Product analysis - Pharmaceutical Biotechnology: Concepts and Aapplications - page 197

GMP requires that, where practicable, process equipment coming into direct contact with the
biopharmaceutical product stream should be rendered endotoxin free (depyrogenated) before
use. Autoclaving, steam or dry heat can effectively be used on many process vessels, pipework,
etc., which are usually manufactured from stainless steel or other heat-resistant material. Such
an approach is not routinely practicable in the case of some items of process equipment, such as
chromatographic systems. Fortunately, endotoxin is sensitive to strongly alkaline conditions; thus,
routine cleaning in place of chromatographic systems using 1 mol l 1 NaOH represents an effec-
tive depyrogenation step. Gentler approaches, such as exhaustive rinsing with WFI (until an LAL
test shows the eluate to be endotoxin free), can also be surprisingly effective.
It is generally unnecessary to introduce specifi c measures aimed at endotoxin removal from the
product during downstream processing. Endotoxin present in the earlier stages of production is
often effectively removed from the product during chromatographic fractionation. The endotoxin
molecule's highly negative charge often facilitates its effective removal from the product stream
by ion-exchange chromatography. Gel-fi ltration chromatography also serves to remove endotoxin
from the product. Although individual LPS molecules exhibit an average molecular mass of less
than 20 kDa, these molecules aggregate in aqueous environments and generate supramolecular
structures of molecular mass 100-1000 kDa.
The molecular mass of most biopharmaceuticals is considerably less than 100 kDa (Table 7.4).
The proteins would thus elute from gel-fi ltration columns much later than contaminating endo-
toxin aggregates. Should the biopharmaceutical exhibit a molecular mass approaching or exceed-
ing 100 kDa, then effective separation can still be achieved by inclusion of a chelating agent such
as EDTA in the running buffer. This promotes depolymerization of the endotoxin aggregates into
monomeric (20 kDa) form.
Additional techniques capable of separating biomolecules on the basis of molecular mass (e.g.
ultrafi ltration) may also be used to remove endotoxin from the product stream.

7.6.3 DNA

The clinical signifi cance of DNA-based contaminants in biopharmaceutical products remains
unclear. The concerns relating to the presence of DNA in modern biopharmaceuticals focus

Table 7.4 The molecular mass of some polypeptide biopharmaceuticals. Many are glycosylated, thereby
exhibiting a range of molecular masses due to differential glycosylation

Molecular mass
(kDa)

Molecular
mass (kDa)

Molecular mass
(kDa)

Protein Protein Protein

IFN- α 20-27 TNF- α 52 a EGF 6

IFN- β 20 GM-CSF 22 NGF 26

IFN-γ 20-25 G-CSF 21 Insulin 5.7

IL-2 15-20 EPO 36 hGH 22

IL-1 17.5 TPO 60 FSH 34

IL-12 30-35 IGF-1 7.6 LH 28.5

a Biologically active, trimeric form.
TPO: thrombopoietin; EGF: epidermal growth factor; NGF: nerve growth factor; LH: luteinizing hormone.