Companies:

Biotechne Simple Western (after preparing reagents they are loaded into a Simple Western plate which is placed with a cpaillary cartridhe into a Simple Western instrument. The Simple Western does protein analysis thorugh separation and immunoprobing in a capillary system.) 

Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS): (see also capillary electrophoresis under Diagnostic Techniques)

Capillary electrophoresis-sodium dodecyl sulfate (CD-SDS) has emerged as a modern equilvalent of SDS-PAGE, offering superior reproductibility, sensitivity and throughput. (Wang WO 2019/152303)

Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is a modern equivalent of SDS-PAGE discussed below and offers superior reproducibility, sensitivty and throughput. During CD-SDS analysis of mAb products, minor peaks with shorter migraton times (LMW forms) than the intact antibody can be routinely observed. Unliked SDS-PAGE analysis, these LMW impurites cannot be extracted or subjected to further analyses. As a result, the idenitites of LMW impurites obersrved in CE-SDS methods are often proposed solely based on empirical knowledge. (Wang WL 2019/152303)

CE-SDS has emerged as a valuable alternative to conventional sodium dodecyl sulfate polycarylamide gele electrophoresis (SDS-PAGE) for the characterizaiton of automatic quantitation of biomolecules and particularly monoclonal antibodies. High separation efficiency, ease of operation and automation and reduced operating costs are the main reasons for this success. As a result, CE-SDS has found wide acceptance within the biopharmaceutical industry for the development, manufacturing and release of therapeutic mAbs. CE-SDS methods are in routine use for in process monitoring, purity assessment, detecing low level impurites and measuring size variant hterogeneity. (Schneider, “Tracking of antibody reduction fragment by capillary gel electrophoresis during the coupling to microparticles surface” J. Pharma. and Biomed. Analysis 53 (2010). 

CE-SDS is widely used for purity analysis of mAb therapeutics for release and stability to demonstrate product consistency and shelf life during the manurfacturing and life cycle of the product. The method separates molecule specific fragments and other impurities based on their electrophoretic mobility under denaturing conditions. Recently, US Pharmacopeia (USP) suggested that a universal mAb system suitability reference standard be adopted for CE-SDS and size exclusion chromatography. During sample prepration, antibodies are denatured in the presence of an ionic detergent which masks the native charge of the protein providing a net negative charge enabling electrophoretic mobility speration based almost exclusively on the hydrodynamic radius of the protein molecule(s). The disulfide bonds which are responsible for the tetrameric structure of mAbs are disrupted (reducing) or protected (non-reducing) during heat denaturation by reducing or alkylating reagents respectively. (Esterman “implementation of USP antibody standard for system suitability in capillary electrophoresis sodium dodecyl sulfate (CE-SDS) for rlease and stability methods” J. Pharmaceutical and Biomedical Analysis 128 (2016))

CE-SDS was used for seperation of denatured prtoein size variants under non-reduced or reduced conditions For non-reduced CE-SDS (nrCE-SDS) samples were denatured with sodium dodecyl suflate at 70C for 5 min in the present of iodoacetamide. for reduced CE-SDC (rCE-SDS), 2-mercaptoethanol (Bio-Rad) was added to the protein denaturation step to reduce the disulfide bonds. After denaturation, both non-reduced and reduced samples were injected int a bar fused silica capilary (Beckman Coulter) and separated based on hydrodynamic size resulting form an applied electric field in which migraiton of smaller sized proteins is inversiy related to overal size. (Katterle “How sable are new biologicals” Pharm, 80(11) 1557-1563 (2018). 

For Activatable Antibodies:

Carman (US 2019/0117789) discloses activatable antibodies that bind CD166. The activatable antibody (AA) includes an antibody that binds to CD166 including a heavy chain and light change, a masking moeity (MM) coupled to the Ab wherein the MM inhibits the binding of the AB to the CD166 when the AA is in an uncleaved state and a cleavable moeity (CM) coupled to the Ab, wehrein the CM is a substrate that functions for a protease. In some embdoiments an agent such as maytansinoid is conjugated to the AA. The anti-CD166 conjugated activatable antibody was activated with matriptase or MMP14 for 2 hours at 37C and mixed with intact conjugated activatable antibody. The mixture was then analysed by the The WES Capillary electrophoresis system using anti-human IgG (American Qualex Catalog #A110UK). Carman showed the ability to separate matriptase-activated or MMP14 activated conjugated activatable antibodies form intact conjugated activatable antibodies. 

