurity profiles.

Analytical readiness. Assays that were fit for purpose or assessed as ‘report result’ on an early phase CoA, need to mature into qualified, and then validated, commercial ready methods with tighter specifications. A phase appropriate framework can still be utilized as you advance through the clinical phases towards commercial. To release a pivotal lot, a validated ddPCR titer assay is typically recommended, while most other product specific analytics may be working through qualifications or pre-validation stages. Regulatory bodies may also provide additional guidance specific to your product.

Raw material program. A phase appropriate raw material program will increase the rigor around sampling and testing of input materials. A risk assessment of all materials on your locked bill of materials (BOM) will guide appropriate testing plans for your process. For a pivotal manufacturing run, identification testing is required for all raw materials used and an assessment of raw materials potentially impacting critical quality attributes is also beneficial. The lesson: planning for pivotal is a large step towards planning for commercial. Whatever you can lock, characterize, and qualify early in development, before Phase 3, will streamline your pivotal readiness and guide you to a safe product that passes comparability assessments.

— Dr. Barrett

July 2026

Dear Dr. Barrett,

We see replication competent AAV (rcAAV) on the standard clinical release panel at many CDMOs. Is there anything we can do early on to ensure this assay passes as we scale and transition to cGMP?

— rcAAV: So Yesterday

Dear rcAAV: So Yesterday

Way to think ahead! You are correct, for safety, every clinical AAV release panel has a line for rcAAV, even though you almost never see it at the bench. That's exactly what makes it challenging. It is vanishingly rare per molecule, but it becomes real once you are transfecting grams of plasmid into hundreds of liters of cells. Let’s quickly cover how rcAAV forms, and why the fix lives in the molecular design of the input plasmids.

How Does rcAAV Form?
For the therapeutic rAAV the genome we package is the GOI flanked by the two ITRs, which carry the packaging signal and origin of replication but none of the machinery to replicate the genome itself. We supply the rep and cap genes in trans, on a plasmid that deliberately has no ITRs. So, a finished particle can transduce a cell, but it cannot make more of itself. rcAAV breaks that guarantee. It is a particle whose ITR-flanked genome also contains functional rep and cap, essentially a pseudo-wild-type AAV that can replicate and re-package itself whenever helper functions are present. It forms when that separation fails at the DNA level. The dominant route is homologous recombination between the cis (ITR) plasmid and the trans (rep/cap) plasmid wherever they share sequence: residual wild-type AAV flanking the ITRs, homology around the p5 promoter, or shared backbone. Any common stretch becomes a landing pad that splices rep and cap into an ITR-bearing molecule.

Why Manufacturing Scale Changes Everything
At analytical scale, the odds of any single event round to zero. But scale up the plasmid mass and the number of transfection events, and a rate you never saw on the bench becomes a countable population in the batch. That is why the durable control lives upstream of the bioreactor, in the sequence editor. Here are some common design strategies to reduce rcAAV risk:

• Start by eliminating homology overlap between the cis and trans plasmids, because no common sequence means no efficient recombination
• Relocate the p5 promoter out from upstream of rep to remove a hotspot and cripple any crossover product
• Split rep and cap so no single event can reconstitute a self-sufficient genome.
• Diverge the coding sequences and trim the backbones to shrink the attack surface

Testing Should Confirm Design, Not Replace It
We still test, of course. Serial passage with adenoviral helper, then qPCR for rep. But there is a real difference between depending on that assay to catch rcAAV and using it to confirm what the design already made improbable. The safest route is the preventing your process from encountering the impurity in the first place.

— Dr. Barrett

June 2026

Dear Dr. Barrett,

We are preparing for our first preclinical AAV manufacturing run and thinking through formulation development activities. What considerations should we make to ensure vector stability when designing our formulation?
— Stability by Design

Dear Stability by Design,

You are thinking of this at the optimal time in your product development!

AAV capsid stability is governed by a complex interplay of ionic strength, pH, cryoprotectants, and non-ionic surfactants, all of which contribute to minimizing aggregation and preserving infectivity.

Here is a brief guide to the common elements within all AAV formulations, with the largest section for the star of the show: salt.

