LC-MS characterization and purity assessment of a prototype bispecific antibody

Bispecific IgG asymmetric (heterodimeric) antibodies offer enhanced therapeutic efficacy, but present unique challenges for drug development. These challenges are related to the proper assembly of heavy and light chains. Impurities such as symmetric (homodimeric) antibodies can arise with improper assembly. A new method to assess heterodimer purity of such bispecific antibody products is needed because traditional separation-based purity assays are unable to separate or quantify homodimer impurities. This paper presents a liquid chromatography-mass spectrometry (LC-MS)-based method for evaluating heterodimeric purity of a prototype asymmetric antibody containing two different heavy chains and two identical light chains. The heterodimer and independently expressed homodimeric standards were characterized by two complementary LC-MS techniques: Intact protein mass measurement of deglycosylated antibody and peptide map analyses. Intact protein mass analysis was used to check molecular integrity and composition. LC-MS^E peptide mapping of Lys-C digests was used to verify protein sequences and characterize post-translational modifications, including C-terminal truncation species. Guided by the characterization results, a heterodimer purity assay was demonstrated by intact protein mass analysis of pure deglycosylated heterodimer spiked with each deglycosylated homodimeric standard. The assay was capable of detecting low levels (2%) of spiked homodimers in conjunction with co-eluting half antibodies and multiple mass species present in the homodimer standards and providing relative purity differences between samples. Detection of minor homodimer and half-antibody C-terminal truncation species at levels as low as 0.6% demonstrates the sensitivity of the method. This method is suitable for purity assessment of heterodimer samples during process and purification development of bispecific antibodies, e.g., clone selection.     

High-throughput laser-mediated in situ cell purification with high purity and yield.

BACKGROUND: Technologies for purification of living cells have significantly advanced basic and applied research in many settings. Nevertheless, certain challenges remain, including the robust and efficient purification (e.g., high purity, yield, and sterility) of adherent and/or fragile cells and small cell samples, efficient cell cloning, and safe purification of biohazardous cells. In addition, existing purification methods are generally open loop and exhibit an inverse relation between cell purity and yield. METHODS: An automated closed-loop (i.e., employing feedback control) cell purification technology was developed by building upon medical laser applications and laser-based semiconductor manufacturing equipment. Laser-enabled analysis and processing has combined high-throughput in situ cell imaging with laser-mediated cell manipulation via large field-of-view optics and galvanometer steering. Laser parameters were determined for cell purification using three mechanisms (photothermal, photochemical, and photomechanical), followed by demonstration of system performance and utility. RESULTS: Photothermal purification required approximately 10(8) W/cm(2) at 523 nm in the presence of Allura Red, resulting in immediate protein coagulation and cell necrosis. Photochemical purification required approximately 10(9) W/cm(2) at 355 nm, resulting in apoptosis induction over 4 to 24 h. Photomechanical purification required more than 10(10) W/cm(2) independent of wavelength, resulting in immediate cell lysis. Each approach resulted in high efficiency purification (>99%) after a single operation, as demonstrated with eight cell types. An automated closed-loop process to re-image and irradiate remaining targets in situ was implemented, resulting in improved purification (99.5-100%) without decreasing cell yield or affecting sterility in this closed system. Efficient purification was demonstrated with B- and T-cell mixtures over a wide range of contaminating cell percentages (0.1-99%) and cell densities (10(4)-10(6)/cm(2)). Efficient cloning of 293T cells based on fluorescence with green fluorescent protein after plasmid transfection was also demonstrated. CONCLUSIONS: In situ laser-mediated purification was achieved with nonadherent and adherent cells on the automated laser-enabled analysis and processing platform. Closed-loop processing routinely enabled greater than 99.5% purity with a greater than 90% cell yield in sample sizes ranging from 10(1) to 10(8) cells. Throughput ranged from approximately 10(3) to 10(5) total cells/s for contaminating percentages ranging from 99% to 0.1%, respectively.     

