Purity/Impurity

SDS-PAGE

SDS-PAGE is a denaturing gel electrophoresis technique used in the development and characterization of protein based products. Sodium dodecyl sulfate (SDS) is a detergent that dissociates and unfolds oligomeric proteins into its subunits. The SDS binds to the polypeptides to form complexes with fairly constant charge to mass ratios. The electrophoretic migration rate through a gel is therefore determined only by the size of the complexes. Molecular weights are determined by simultaneously running marker proteins of known molecular weight.

Samples are typically treated with sample buffer containing SDS at higher temperatures (<90 deg C) in the presence or absence of reducing agents (depending on type of analysis required). Samples are loaded on to either a gradient 4-20% Tris-Glycine gel or normal 10% Tris-Glycine gel (depending on the size of the protein). Mark12 molecular weight marker is used to compare and identify the molecular weight of sample bands. The gel is stained by silver staining using either SilverXpress or SilverQuest silver staining kits or Colloidal blue using Colloidal blue kit. The gels are scanned and analyzed using a densitometer to identify the sample bands.

• Sample required: 100 µg -500 µg protein

SDS-CE

Capillary methods to determine molecular weight are performed with non-reducing CE-SDS using a ProteomeLab PA 800 Capillary electrophoresis system. Separation is performed on a bare fused silica capillary with -15 kV separation voltage applied over 35 minutes. Intact antibody (main peak) and impurities (if any) can be detected at 220 nm. The migration time / purity of the sample is correlated with designated assay control.

Residual testing

Residual DNA (qPCR)

• Validated assays for CHO, BHK, E. coli, Human, and Mouse DNA

Host Cell Protein (ELISA)

• Cygnus Kit and custom assays

Residual Antibiotics

• Kanamycin, ampicillin, tetracycline, penicillin, chloramphenicol, gentamicin sulfate, amphotericin B, hygromycin B, plasmocin

Surfactants & Antifoams

• Triton-X, Pluoronic, Tween 20 & 80,Antifoam A,B,C

Process Enhancing Agents

• IPTG, DTT, Urea, Guanidine

Residual Solvents

• USP <467> and custom methods

Extractables & Leachables

• Design and execution of forced extraction, controlled extraction and leaching studies
  Metals
• 32 element scan, single or multiple element determinations

Fraction collection for isolation of impurities and residuals