Differential scanning calorimetry (DSC) can be used to examine the stability of the tertiary structure of antibody preparations. (Brass, Pharm Acta Helv. 1996, 71(6): 395-403) 

Immunoassay (ELISA): can be used to determine the binding activites of antibody preparations. 

Biospecific Interaction Analysis:

Biosensor Technology:

–BIAcore biosensor-based analytical system: (BIAcore, Pharmacia Biosensor AB, uppsala, Sweden) is designed for functional chacterization of protein-protein, protein NA and ligand-receptor interaction in real time. The adsorption of biomolecuesl to an immobilized ligand on a sensor chip is measured in the same time and place as it occurs. BIAcore is baswed on a bisensor that uses surface plasmon resonance (SPR) to monitor the adsorption of biomolecuels on a sensor chip. This optical techniques measures changes in refractive index in the vicinity of the surface. Such changes are directly proportional to the change in adsorbed mass,. The system includes a sensor chip to which the lgiand can be immobilized in a 100 nm thick hyrophilic matrix composed of 2-3 per cent flexible desxtran, a miniaturized fluidics cartridge for the transport of analytes and reagents to the sensor chip, a SPR detector, an auto inejector and sotware for the system control and evaluation of resutls. Specific ligands are iimmobilized to the sensor chip at low concetnraiton through biotin-avidin interaction or covalently thorugh amine, thiol or aldehyde chemistry. The stable binidng allows regeneration of the sensor surface of 50-100 analytical cycles. The analysis can also be performed in tissue culture media or bacterial browth without the need for purificaiton in contrast to other techniques used for kinetics analysis. The technology was originally developed using mAb-antigen interactions but is not rapidly being transferred to ther areas of biological significance. There are many applications such as epitope mapping. For example eptiope mapping of HIV-1 core protein p24 by pairwise mapping of 30 different mAbs using the BIAcore system has been performed. Both identifciation of peptides inhibiting the mAb binding and mltiple binding of several mAbs in sequence forming a hexamolecular complex on the surface were shown. (Malmqvist “Biospecific interaction analysis using biosensor technology”)

Analysis of DNA:

DNA quantification: (Brass, Pharm Acta Helv. 1996, 71(6): 395-403)

Total DNA as a contaminant in biopharmaceutical products if of concern becasue of its potential health ris. In the past, the analytical method to quantitate DNA contamination was DNA probe hydridization whcih can detect less than 10 pg of specific DNA. Merrick (“A complete system for quantitative analysis of total DNA, prtoein impurities and relevant proteins” BFE, vol 9, no.6, 1992) discloses a threshold system which includes an instrument, computer and softwarefor quantitative asss for picogram amounts of total DNA. After denaturation of DNA in a sample, a single labeling reagent is added, containing streptaidin plus two non sequence specific DNA binding proteins with high specificity fo single stranded NA. E. coli single stranded binding protein conjugated to biotein and a monoclonal anti-DNA antibody conjugated direclty to urease. After formation of reaciton complexes in liquid phase and active cpature on the giotinylated nitrocellulose membranes, urease actiity is measured in the sensor. 

–Real Time PCR (CHO DNA): can be quantified using a real time PCR. First DNA is extracted using Qiagen’s Virus biorobot kit and then samples/controls subjected to TaqMan real time PCR using a 110 base pair segment of repetitive DNA sequence in the Cricetulus griseus genome. (Nadarajah, US 14/355818)

Ion Exchange

Cation Exchange:

–% iimpurities:

Brige (US 9,884,117) discloses a purity assay of an immunogloublin single domain using a Dionex ProPac WCX-10 weak cation exchagne columne and mobile phase consisting of citrate buffer. After loading the protein(s) on the olumn, bound materials were eluted by a sodium chloride graient. The relative amount of the specific protein variant or impurities, expressed as area %, was calculated by dividing the peak area corresponding to the specific protein or to any protein impurity by the total area of all integrated peaks. 

SDS-PAGE: 

SDS-PAGE performed under non-reducing conditions is one of the most commonly used techniques to access mAb product purity. During analysis, minor bands corresponding to LMW species can be routinely observed and quantified include H2L (2 heavy chains and 1 light chain), H2 (2 heavy chains), HL (1 heavy chain and 1 light chain), HC (1 heavy chain) and LC (1 light chain) species, with respect to antibodies (Wang US 2019/152303)

During analysis, minor bands corresponding to LMW speceis can be routinely observed and quantified including H2L (2 heavy chains and 1 light chain), H2 (2 heavy chains), HL (1 heay chain and 1 light chain), HC (1 heavy chain ) and LC (1 light chain species, with respect to antibodies). (Wang, US 16/223,463, published as US 2019/0194298)

For Activatable Antibodies:

Chen (“Selective antibody activation through protease-activated pro-antibodies that mask binding sites with inhibitory domains” Scientific Report (2017) discloses development of protease-activated pro-antibodies by masking the binding sites of antibodies with inhibitory domains that can be removed by proteases. The latenecy-assocatied peptide (LAP) was linked through a substrate peptide of matrix metalloproteinase 2 (MMP-2) to an anti-epidermal growth factor receptor (EGFR) antibody and anti-tumor necrosis factor alpha antibody. Results of reducing SDS-PAGE showed that all of the inhibitory domains could be removed by MMP-2 to restore the binding activities of the antiobides. 