New procedures for glycophorin A purification with high yield and high purity

Glycophorin A (GPA) is the major glycoprotein of the human erythrocyte membrane. It is known to form, in SDS gels as well as in a membrane environment, homodimers, and also heterodimers with the homologous molecule Glycophorin B (GPB). It is shown in this report that the propensity of GPA to dimerize with GPB precludes satisfactory preparation with high yield of pure GPA using classical techniques including SEC and RPLC. It was demonstrated using multiple angle light scattering that GPA is eluted from RPLC columns as dimers. A convenient procedure was devised which allowed us to get pure GPA with high yield. This procedure consists of selectively blocking GPA–GPB heterodimer formation by selective modification of Cysteine 50 of GPB before RPLC.     

Monoclonal antibody purification using cationic polyelectrolytes: an alternative to column chromatography

The potential of cationic polyelectrolytes to precipitate host cell and process related impurities was investigated, to replace one or more chromatography steps in monoclonal antibody purification. The impact of antibody isoelectric point, solution properties (pH and ionic strength), and polyelectrolyte properties (structure, molecular weight and pK(a)) on the degree of precipitation was studied. At neutral pH, increasing solution ionic strength impeded the ionic interaction between the polyelectrolyte and impurities, reducing impurity precipitation. Increasing polyelectrolyte molecular weight and pK(a) enabled precipitation of impurities at higher ionic strength. PoIy(arginine) was selected as the preferred polyelectrolyte in unconditioned cell culture fluid. PoIy(arginine) precipitation achieved consistent host cell protein clearance and antibody recovery for multiple antibodies across a wider range of polyelectrolyte concentrations. Poly(arginine) precipitation was evaluated as a flocculant and as a functional replacement for anion exchange chromatography in an antibody purification process. Upstream treatment of cell culture fluid with poly(arginine) resulted in flocculation of solids (cells and cell debris), and antibody recovery and impurity clearance (host cell proteins, DNA and insulin) comparable to the downstream anion exchange chromatography step.     

Antibiotic purification from fermentation broths by counter-current chromatography: analysis of product purity and yield trade-offs

Counter-current chromatography (CCC) is a low pressure, liquid–liquid chromatographic technique which has proven to be a powerful purification tool for the high-resolution fractionation of a variety of active pharmaceutical compounds. The successful integration of CCC into either existing or new manufacturing processes requires the predictable purification of target compounds from crude, fermentation-derived, feed streams. This work examines the feasibility of CCC for the purification of fermentation-derived erythromycin A (EA) from its structurally and chemically similar analogues. At the laboratory scale, the effect of feed pre-treatment using either clarified, forward extracted (butyl acetate) or back extracted broth on EA separation was investigated. This defined the degree of impurity removal required, i.e. back extracted broth, to ensure a reproducible elution profile of EA during CCC. Optimisation and scale-up of the separation studied the effects of mobile phase flow (2–40 mlmin^–1) and solute loading (0.1–10 g) on the attainable EA purity and yield. The results in all cases demonstrated a high attainable EA purity (>97% w/w) with throughputs up to 0.33 kgday^–1. Secondly, a predictive scale-up model was applied demonstrating, that from knowledge of the solute distribution ratio of EA (K_EA) at the laboratory scale, the EA elution time at the pilot scale could be predicted to within 3–10%, depending upon the solute injection volume. In addition, this study has evaluated a fractionation diagram approach to visually determine the effects of key operational variables on separation performance. This resulted in accurate fraction cut-point determination for a required degree of product purity and yield. Overall, the results show CCC to be a predictable and scaleable separation technique capable of handling real feed streams.     

Rapid purification of EGFP, EYFP, and ECFP with high yield and purity

Most current high throughput purification procedures for the green fluorescent protein (GFP) suffer from poor yields and low purity. An improved purification procedure that delivers highly pure protein (>95% homogeneity) in high yields (>70% of the initial fluorescent protein content) has been developed. The purification procedure requires only two steps: the cell lysate is heated to 60 degrees C for 4 min in ammonium sulfate and triethylamine, followed by hydrophobic interaction chromatography using isopropanol during the elution phase. The resulting pure product exhibits the same fluorescence profile as the crude sample. This procedure has been demonstrated on three commercial variants of GFP from Aequorea victoria, enhanced green, enhanced yellow, and enhanced cyan fluorescent protein (Becton-Dickinson). The yield and purity of material are superior to other recently described methods.     

