Downstream processing - Pharmaceutical Biotechnology: Concepts and Aapplications - page 143

1. Apply protein-containing sample
2. Irrigate with buffer (wash out unbound material)
3. Apply elution buffer and collect fractions

Assay for
target protein

A 280

Collect fractions
when elution buffer
is applied
(a)

5 10

15 20 25 30

Fraction number

(b)

Figure 6.7 (a) Typical sequence of events undertaken during an (adsorption-based) protein purifi cation
chromatographic step. Note that the chromatographic beads are not drawn to scale, and in reality these
display diameters 0.1 mm. Fractions collected during protein desorption are assayed for (i) total protein,
usually by measuring absorbance at 280 nm, and (ii) target protein activity. (b) In the case illustrated, two
major protein peaks are evident, only one of which contains the protein of interest. Thus, desorption and
adsorption steps can result in selective purifi cation

processing columns are available that are manufactured from stainless steel. Process-scale chroma-
tographic separation is generally undertaken under low pressure, but production-scale high-pressure
systems (i.e. process-scale HPLC) are sometimes used, as long as the protein product is not adversely
affected by the high pressure experienced. An HPLC-based 'polishing step' is sometimes employed,
e.g. during the production of highly purifi ed insulin preparations (Chapter 11). Next, we will consider
individually the most common forms of chromatography used to purify therapeutic proteins.

Table 6.2 Chromatographic techniques most commonly used in protein purifi cation protocols. The basis of
separation is listed in each case

Te ch n ique Ba sis of sepa r at ion

Ion-exchange chromatography Differences in protein surface charge at a given pH

Gel-fi ltration chromatography Differences in mass/shape of different proteins

Affi nity chromatography Based upon biospecifi c interaction between a protein and an

appropriate ligand

Hydrophobic interaction chromatography Differences in surface hydrophobicity of proteins

Chromatofocusing Separates proteins on the basis of their isolectric points

Hydroxyapatite chromatography Complex interactions between proteins and the calcium

phosphate-based media; not fully understood