Moore (US Patent No: 2015/0087810) discloses multispecific activatable antibodies that engage immune effector cells, also referred to as immune effector cell engaging multispecific activatable antibodies. In some embodimetns, the antibodies are designed to engage leukocytes. Moore demonstrate protease activation of the multispecific activatable antibodies with SDS-PAGE. Briefly, the samples were denatured at 70C, six ug of antibody was loaded onto a NuPAGE 10% Bis-Tris gel (Invitrogen) and proteins were separated by sie using the MOPS electrophoresis buffer. Folloiwing electrophorosis the gel was stained with Coomassie blue. The change in mobility of anti-EGFR activatable antibody and ant-EGFR multispecific activatable antibody demonstrated proteolytic activation of the multispecific activatalbe antiobdies. the lack of any change in the moility of the H chain fusions demonstrates the resistance to protease cleavage. 

Size exclusion chromatography (SEC): 

High performance size exclusion chromatography such as the TSK G3000SWXl SEC column can be used to determined relative levels of mAb monomer (Nadarajah, US 14/355818)

Boschetti (WO/2003/042233) discloses using SELDI to identify and/or quantify non-degraded target polypeptides and distinguish impurities during the expression of target polypeptides.

O’Connor (US 2016/0251441) discloses injecting a gest sample onto a TosoHaas G300SWXL column and eluting isocratically with 0.1 M disodium phosphate containing 0.1 M sodium sulfate and 0.05% sodium azide, pH 6.8 at a flow rate of 1.0 ml/minute. The eluted prtoein was detected using UV absorbance at 280 nm. Peaks eluting elarier than the nomer peak were recorded as percent aggreate and peaks eluting after the monomer peak were recorded as percent other. 

SEC-MS:

Haberger (MABS, 2016, 8(2) 331-339) discloses detetermining HMW aggregates such as dimers as well as fragment variants in a CrossMap preparation using ultra-pressue liquid chromatography size exclusion separation combined with native electrospray ionization mass spectrometry. 

Dixit “LC-MS characterization and purity assessment of a prototype bispecific antibody” mAbs 5-6, 711-722 (2013) discloses a liquid chromatography mass spectometry 9LC-MS) based method for evaluating heterodimeric purity of a prototype antibody containing two different heavy chains and two identical light chains. The LC-MS based assay used an advanced ESI-Q-TOF mass spectromer that evalutes heterodimeric purity of MAb1, a prototype ehterodimeric antibody after deglycosylation by detecting and quantifying homodimeric and half-antibody (H+L chain) impurities. MAb1 was based on the IgG1 trastuzumab and is an Fc heterodimer composed of two different half antibodies sharing a common light chain. H chains have different amino acids at 6 sites in the Fc region (resulting in a 172 Da mass difference). MAb1 and the independently expressed homodimeric standards (contianing only the A or B heavy chains plus a common light chain were first characterized by two complentary LC-MS techniques, intact protein mass analysis after deglycosylation and LC-MS peptide mapping of Lys-C digests prepared form reduced and alkylated samples. (1) First, the homodimeric standards were purified by siz-exclusion chromatogrpahy (SEC) HPLC to separate out half antibodies and enrich full lenght homodimeric antibody in the sample. (2) Deglycosylation was performed prior to intact protein mass analysis to reduce sample heterogeneity and simplify detection and quantificiton of heterodimer and homdimers. Although half-antibodies can be detected and quantified by purely seperation based methods such as SDS-PAGE, evaluation of half anitobdies in conjunction with homodimer evaluation will save time and resources. 

Mass Spectrometry (MS) (see also MS in Diagnostic Techniques)

Light Scattering: (See outline)

RP-HPLC: 

O’Connor (US 2016/0251441) discloses intecting a sample onto a PLRP-S column. Mobile phase A was 0.05% TFA in water and mobile phase B was 0.05% TFA in acetonitrile. The protein was eluted using a gradient of 34-40% TFA in acetonitril (mobile phase b) form 5-12 minutes and was monitored using UV absorbance at 280 nm. The reproted fragment percent was the total area of all the fragments divdied by the total peak area of all the fragments and the intact antibdoy.