MedScope - BioProcess Consultants
        QbD/CMC/cGMP Consultants in Gene Therapy, Immunotherapy, Small Molecule, LentiVector Development: Inception to Launch
       802-236-8650     info@medscope.com

Commercial Operations
 - LentiVector Development
 - Mnftr
 - Cell Line Development
 - Continuous Manufacturing
 - Process Analytics (PAT)
 - DOE, Multivariate Design
 - Scale-Up,-Out,-Down
 - Development Protocols
 - Equipment
 - Single Use
 - Strategic De-risk

Regulatory Science
 - CMC Development
 - Comparability Studies
 - Device Design Controls

Analytical Development
 - Bioanalytic Assays
 - Immuno/PK
 - Product/Impurity
 - SU Extractables/Leachables

Medical
- CMO/Med Affairs
 - Preclinical Development
 - Clinical Development
 - CMC/Commercial 
 - Strategic Develop
 - Rare Disease
 - Pediatric 
 - Breakthrough Therapies

Manufacturing and ComOps
  - Capital Equipment
  - BSL-2, ISO7
 - Closed Manufacturing
 - Suppliers
 - Tech Transfer

Facilities & Locations
 - 100,00 ft2 Mnftr/Lab Space
 - 50,000 ft2 Office Space 
 - Cambridge, MA
 - Burlington, VT

Quality
  - Documentation
 - WIFI Controls 
 - QBD, SOP, WI
 - BPR, Protocols, 
 - Audits, PAI, 

Sales & Marketing
 - Global Strategic Pricing
 - General Management
 - Strategic Plan
 - Market Plans
 - Alliance Management

Political Advocacy, Gene Therapy
 - Gene Therapy Cost Models
 - FDA Realignement
 - Workforce Expansion
 - Workforce Development
 - Regs Patient Bedside Mnftr

U.S. Defense
 - Biologics Rapid Monitor
- Detection
 - Biologics Rapid Response
 - DARPA, SBIR

Computation
 - Protein/DNA Modelling
 - Process Capability
 - Remote RTOPC/OLE










Process Development - Lenti Vector Manufacture at MedScope Biologics
Lentiviral vectors (LVs) have emerged as potent and versatile vectors for ex vivo or in vivo gene transfer into dividing and nondividing cells. Robust phenotypic correction of diseases in humans has now been achieved in many clinical trials. LVs can deliver genes ex vivo into bona fide stem cells, particularly hematopoietic stem cells, allowing for stable transgene expression upon hematopoietic reconstitution. They are also useful to generate induced pluripotent stem cells. LVs can be pseudotyped with distinct viral envelopes that influence vector tropism and transduction efficiency. Third-generation, self-inactivating lentiviral vectors have been used in multiple clinical trials to introduce genes into hematopoietic stem cells to correct primary immunodeficiencies and hemoglobinopathies. These vectors have also been used to introduce genes into mature T cells to generate immunity to cancer through the delivery of chimeric antigen receptors (CARs) or cloned T-cell receptors. CAR T-cell therapies engineered using lentiviral vectors have demonstrated noteworthy clinical success in patients with B-cell malignancies leading to regulatory approval of the first genetically engineered cellular therapy using lentiviral vectors. Contact MedScope Biologics today and learn how MedScope can help support and/or lead development of your Lenti Vector program. MedScope is a rapid development organization, and in most instances can initiate value-based discussions within 24 hours. Please contact us at 802-236-8650 or info@medscope.com.

Process Development - Clinical/Commercial Operations with Lenti Vectors
MedScope is a leader in developing state-of-the-art cGMP Lenti Vector manufacturing tecchnologies, and we have successfully lead development and launch of numerous QbD products, including continuously manufactured products with integreated PAT feedback and feedforward controls, using both single use and stainless components. MedScope has vast QbD CMC scale up experience (Tech Transfer (Scale -up, -down, -out), Quality, Regulatory/CMC submission, PAI, and product launch experience, and on a global scale can efficiently guide your company through this very complex technical, quality, regulatory space.

Process Development - Plasmids
In order to accomplish transfection of HEK 293T producer cells to create Lenti Viral Particles, MedScope first creates three lentiviral helper plasmids which can be produced under cGMP conditions based upon the stage of the program. For each plasmid, a bacterial master cell bank (bMCB) is generated and release tested (including plasmid DNA sequencing) utilizing E.Coli cell-plasmid manufacturing technologies. Once the master plasmid seed culture has been created and qualified, a single vial of the bMCB can be used for future batch manufacture by bMCB expansion using a variety of available expansion technologies (eg. seed culture to inoculate a Wave Bioreactor unit (GE Healthcare), Bioflo 3000 bioreactor (New Brunswick Scientific), MiltenylCliniMACS Prodigy, etc.).

Once seed and expansion is complete, harvested bacteria are processed to purify the plasmids, typically first by lysing via alkaline lysis methods, followed by clarification of the the lysate with centrifugation and depth filtration, followed by ultrafiltration10- to 15-fold (for example using a hollow-fiber cartridge (GE Healthcare) with a 30-kDa nominal molecular-weight cutoff (NMWC)). Next, the supernatant is concentrated and passed through a bed of USP-grade Celite diatomaceous earth from Celpure (Sigma Aldrich) to reduce endotoxin content.

