UNIT OPERATION / PRODUCTION PROCESS


Downstream Processing

Introduction

Downstream processing is the initial step of a biomanufacturing process to harvest cell culture containing highly-expressed active pharmaceutical ingredients. This is then purified and concentrated for final product formulation and commercialization.

The entire phase requires cGMP compliance to ensure that these living organisms are maintained under safe and sterile conditions. The equipment to be used in isolation, purification, and filtration can be enclosed in a cell therapy aseptic isolator during the whole process. This technology will prevent occupational contamination and unwanted air particles to mix with the isolates.

The CradlePro-Iso is an integrated system that combines the Esco Versati™ Centrifuge, Esco CO2 incubator, and CelCradle™ benchtop bioreactor system inside the Esco HPI G3. The CelCradle™ system provides cells with an environment of low shear stress, zero foaming, and of high rates of aeration and nutrition, while the CO2 incubator controls the cells surrounding conditions.

Goals of the Downstream Process
  • Capture of Recovery – This involves the accelerated segregation of the product of the lead compound from the cells. The goal is:
    •elimination of all micro-particulates and colloidal materials (a mixture with one substance uniformly dispersed throughout another).
    • elimination of the majority of water, growth medium supplements, and small molecule solutes via product concentration.
    • separation of product away from proteolytic enzymes or other degradative elements.
  • Intermediate Purification – This involves the elimination of bulk contaminants, including host cell proteins and adventitious viruses, as well as any potential leaching foreign bodies from other in-process materials. Often there are microscopic (but finite) specific level of leached ligand from the capture resin that can be moderately bound or otherwise co-eluted with the lead compound.
  • Polishing – This involves the elimination of trace contaminants and impurities, including inactive or unwanted isoform of the desired therapeutic or common impurities, including fragments or other chemical modifications thereof.
Downstream Process Unit Operations

• Process filtration

Filtration is used at several stages in the downstream processing of the bioreactor harvest, as well as for the preparation of purified water and other processing fluids (buffers, sanitizing agents, etc.). Several filtration steps are integral to the Capture, Intermediate purification and Polishing stages; these types of filtration fall into one of three general types:

Filtration is a crucial step in the downstream processing of any harvest cells since it is also integral to the capture, intermediate purification, and polishing stages. Moreover, filtration is utilized for the preparation of purified water and other liquids for buffering and sanitizing purposes. Below are the different types of filtration process:

  • Microfiltration can be used at the start of the downstream process to clarify the feed beyond what was accomplished in the upstream harvest and centrifugation/clarification.
  • Ultrafiltration is used between chromatography steps to concentrate the product and change the buffer conditions to prepare it for subsequent chromatography steps.
  • Sterilizing grade direct flow filtration involving the use of nanofiltration cartridges, eliminates microbial organisms and insoluble proteins, removes adventitious and endogenous viruses, and sterile filters the product in preparation for final formulation.
Flow-through devices, assembled with the above membrane media, are formatted to affect two general flow types:
  • Direct Flow Filtration devices allow the process fluid to cross the membrane in essentially a perpendicular flow direction; this provides little or no prevention of particulate build-up or the concentration of other elements that do not fit through the pore structure.
  • Tangential Flow Filtration devices orient the membrane so that process flow sweeps across the active filtration surface, which minimizes pore plugging and surface fouling by concentrated reject elements of the feed.
Direct Flow Filtration (DFF)

A flat membrane disk is the simplest arrangement of the filter. However, as membrane surface area requirements increase, a pleated sheet either with or without a secondary supporting layer is formed. The combined filtration layer is then wrapped around a perforated collecting core. An advanced version of this type fits an even greater membrane area into the same cartridge volume by folding the pleats over, allowing a greater length of membrane along each fold.

In the typical pleated membrane DFF cartridge, flow is directed from the inlet (through the media in a perpendicular or direct path across the membrane) to the outer surface of the media. The flow the collects in the core and exits the holder to the device’s downstream port.

Tangential Flow Filtration (TFF)

TFF is rapid and efficient method for separating and purifying process flow. It can be used to recover and purify solutions from small volumes (10 mL) up to thousands of liters. With TFF the feed flows tangentially over the surface of the membrane, where a portion flows through the membrane as permeate.

Chromatography

Liquid chromatography is purification method employing a packed resin bed, through which a solution containing a mixture of solutes is flowed; specific solutes are differentially bound or slowed as they contact the bed, while others pass through without interacting with the packed resin. The large majority of chromatography steps used in the purification of mAbs and other protein-based biotherapeutics are those in which some constituents bind or interact with a ligand of the stationary phases (packed resin) absolutely, while others pass through with no interaction. The bound components are then removed or eluted by the gradual or step-wise change of the composition of the mobile phase run through the packed resin bed, such that bound feed constituents are ideally eluted separately from the product of interest. In bind-elute methods the product is what binds, while in flow-through methods the product passes through while other unwanted elements of the feed are temporarily bound.