AAV Production – How To Improve Transfection Efficiency and Titer Yield

Published on 18 October 2021

Production of Adenoviral Associated Viral Vectors

 

Introduction

Adenoviral associated viral (AAV) vectors used in gene therapy have received notable attention considering the recent clinical successes. Traditionally, production of these vectors is achieved by triple transient transfection using the adherent HEK293 cell line in fetal bovine serum (FBS)-supplemented basal media, such as DMEM. The basal media provides nutritional support as well as buffering capacity for the cells while FBS provides the appropriate mitogenic and survival signaling. Collectively, this media/supplement combination supports HEK293 cell growth to achieve densities appropriate for transfection and subsequent vector production.

Although proven successful at small scale in terms of commercially viable AAV titers, use of this substance for large scale clinical manufacturing applications represents a significant safety risk due to potential adventitious agent contamination, unreliable supply chain from limited global supply, and considerable variability in vector yield due to lot-to-lot composition variations. Though FBS composition has considerable variability, several key protein components have been thoroughly characterized and the functional contribution in cell culture media is well understood.

Serum albumin provides multiple functions in vitro, including antioxidant, macromolecule delivery, and free radical scavenger functions. Serum transferrin delivers iron to cells via catalytic cycling. Cytokines deliver the necessary mitogenic signaling to maintain the proliferation rate and promote survival of HEK293 cells.

To enhance the degree of chemical definition in methods used in AAV manufacturing, a recombinant human transferrin and recombinant human albumin were used to formulate a chemically defined serum-free media that is optimized specifically for production of viral vectors, including AAV, in HEK293 cells. The growth kinetics, transfection efficiency, and productivity of this chemically defined media are compared to those of DMEM + 10% FBS in 2D culture.

Materials & Methods

Cells and Reagents 

HEK293 (CRL 1573) and HT-1080 (CCL 121) cells were obtained from American Type Culture Collection. Working cell banks were established in high glucose DMEM supplemented with GlutaMAX (Gibco, Grand Island, NY) and 10% US-sourced fetal bovine serum (Gibco, Grand Island, NY). All serum-supplemented cultures were maintained at 5% CO₂ and 95% humidified incubators in the presence of 100 U/mL Penicillin-Streptomycin (Gibco, Grand Island, NY).

Adaptation of HEK293 cells to OptiPEAK Serum-Free Media

Transition of HEK293 cells to OptiPEAK HEK293t serum-free media, referred to as OptiPEAK in this paper, (InVitria, Junction City, KS) consisted of direct adaptation from cells cryopreserved in DMEM+10% FBS. Serum-free cells were maintained in a 10% CO₂ and 95% humidity environment on CellBIND-treated flasks (Corning, Corning, NY). All serum-free cultures were maintained in the presence of 10 U/mL Penicillin-Streptomycin (Gibco, Grand Island, NY). Cells were adapted for at least three passages prior to starting transfection studies.

Transfection Protocol

Transfection methods were performed as previously described in the white paper “Enhancement of Transient Transfection with Serum-Free and Blood-Free Transferrin.”¹ HEK293 cells were plated for transfection 20-24 hours prior at an initial cell density of 50,000 cells/cm2. For transfection efficiency with a green fluorescent protein (GFP) reporter, the plasmid Monster Green® Flourescent Protein phMGFP Vector (Promega, Madison, WI) was used at a concentration of 0.2 µg/cm².

Plasmids for AAV-2 were sourced from Takara Bio (Mountain View, CA) and used at a final concentration of 0.21 or 0.30 µg/cm² for FBS- and OptiPEAK-expanded cells, respectively. For DMEM-expanded cultures, 0.084, 0.074, and 0.052 µg/cm² of pHELP, RepCap, and ZSGreen were used. OptiPEAK-expanded cells were transfected with 0.105, 0.104, and 0.073 µg/cm² of pHELP, RepCap, and ZSGreen.  PEIpro® (Polyplus, Alsace, France) was used at a ratio of 1:1 or 1.1:1 with plasmid DNA for formation of DNA complexes for FBS- or OptiPEAK-expanded cultures, respectively.

For serum-free conditions, complexes were formed in complete OptiPEAK HEK with additional transferrin, and, for serum conditions, complexes were formed with OptiMEM (ThermoFisher, #31985070). Transferrin supplementation was performed by adding reconstituted Optiferrin® recombinant human transferrin (InVitria, Junction City, Kansas) to complete OptiPEAK HEK at a final concentration of 1 mg/mL. Complexes were formed by separately diluting DNA and PEIpro 40-fold with the appropriate media. Diluted DNA was then added to the diluted PEIpro. Complexes were mixed gently and incubated for 15 minutes at room temperature and subsequently diluted to a final volume of 0.08 mL/cm² with additional complexation media.