Finally, separation of pDNA from bacterial RNA and other impurities can be accomplished in various ways, such as using size-exclusion column chromatography with Sepharose 6 Fast Flow resin (GE Healthcare) in the presence of 2M ammonium sulfate (JT Baker), followed by collecting the first peak containing pDNA, then passing it through a second Celite bed to further reduce endotoxin. Finally, column-purified material is ultrafiltered and diafiltered into Tris-EDTA buffer (Fisher Scientific), yielding purified bulk cGMP pDNA. Careful control over process and environmental conditions is critical to successfull high yield production of the plasmids.

Process Development - HEK 293T Master Cell Bank and Culture of 293T Cells
The 293T cells typically used for lentiviral vector production are expanded from the master cell bank (MCB). That bank was fully release tested according to FDA guidelines for releasing cell banks and products (19). Cells grew in culture at ~2 × 104 cells/cm2 in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose, 1% sodium pyruvate, and 1% glutamine (Biowhittaker) supplemented with 10% Hyclone fetal bovine serum (FBS) (Thermo Fisher). We used a 37 °C incubator with 5% CO2.

Every three to four days, we trypsinized and counted the cells before reseeding them at 2 × 104 cells/cm2. After removing media from the cell culture flasks, we washed them with phosphate-buffered saline (PBS) from Mediatech and then removed that as waste. Trypsin (Irvine Scientific) was diluted 1:10 in PBS to a final concentration of 0.05% and then added to the cell culture flasks, which were returned to the 37 °C incubator for 3–15 minutes. After cells were dislodged, we collected the cell suspension and inactivated the trypsin by adding 293T medium (roughly equal volumes of cell suspension and medium), then collected cells using centrifugation. Those cells were expanded until we had sufficient numbers for transfection.

Process Development - Lentiviral Transfection 
​Numerous cell culture LV transfection DOE's are required to develop and optimize cell culture process conditions, typicall starting with plating 293T cells in an appropriately sized vessel at ~1.0 × 105 cells/cm2 in 293T growth media (DMEM supplemented with 10% FBS, 1% sodium pyruvate, and 1% glutamine). This is typically followed approximately two days later by transfection of the 293T cells with four lentiviral plasmids: the pCgp plasmid containing the gag/pol gene, the pCMV-Rev 2 plasmid containing the rev gene, and the pCMV-G plasmid containing the VSV-G gene — all under the guidance of cytomegalovirus (CMV) promoters — and a transfer plasmid (HIV-CMV-EGFP) in a ratio of 20:13:5:20 respectively with 0.8 µg DNA/cm2.

There are multiple ways to develop and scale up transfection. For example, to make a liter of transfection mixture (eg. transfecting a 10-layer tray system), we typically mix DNA from three helper plasmids and the transfer plasmid with TE buffer at pH 7.9 (~50 mL), then add 2 M CaCl2 (7 mL) from Fluka (Sigma) and 2× HEPES-buffered saline (HBS) at pH 7.2 (58 mLs) in sequential order before mixing (HEPES from Roche; NaCl and Na2HPO4 from Sigma). These volumes can further be scaled down for smaller-scale experiments to improve yield and quality of the LV product through multiple small scale DOE studies. Once optimized, process develoopment continues with removal the medium from each vessel and replacement with the DNA mixture in growth media with 1 L 2% FBS. Cells are then returned to the 37 °C incubator with 5% CO2. Three to five hours later, removal of the transfection solution and addition of fresh 2% FBS-media with 6 µM sodium butyrate occurs (from Fluka (Sigma)) and moved to the Cell Factory trays. About 72 hours after transfection, virus-containing crude supernatant can be collected. 

Process Development - Separation, Purification, and Resuspension of Lentiviral Pellets
At this point, the viral supernatant is further ultrafiltered (UF) to ~500 mL while simultaneously diafiltering (DF) with 8 L chilled DF buffer (4 g/100 mL lactose from JT Baker in DPBS from MediaTech). While maintaining the volume at ~500 mL, diafilter the viral supernatant with an additional 2 L of DF buffer. After UF/DF, the viral suspension is centrifuged for 16–20 hours at 6,000g. The resulting viral pellet is resuspended and then centrifuged at 1,000 rpm for one to two minutes to pellet and remove insoluble material. We then collect the supernatant and keep the concentrated viral vector material at −80 °C until all subbatches are pooled.

​Process Development - Transduction Unit Titer Assay
A key CQA for LV pellet manufacture is of course viral titer. One mechanism for accomplishing this measurement is  use of lentiviral vector expressing green fluorescent protein (GFP) in process development/clinical studies. In this way the viral titer of each lentiviral sample (transduced using HT-1080 cells for example) can be determine by measuring the expression of GFP using fluorescence-activated cell sorting (FACS) analysis. 