Salt: The AAV capsid is composed of 60 protein subunits with a mix of positive and negative surface charges. Salts provide the ionic strength needed to reduce particle-to-particle interactions and prevent aggregation. Unlike the simplified textbook view that increased salt promotes aggregation, AAV formulations generally benefit from higher ionic strength due to the complex charge distribution of the capsid and the presence of nucleic acid impurities. Maintaining approximately 150–200 mM salt is widely recognized as critical for stability during storage and handling. Common choices include NaCl, MgCl₂, and KCl, with overall ionic strength often being more important than the specific salt selected.

Buffer: Pairing your chosen salt(s) with a buffering agent that will hold the product in a narrow neutral window (pH 7-8) helps keep the capsid away from its aggregation prone isoelectric zone. Buffering agents such as Tris or phosphate-based solutions are common choices.

Cryoprotectant: Sugars and polyols protect capsids during freezing and thawing while helping offset freeze-induced pH changes. Common selections are sucrose, trehalose, sorbitol and glycerol.

Non-ionic surfactants: Surfactants reduce vector loss to vials, tubing, and syringes while limiting aggregation caused by interfacial stress. Poloxamer 188 and polysorbate 20/80 are commonly used.

A good starting point is reviewing formulations reported for similar AAV products or target tissues. Here is a handy cheat sheet created by Philipp Beck. Good luck with your studies, and remember, your pre-emptive approach to stability means you are early enough to pivot if needed!

— Dr. Barrett

May 2026

Dear Dr. Barrett,

My team of early-stage AAV developers is heading to Boston to attend ASGCT in a couple of weeks. How do you suggest we tackle this conference to maximize our time there and help us select our CDMO?

—Information Overload

Dear Information Overload,

ASGCT is one of the best meetings of the year for gene therapy developers to gain the latest technical AAV insights (over 1,000 posters!), industry trends from key leaders (750 expert speakers!), regulatory updates, and more networking opportunities than you can count. If you are beginning your search for a CDMO, focusing on new manufacturing technologies and analytical techniques can help you “talk the talk” in your upcoming meetings. Below is my strategy and list of top sessions AAV developers should attend.

1. Prioritize ruthlessly.
   1. Refresh on AAV history with: Founders Award: R. Jude Samulski, Ph.D., “Development of AAV vectors, a journey approaching 50 years: Where are we now?”
   2. Gain insight into what your cells are thinking during triple transfection: Niklas Kraemer, "Nutritional Counselling at the Cellular Level – Combining Ambr® 15 and Orbitrap Mass Spectrometry to Analyze the Metabolome of HEK293 During AAV Production"
   3. Prioritize and evaluate new analytical technology with: Qimin Quan, Ph.D., NanoMosaic, "Accelerating AAV Development with an FDA/CBER AMT-Designated Nanoneedle Analytics Platform"
2. Divide and conquer but make it fun.
3. Treat poster sessions as prime time.
4. Book meetings ahead but leave room for serendipity.

You will not see everything, and that’s okay. Identify a handful of “must-hit” sessions aligned to your goals and build your schedule around those. In 2026 we all know that AAV production yields are increasing and commercial cost of goods matter. Here are my top picks to learn something new:

Don’t move as a pack. Split your team across sessions and poster halls, then regroup daily to share insights. At Forge, we’ve even turned this into a challenge, assigning topics and gaining internal spotlight opportunities for the most valuable takeaways. It keeps everyone engaged and ensures you cover far more ground.

The 5:00–6:30 p.m. window is where you can get into the real data. My pro-tip: walk the floor and read all the titles. You will walk away with the key themes of 2026. While there are over 1,000 posters to choose from, this newsletter lists the Forge posters, presented by our scientists!

An overpacked calendar can crowd out the unexpected conversations that often matter most. I have seen this firsthand at the Forge booth. Stop by #1349 to swap ASGCT insights, visit our Forge School Store to win cool merch, and discuss The Devil Wears Prada 2, if there's time 😀.

Go in with intention, stay flexible, and you’ll leave with far more than just a stack of business cards.

—Dr. Barrett

April 2026
Dear Dr. Barrett,

We've been running our AAV5 process in adherent HEK293T cells in cell factories for two years with acceptable titers. We're now scaling into a 50L stirred-tank bioreactor (SUB) using suspension-adapted HEK293 cells. Our process development lead insists we can directly transfer our transfection conditions using the same PEI:DNA ratio, same plasmid:viable cell ratio, and same harvest time. Is there anything else we should be considering before initiating this batch?

