Industrial purification of Biopharmaceutical (downstream)

downstream process includes all the steps required to produce what

final purified product

the upstream process included the fermentation product, which includes your protein products

true

downstream process includes all steps required to purify a biological product from cell culture broth to a final purified product. it involves multiple purification steps

true

specific downstream steps and their number depend on the product and production system, cost, and challenges you have with every system

true

what are the steps of downstream

initial purification

intermediate purification

final purification

goal is to concentrate the protein in a fast/quick way (rough purification) which step

• includes removal of cell and cell debris

• includes 1 or more steps

initial purification

steps in initial purification

• centrifugation

• cell lysis

• filtration

• dialysis

• precipitation

• expanded bed absortion

• lysates/cells are used directly from the fermenter is what technique

expanded bed adsorption

• includes multiple steps of purification. choice of purification methods depends on the type of other proteins is what purification

intermediate and final purification

what technique is used in intermediated and final purification

• ion exchange chromatography

• adsorption (normal phase) chromatography

• hydrophobic interaction chromatography

• affinity chromatography

• immuno affinity chromatograph

• size exclusion chromatography

stationary phase is what

polar

mobile phase is relatively what

non-polar

sample molecules that have high affinity to the stationary phase will stay longer on the column and elute later

true

• hydrophilic amino acids attract water molecules and are expose to the surface while most hydrophobic amino acid residues are located inside the protein core is what chromatography

hydrophobic interaction chromatography

high salt will attract the water from the protein causing exposing of hydrophobic residues

true

• based on strong interaction between protein of interest and another substance is what chromatography

affinity chromatography

• type of affinity chromatography

• based on affinity of antibodies with their epitopes

what chromatography

immuno affinity chromatography

• separation based on their shape and size

• aka gel permeation

• slow and thus is commonly used late in the purification step when protein is more concentrated

size exclusion chromatography

different charge, same hydrophobicity what purification we use

ion

what kind of contaminations are there

• viral contamination

• bacterial contamination

• pyrogen contamination

• cellular DNA contamination

heat treatment

nanofiltration

ion exchange chromatography

immuno affinity chromatography

viral contamination

filter sterilization

sterilization of raw material at 121C for 15 mins

strict aseptic condition

antibiotics

bacterial contamination

heating materials

ion enchange chromatography of the product

pyrogen contamination

detection using dye binding fluorescence or PCR

cellular DNA contamination

what needs to adopt a distinct tertiary structure where they expose certain region for recognition by other receptors or substrates and hence become function

proteins

what are occasionally produced as insoluble in the form of inclusion bodies

recombinant proteins

detection of protein folding are what 3 types

1. circular dichroism

2. fluorescence

3. fourier transform infrared

CD in the far UV region (180-260 nm), however occasional overlap from β-sheets (weak signals)

FTIR, however, occasional overlap from loop structures

a-helices

CD: weak variable signals due to twists of interacting β-strands

FTIR: efficiently estimates β-structure and differentiates between parallel and antiparallel forms

B-sheets

CD in the near UV region (250-340 nm). Can also provide information on disulfide structures

Fluorescence

aromatic amino acids

requires less protein concentration

fast

give no information on full protein structure or the exact location of each amino acid

CD, FTIR, anf fluorescens spectroscopy

techniques that can allow proper folding of misfolded proteins

1. dilution

2. dialysis

3. chromatography

4. high hydrostatic pressure

• easy

• requires huge vessels

• requires reconcentration

which technique to misfiled proteins

dilution

• different chromatographic techniques are used to enhance purity based on the properties of the protein as well as impurities associated with the product

true

contaminants could be host-related, product related or process related. it is essential to remove all sources of contaminants from the final product

true

severe techniques exist to determine the proper folding of the proteins

true

several methods can be used to refold a partially or unfolded proteins

true