Downstream processing
Thick, porous pads of cellulose fibres, filter aids and charged resins catch particles throughout their depth rather than on a surface, and adsorb some DNA and HCP. Used for harvest clarification after centrifugation, and sized by Vmax tests on the real feed.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Depth filtration
Thick, porous pads of cellulose fibres, filter aids and charged resins catch particles throughout their depth rather than on a surface, and adsorb some DNA and HCP. Used for harvest clarification after centrifugation, and sized by Vmax tests on the real feed.
How it differs from the standard method
- Flush before use to remove extractables and wet the media evenly.
- Capacity falls with feed turbidity and flux; size with a margin.
- Chase the hold-up with buffer at the end.
1From broth to drug substance
Downstream processing is everything between the end of the culture and a purified, formulated product: removing cells and debris, capturing the product, removing host-cell proteins, DNA, aggregates, viruses and process additives, and putting the product into its final buffer at its final concentration. Each step trades a little product for a lot of impurity, so the train is judged by two kinds of number that behave differently: step yields, which multiply (five steps at 90% leave 59%), and clearances, which add in logs (a step that removes 99% of host-cell protein contributes two logs).
Centrifuge and depth filter
Cells come out by centrifugation; fines, colloids and some DNA and HCP are caught in thick, charged depth filters; a 0.2 µm membrane finishes the job.
Process chromatography
An affinity step (Protein A for antibodies) captures and concentrates the product; ion-exchange or mixed-mode steps remove aggregates, HCP, DNA and viruses; a low-pH hold and a virus filter inactivate and remove viruses.
Tangential flow filtration
Ultrafiltration concentrates the product and diafiltration exchanges it into the formulation buffer, with the liquid swept across the membrane so that it does not cake.
2Filters: capacity, pressure and the knee
A depth filter run at constant flux plugs gradually: the pressure rises slowly at first and then steeply as the throughput approaches the filter's capacity, Vmax. Capacity falls for dirtier feeds (a low-viability harvest is full of fines) and at higher flux, so filters are sized from small-scale Vmax tests with a safety margin.
3Chromatography: capacity, elution and pooling
A capture column holds only so much: its dynamic binding capacity, measured at breakthrough, rises with residence time because protein needs time to diffuse into the beads. Loaded past it, product appears in the flow-through.
Key terms
Practise these as flashcards →The throughput per filter area at which a filter would block completely; used to size depth and membrane filters.
The average pressure difference across a membrane between the feed side and the permeate side.
Build-up of retained solute at the membrane surface; it limits flux and, if compacted, becomes fouling.
A volume of new buffer equal to the retentate volume passed through during constant-volume diafiltration.
Concentration in the permeate divided by that in the retentate; near 0 for a retained product, near 1 for salts.
Product bound per litre of resin when the flow-through reaches a set breakthrough (often 10%) at a given residence time.
log₁₀ of impurity in over impurity out across a step.
Proteins from the production cells; reported in ng per mg product (ppm).
Chromatography in which the product passes and impurities bind.
Holding the Protein A eluate at about pH 3.5–3.7 for an hour to inactivate enveloped viruses.
When to use it
- Recovering and purifying a recombinant protein or antibody from a large culture to a defined purity, as for any biotherapeutic or reagent made at scale.
- Designing or troubleshooting a purification train: sizing filters, loading columns, choosing pool cuts and buffer-exchange conditions.
- Showing that a process clears host-cell protein, DNA and viruses to specification.
Compared with alternatives
The bench-scale version of capture, for milligrams in a lab rather than grams in a process.
Size-exclusion and ion exchange at bench scale, with the same separation physics.
Primary clarification: cells and large debris come out before depth filtration.
Dead-end membrane filtration, the final 0.2 µm step after depth filters.
Buffer exchange for small volumes where a TFF system is not worth setting up.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Thick, porous pads of cellulose fibres, filter aids and charged resins catch particles throughout their depth rather than on a surface, and adsorb some DNA and HCP. Used for harvest clarification after centrifugation, and sized by Vmax tests on the real feed.
- Flush before use to remove extractables and wet the media evenly.
- Capacity falls with feed turbidity and flux; size with a margin.
- Chase the hold-up with buffer at the end.