Growth medium was removed from the cell monolayer and complexes were added by gentle pipetting down the side of the flask and allowing the media to fall onto the cells to avoid excessive cell dislodging during the media change. Cells were incubated in normal growth conditions for 2.5 hours ± 30 minutes, after which transfection complexes were aspirated off of the cells.  Following aspiration, either OptiMEM (FBS-expanded cells) or complete OptiPEAK (without additional transferrin for transfection enhancement) was added and cells were incubated for an additional 48 hours at standard growth conditions.

For evaluation of transfection efficiency of the Monster Green Fluorescent Protein, cells were harvested via 1x TrypLE + 1 mM EDTA and subsequently pelleted. Cells were resuspended in DPBS + 10% FBS, 0.1% F-68 and analyzed for GFP expression via flow cytometry. To harvest the AAV, 20 mM MgCl₂, 10% Tween 20, 50 mM HEPES, pH 8.0 was added to the growth media at a 1:10 dilution and cells were incubated for two hours at 37 °C to ensure complete lysis. Lysates were immediately harvested for functional titering.

AAV-2 GFP Functional Titering

HT-1080 cells were used for functional transduction assays and were plated at a density of 30,000 cells per well in 12-well plates 24 hours before the assay in DMEM + 10% FBS. HEK293 lysates containing vector were diluted 100-10,000 fold in DMEM + 10% FBS and 0.1% F-68.

Growth medium was removed from the HT-1080 cells and 300 µL/well of diluted virus was added. Cells were incubated for four hours at standard growth conditions and cultures were subsequently overlayed with 1 mL of DMEM + 10% FBS and further incubated for 48 hours to allow for GFP transduction.

Cells were harvested by trypsinization and analyzed for GFP transduction by flow cytometry. A positive control cell lysate containing a known concentration of AAV-2 GFP was utilized to establish a standard curve and the linear portion of the curve was used to estimate the functional titer of the unknown samples.

Statistics

Transfections were performed with at least an n = 3 independent experiments. Difference in means was determined using a Student’s t-test with a 95% confidence interval. Differences were considered significant with p values ≤ 0.05.

Results

Cells expanded in OptiPEAK demonstrated a significant increase in transfection efficiency with phMGFP when compared to cells cultured in DMEM + 10% FBS and transfected in OptiMEM (p ≤ 0.001). Figure 1 shows representative flow cytometry data that demonstrate cell response to transfection. At the point of analysis, HEK293 singlets were determined based on forward scatter area and doublets were excluded from analysis.

After 48 hours, cells transfected in OptiPEAK + TF exhibited a GFP positive percentage of 85.52 ± 1.18% of the population while DMEM/OptiMEM cells were 61.23 ± 1.01% positive for GFP expression (Figure 2).

These results indicated that HEK293 cells cultured in OptiPEAK were receptive to transfection that mirrored cells expanded in serum and transfected in the presence of human serum-derived transferrin present in OptiMEM. Further evaluation focused on the production capacity of AAV-2 carrying GFP as the gene of interest (GOI).

Using the optimized transfection parameters, HEK293 cells expanded and transfected in OptiPEAK demonstrated similar morphological changes post transfection as cells expanded in DMEM + 10% FBS and transfected in OptiMEM (Figure 3).

Cells appeared rounded with significant expansion of the cytosolic volume with increased detachment cells from the growth surface. Slight manifestations were apparent by 24 hours post transfection and even more so by 48 hours post transfection. These morphological changes are presumably from the intracellular accumulation of viral particles.

Functional titering was determined using GFP transduction of HT-1080 cells to quantify the amount of AAV-2 GFP present in the HEK293 lysates. Functional titer exhibited similar results observed in the GFP reporter transfection assay. Cells expanded in DMEM + 10% FBS and transfected in OptiMEM produced 3.64 ± 2.09 x 10⁹ functional capsids/cm² while OptiHEK produced 4.28 ± 2.88 x 10⁹ functional capsids/cm² (Figure 4).

These results suggest that mitigation of undefined components, which may hinder the transfection process, by removing FBS from the culture system can enhance the transfectability of cells. This enhancement in transfection has the potential to lead to more productive culture systems. HEK293 cells cultured in the OptiHEK Blood-Free and chemically defined media exhibited robust transfectability compared to cells cultured in DMEM + 10% FBS. This enhanced transfection translated to overall higher, though statistically insignificant, titers using an AAV-2 GFP system. These data provide proof of principal that poorly defined and blood-derived components can be replaced for chemically defined and blood free solutions in adherent HEK-based AAV production.