Harvesting and Preservation of Cells for Therapy

Flashcards on the topics of Harvesting and Preservation of Cells for Therapy

What is Upstream processing?

From gene/cell to cell cultivation processes resulting in the initial generation of a target product.

What is Downstream processing?

Cell harvesting, lysing, isolating, purification of a target product, and generation of finished product format.

What is Quality Control?

Product safety and efficacy.

What are Unit operations?

Individual operations or steps within the process that change or separate components.

Name some examples of Unit Operations.

Centrifugation, chromatography, cooling, crystallisation, dialysis, distillation, drying, evaporation, filtration, heating, humidification, membrane separation, milling, mixing, precipitation, solids handling, solvent extraction.

What is the purpose of volume reduction and cell concentration in cell therapy DSP?

Achieves high cell concentration

What is the purpose of purification (diafiltration) in cell therapy DSP?

Removes impurities (e.g., serum, trypsin, and undifferentiated cells)

What is the purpose of formulation in cell therapy DSP?

Combine freeze media and cell suspension; achieves desired cell concentration for filling

What is the purpose of fill process in cell therapy DSP?

Accurate fill volumes and consistent.

What is the relation to DSP requirement for >80% viability at the clinic after thaw?

Frozen at >85% viability, well-controlled freezing profiles, controlled processing times, same-day processing.

What is the relation to DSP requirement for a specified number of viable cells at the clinic after thaw?

Consistent and accurate fill volumes, minimal volume loss during thaw, robust formulation step.

What is the relation to DSP requirement for >80% viable cell recovery at the clinic after thaw?

Achieve high concentration factor (C) during volume-reduction step with minimal cell loss or damage, low system volume in the volume-reduction unit step.

What is the relation to DSP requirement for 10-100 million cells/mL, Single-cell suspension at the clinic after thaw?

No/minimal clumps of cells at fill, no pelleting, high-efficiency wash step, cells in suspension during wash step, low system volume, no cell damage, low-shear processing.

What is the relation to DSP requirement for impurities <1 ppm at the clinic after thaw?

Low system volume, no cell damage, low-shear processing.

What is the relation to DSP requirement for product quality attributes are maintained at the clinic after thaw?

Low-shear processing.

What type of technology is often used for volume reduction and wash when the estimated harvest volume is <10L?

Bench-top systems (centrifugation, COBE)

What type of technology is often used for volume reduction and wash when the estimated harvest volume is 10-100L?

Automated, closed, and scalable entry-level systems (TFF, small kSep)

What type of technology is often used for volume reduction and wash when the estimated harvest volume is 100-1,000L?

Large, fully automated bioprocessing systems: TFF skid, multiple large kSep

What is the applicability of TFF-Bioprocessing in cell-therapy downstream processing?

Both autologous and allogeneic

What is the applicability of kSep systems in cell-therapy downstream processing?

Allogeneic

What is the applicability of Conventional Centrifugation in cell-therapy downstream processing?

Allogeneic

What did Nienow et al. research?

A potentially scalable method for the harvesting of hMSCs from microcarriers.

What is the strategy to get cells off of microcarriers?

Brief period of intense agitation to aid detachment of cells whilst in contact with dissociation reagent.

What is the SoloHill Harvesting Procedure?

Aspirate media, PBS Wash (agitate at 40rpm for 15 min), Add dissociation reagent for 15 min, 37°C, Quench dissociation reagent with growth medium, Pipette microcarrier-cell suspension to dislodge the cells, Separate cells from microcarriers via vacuum filtration.

What is Kolmogorov’s theory of microscales of turbulence?

Provided the size of the biological entity suspended in flow < Kolmogorov scale, then the entity should not be damaged

What is the Novel harvesting procedure?

Aspirate media, PBS Wash (agitate at 40rpm for 15 min), Agitate at 150 rpm with dissociation reagent for 7 min, 37°C, Quench dissociation reagent with growth medium, Separate cells from microcarriers via vacuum filtration, Cell concentration via centrifugation

Why Preserve Cells?

Transport; Banking; Uncouple expansion/manipulation of cells from the clinic or cell- based assays

What are deciding factors when considering preservation of cell therapies?

Autologous vs Allogeneic, Scale, Business model, Preservation method

What are challenges with cell therapy preservation?

Storage of product, Short-shelf life, Back up systems, Contamination

What are transport challenges with cell therapy preservation?

Refrigerated transport, Frozen transport

What are the challenges at the clinic for cell therapy preservation?

Can any on-site processing be done? Matching cell, surgeon and patient availability; Levels of investment required in equipment and staff training.

What are ways to preserve cells?

Cryopreservation, Desiccation, Hypothermia

When to preserve cells?

Cell isolation, Cell-based therapies, Cell banks, Product shelf-life

What makes an ideal preservation method?

Widely applicable; maintains cells/organisms’ key characteristics; GMP compatible; output is easily stored and transported; cell recovery requires minimal manipulation; Is cost effective

What is Cryopreservation?

Preservation of cells below 0ºC typically in liquid nitrogen or its vapour phase

What are 2 options of Cryopreservation?

Slow / conventional / controlled-rate freezing and Vitrification

What is the Slow Freezing process?

Harvest Cells, Suspend Cells in Freezing Solution, Freeze at ~1ºC/min, Thaw, Wash cells

What is cell damage during cryopreservation?

Formation of extracellular ice leads to creation of an osmotic imbalance, drawing water out of the cell.

What are the 2 types of Cryoprotective Agents (CPAs)?

Penetrating and Non-penetrating

What are the benefits of vitrification?

Avoids formation of extra and intracellular ice crystals; Generates amorphous ice

What is the Vitrification process?

Harvest Cells, Suspend Cells in Freezing Solution, Plunge into LN2, Thaw, Wash cells

What is the issue with CPA toxicity as it related to DMSO?

Found in most freezing solutions and is widely believed to be cytotoxic at temperatures > 0°C.

What is Dessication?

Based on inherent capacity of some cells to survive almost complete dehydration

What is Desiccation - lyophilisation?

Removes moisture by sublimation of ice into water vapour and requires the use of lyoprotectants

What is Desiccation – vacuum desiccation?

• Also requires the use of a protective agent e.g. trehalose
• Very limited number of studies!

Gordon et al
• hMSCs
• Dehydrated and stored for 24 hours
• Retained morphology, adhesive capacity, >90% viability, proliferated and expressed key surface markers.
• Authors admit large inconsistency between runs

What is Hypothermia also known as?

Cell pausing

What is Hypothermia?

Based on cold storage of organs - Cells are preserved at >0ºC

What does Hypothermia- specialised media generally contain?

Mimic ionic composition of cells at cold temperatures; Contain antioxidants; Provide pH buffering system; Some nutrients

What are concerns with Hypothermia?

Impact on cell function; Is a few days long enough?; Impact of variation in storage temperature/humidity?; Lack of research into rewarming process