— Polyplex Doubts

Dear Polyplex Doubts,

Your adherent and suspension processes are not the same biological system in different clothing. These two systems are fundamentally different contexts for transfection, and PEI cares enormously about context. In adherent culture, PEI-DNA polyplexes sediment passively onto a stationary monolayer allowing gravity to work for you. In a stirred-tank bioreactor, that same polyplex must find a cell in a turbulent, impeller-mixed environment. Polyplex size, stability, and endosomal escape kinetics all shift under shear. A PEI:DNA ratio optimized for a static cell stack may aggregate, underperform, or become cytotoxic at bioreactor scale.

"Your adherent process is a starting hypothesis for suspension.”

At minimum, I would recommend reoptimizing your PEI:DNA ratio across a range in a benchtop bioreactor or high throughput system such as the Ambr® 250, recheck your cell density at transfection and run a harvest time course from 48 to 120 hours post-transfection. Suspension optimums for cell density are typically tighter than adherent, often 1–3×10⁶ viable cells/mL, and this value is the basis for most transfection calculations.

A path to scaling up from adherent to 50L suspension may include parameter optimization design-of-experiments (DOE) in Ambr® 250, process confirmation in 3 x 5 L benchtop bioreactors, then transfer to a 50L SUB. The cost of one failed production run poses a greater risk than the cost of a proper scale-down DOE. Trust your instinct and choose the path that best positions your company for speed and precision.

—Dr. Barrett
Rooting for your polyplexes

March 2026

Dear Dr. Barrett,
Purity is a top priority for our AAV program. We have a low dose, low volume, intrathecal (IT) delivery planned. Therefore, the full capsid percentage is our focus even if it comes with a slight yield decrease. What purification options should we be considering to maximize full capsids?
— Prioritizing Purity

Dear Prioritizing Purity,

Assuming you have confirmed manufacturability and are already at a commercially viable yield for your program, we can get creative with the purification strategy.

Two common approaches are anion exchange chromatography (AEX) and CsCl ultracentrifugation. These processes separate capsid species by charge or buoyant density, respectively. AEX offers a fully closed, chromatography-based process, but requires upfront time for process development and product purity goals may still prove unattainable. To reach a high percentage of full AAV capsids, with no upfront development time, CsCl ultracentrifugation coupled with additional controls to enhance reproducibility is a great choice.

While CsCl has traditionally been viewed as a research-scale method, at Forge we’ve successfully scaled this to support manufacturing runs up to 1,000L, making it viable for programs where purity is the primary goal. Notably, high step recovery is also achievable with a CsCl based full enrichment process!

Of course, separation is only half the story! Developers should confirm results using analytics such as mass photometry (MP), analytical ultracentrifugation (AUC), and next-generation sequencing (NGS) to fully characterize capsid species in their process and in their final drug product.

Hope this information helps for those prioritizing low dose, high purity AAV therapeutics!

— Dr. Barrett

February 2026 

Dear Dr. Barrett,
I have a lead AAV candidate and we’re getting ready to engage a CDMO. What are the first manufacturing decisions we should be thinking about?
— Trying to Start Right

Dear Trying to Start Right,

The right first step depends on your program’s goal.

If you’re still unsure how your AAV will perform during manufacturing, start with a molecular development consultation. An early review of construct design can identify risks to productivity, quality, or safety before they surface at scale.

If maximizing yield is the priority, a transfection Design of Experiments (DoE) is often the most effective place to begin. It helps to pinpoint optimal transfection conditions and this has historically led our clients toward substantial yield gains (up to 22-fold 🥳) when approached methodically. This starting point can avoid rework later.

Many developers pair one of these approaches with a small-scale platform pilot run to generate early performance data. Even a quick pilot run alongside strong analytics helps gain insight into future product critical quality attributes (CQAs) that will guide development through cGMP.

Starting with clear goals and early data is one of the most tried and true reliable ways to reduce risk and set your program up for success. Happy evaluating, and may your data guide the way!

— Dr. Barrett