Desnoyers (US Patent Application No: 16/632265) discloses a method of quantitating a level of activation of an activatable antibody which includes the steps of 1) loading at least one capillary with a stacking matrix and a separation matrix. the “stacking matrix” refers to a highly porous (relative to the separation matrix) material that functions to concentrate proteins present in the sample and “stack” them at the interface with the separation matrix so that the proteins start migration under electrophoresis conditions form the same physical starting point. Suitable stacking matrices include those used to prepare stacking gels for Western blotting such as acrylamide, SDS. Capillaries pre-loaded with stacking matrix and separation matrix are available commercially as Wes Separation Module. Electrophoresis causes the compounds in the sample to migrate through the separation gel at differential rates according to molecular size/weight. 2) the HMW and LMW compounds are immobilzied within each capilalry as by using UV light. 3) Each capilarry is immunoprobed with a first reagent such as an idiotypic antibody that is specific for the activatable antibody. 3) Detection of the first reagent can be accomplised for example with a second reagent that binds to the first reagent. Exemplary detectably labeled second r agents include HRP-conjugate anti-mouse secondary antibody. 

Vasiljeva (US Patent Application 2020/0377602) discloses polypeptides that include at least a first cleavable moiety (CM1) that is a substrate for at oast one matrix metalloprotease (MMP) and at leaset a second clevable moeity (CM2) that is a substrate for at least one serine protease to activatable antibodies which include these polypeptides. Vasiljeva disclosing measuring cleavage by capillary electrophoresis and analyzation with the Wes Western Blot protocol using goat anti-human IgG antibodies and anti-goat secondary anitobodies. The fraction of the cleaved activatable antibody was determined by quantifying the mobility of the higher mobility polypeptide corresponding to the cleaved antivatable antibody. 

Capillary electrophoresis-mass spectrometry (CE-MS):

CE-MS is a pwerful orthogonal technique capable of filling in gaps int he dientification, quantitation and isomeric resoluion of many small hydrophilic and charged metabolites. The metabolome is a large complex mixture of molecuels for which not one technique can optimally identify and measure it in it’s entirety. LC-MS, GC-MS and NMR have been widely used for metabolomics for teh past 20 years for a wide range of applications. CE-MS captures a unique metabolic chemical space beyond these standard methods pvoiding another window into metabolomics profiling. CE-MS is the most suited technique for analyzing posphorylated metabolites, amino acids or metabolites form teh TCA cycle and glycolytic pathways. A fused silica capillary contains surface charges of sianol groups present on the inner walls. The silanol groups on the capillary inner wall are ionized presenting an overall negative surface. Opposing ions in the electrolyte solution are attracted to the inner wall surface to achieve a balance of electric charges, resulting in the formation of a double layer with ionized silanol groups. Under these conditions, a potential diffference is created very close to the inner wall. The applicaiton of a voltage to both ends of the capillary attracts the positively charged ions of the diffuse double alyer to an anode. In contrast, the silanol groups cannot move due to the fixation on the wall surface and the entire electrolyte solution in the capillary is directed toward the anode with the migration of the positively charged ions, thereby generating a flow. The degree of mobility of any copound relative to tothers is due to variation in their ionic radius and size and charge of the electrolyte filing the capillary. A compound or metabilite with a larger ionic radius and smaller charge would ahve limited mobility compared to small, more polar species. Compounds with a small ionic radius and higher charge would have high obility. Hence controlling the electrical gradient across the capillary and pH of teh electrolyte solution are two of the most important parameters in controlling metabolite separation into the mass spectrometer. (Buko “Capillary electrophoresis mass spectrometry based metabolomics” J Applied Bioanalysis, 2017). 

Imaged capillary isoelectric focusing (iCIEF):

Charge profiels/isoelectric points (pI) of analytes can be determeind by imaged capillary isoelectric focusing (icIEF) analysis using an iCE3 system (ProteinSimple) equipped with a 5 cmx100 uM ID flurocarbon coated capillary Sampels are spearated according to theri pI and detected by using a whole column UV adsorption detector (280 nm) that avoids distrubing focused protein zones. Imaged capillary isoelectric focusing fo tthe analysis of therapeutic antibodies and antibody drug conjugates provides a fast separation and resolution of acidic, neutral and basis variants of an antibody. Katterle “How sable are new biologicals” Pharm, 80(11) 1557-1563 (2018).