For example, 5 × 104 HT-1080 cells (ATCC) can be seeded in each well of a 12-well plate using standard growth medium (DMEM supplemented with 10% FBS) and incubated for ~24 hours in a tissue culture incubator at 37 °C with 5% CO2. A single well of cells can then be counted to approximate the number of cells transduced in each well. Cells can then be transduced with various amounts of test samples in the presence of 4 µg/mL polybrene (Sigma). A previously titered viral sample of HIV-CMV-EGFP lentivirus can then be used to serve as a standard to control for assay variations. To quantify the number of cells transduced by the vector, simply remove the HT-1080 cells from their plates ~48 hours after transduction and fixed them with 3.7% formaldehyde (Sigma). For complementary testing, these same fixed cells anc also be analyzed using an EPICS XL-MCL flow cytometer (Beckman Coulter) to detect GFP expression. Based on the following formula, lentiviral titers can be determined as follows:

Titer (Transduction Unit/mL) = % of GFP-positive cells × number of cells transduced ÷ volume (mL) of sample used for transduction.

Additionally, a p24 Titer Assay can be performed using the HIV-1 p24 ELISA kit from PerkinElmer (catalog #NEK050). Briefly, a test article is incubated in microplate wells coated with a highly specific mouse monoclonal antibody to HIV-1 p24. Captured antigen is complexed with a biotinylated polyclonal antibody to HIV-1 p24, followed by a streptavidin-HRP (horseradish peroxidase) conjugate. The resulting complex is detected after incubation with ortho-phenylenediamine-HCl (OPD), which produces a yellow hue that is directly proportional to the amount of HIV-1 p24 captured. Absorbance (490 nm) of each microplate well is determined using a microplate reader. From this, a p24 concentration can be calculated based on a standard curve generated by p24 standards with known concentration.  

Process Development - Equipment available for scaled-up GMP lentiviral manufacture
1. Biotek 3D Perfusion Bioreactor. 4 independent, autoclavable polycarbonate chambers. Cell culture medias
are perfused through the open porousn structure of scaffolds using a pulsatile pump. 3D Insert scaffolds with various sizes ranging from 96-well to 6-well. Canbe used as a single-use bioreactor system. Small-scale.

2. Corning CELLSTACK Culture Chambers. Cell yields in the 107 –109 range and are useful alternatives to multiple roller bottles or spinner flasks. A more hydrophilic surface giving more consistent, even cell attachment, increased cell growth and yields. 200 mL, 1 L, 2 L, or 8 L.

3. Terumo Quantum Bioreactor. Hollow-fiber bioreactor system allows seeding of producer cells on high-surface areas using small volumes for continuous flow. High yields with a small footprint of 2.1 m2. Surface area occupying a total volume of 180 mL.

4. Sartorius l-bioreactor (small-scale) to BIOSTAT (large-scale). Bioreactor platform from cell line development to commercial manufacturing. Simple transition between scales. 15 mL (MBR) or 2,000 L (BIOSTAT).

5. Pall iCELLis A closed-system, fully integrated, singleuse, fixed-bed bioreactor. Uses microcarriers to immobilize continuously perfused cells. Excellent large-scale production, and simple transition between scales. No oxygen sparging required. 1–70 L in bioreactor; 40 mL to 25 L infixed beds with diameters 110–860 mm.

6. GE Xcellerex XDR-10 A closed-system, fully-integrated, singleuse bioreactor which uses microcarriers to immobilize continuouslyperfused cells. Excellent large-scale production. Automated data management, low-shear
impeller for gentle efficient mixing ofmedium. 4.5–10 L, available in single, twin, triple, and quad vessel configuration.


Process Development - Equipment - Tangential-flow filtration (TFF) for lentivirus particles concentration
1. GE Healthcare Life Sciences. UniFluxTM system with a 750 kDa hollowfiber ultrafiltration cartridge. Available in four sizes (5–600 L) Merten et al.17; Ausubel et al.16 Fully automated Single-use cartridges Real-time data logging and reporting function.

2. Spectrum Laboratories. KrosFlo Research II TFF System. Volumes ranging from 1 mL to 10 L Cooper et al. 201131 Automated process control and data collection software.

3. Pall Minim. Centramate LV holder and a 100 or 300 kDa Omega Screen channel-cassette Scalable Geraerts et al. 200532 Ready-to-use Semi or full automation.  

​Process Development - Raw Materials
1. Packaging and Transfer Plasmids. Release includes sequence verification, amplified to cGMP acceptable levels per BPR, sterile and free of bacteria or fungi, endotoxin free. Approved COA.
2. Reagents. Produced under cGMP, verifed via quality documentation and test and release. Approved COA.
3. Working Cell Bank. Identification. Expanded under cGMP, virus free, sterile and free of bacteria and fungi, mycoplasma free. Approved COA.

Process Development - Batch Production Downstream
1. Transfection of cells, collection of virus particles.
2. Particulate Filtration.
3. Ion exchange purification.
4. Concentration and Desalting by TFF.
5. Fill finish.
6. Storage and controlled transport.

Process Development - Batch Release Testing
1. Sterility.
2. Viral Titre.
3. Vector Integrity.
4. Genomic Integration. 
5. Replication Incompetence.
6. Cytotoxicity.
7. Expression and Functionality.












802-236-8650, info@medscope.com