Functional phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv: rapid purification with high yield and purity

Phosphoglucose isomerase (PGI) EC 5.3.1.9, is a housekeeping enzyme that catalyzes the reversible isomerization of d-glucopyranose-6-phosphate and d-fructofuranose-6-phosphate. We have previously reported expression and multistep purification of recombinant PGI from Mycobacterium tuberculosis using conventional methods. We now describe an improved and simplified single step approach for purification of functionally active mycobacterial rPGI. The gene encoding PGI from M. tuberculosis H37Rv was cloned in bacterial expression vector pET22b(+). Expression of recombinant PGI with six-histidine-tag protein was observed both in the soluble fraction and inclusion bodies. Approximately 116mg of recombinant enzyme was purified to near homogeneity with approximately 80% yield from the soluble fraction of 1L culture at shake flask level using one step Ni-NTA affinity chromatography. The specific activity of the purified six-histidine-tagged recombinant PGI (rPGI-His(6)) was approximately 800U/mg of protein. The apparent K(m) value of the active recombinant protein followed Michaelis-Menten kinetics and was 0.27+/-0.03mM. K(i) for the competitive inhibitor 6-phosphogluconate was 0.75mM. The enzyme had pH optima in the range of pH 7.6-9.0 and was stable up to 55 degrees C. rPGI-His(6) exhibited enzyme activity almost equal to that of enzyme without histidine tag.     

Monoclonal antibody assessment of tissue- and species-specific myosin light chain kinase isozymes

Monoclonal antibodies raised against chicken gizzard smooth muscle myosin light chain kinase were used for immunological and structural studies of this enzyme. Epitope mapping of trypsin-digested chicken gizzard enzyme showed that MM-1, 2, 3, 4, 5, 6, and 7 bind to 65 kDa (trypsin-digested) and 60 kDa (chymotrypsin-digested) fragments which contain the catalytic domain of the kinase. Kinetic analysis demonstrated that MM-7 inhibited kinase activity competitively with respect to ATP and noncompetitively with respect to myosin light chain, thereby indicating that MM-7 binds at or near the ATP binding site of the enzyme. Immunoblot analysis revealed that all these antibodies (MM-1 to 12) reacted with the enzyme (130 kDa) from intestinal and vascular smooth muscles, whereas 5 (MM-1, 3, 4, 6, and 9) or 3 (MM-1, 3, and 4) of 12 antibodies did not cross-react with chicken cardiac muscle or with blood platelet myosin light chain kinase (130 kDa), respectively. None of these antibodies showed cross-reactivity against skeletal muscle myosin light chain kinase. As for mammalian species, MM-11 and 12 reacted with myosin light chain kinase of vascular smooth muscle (140 kDa) and MM-11 cross-reacted with the enzyme (140 kDa) from cardiac muscle of rat and rabbit. These data suggest the existence of at least 4 subspecies of myosin light chain kinase in chicken tissues and the heterogeneity of tissue- and species-specific isozyme forms.     

Monoclonal antibody immunocytochemistry: novel method extending usefulness of monoclonal antibodies for antigen visualization

We describe a novel procedure combining the multiple-site reactivity of polyclonal antibodies with the defined single epitope-specificity of monoclonal antibodies. The method is based on previous findings that IgG molecules often only react with tissue-bound antigens with one of their two antigen-combining sites; thus, the remaining site is free to bind subsequently added antigen. In the procedure devised, such (undenatured) antigen is subsequently detected by a specific monoclonal antibody and the reaction is finally revealed by immunogold-silver staining. Antibody subpopulations to contaminating antigens may well be present in the polyclonal antiserum and may well bind first to tissue and then to the corresponding contaminants in the crude antigen preparation applied as second layer. Such contaminants will, however, not react with the monoclonal antibody and will therefore not be immunocytochemically detected. The method has been evaluated with one antigen which cannot be detected by monoclonal antibodies in paraffin sections (glial fibrillar acidic protein) and with another antigen (human chorionic gonadotropin) which can only be detected by the monoclonal antibody when occurring in high concentrations. In both cases the procedure resulted in strong specific staining of the antigens with no background.     

New method to prepare single-headed heavy meromyosin with high purity and a high yield

We have developed a new method to prepare single-headed heavy meromyosin with high purity and a high yield. To examine whether the two heads on the same myosin molecule work cooperatively or not, it is important to prepare pure single-headed heavy meromyosin. Myosin was extracted from myofibrils treated with a solution containing CyDTA, a strong divalent cation chelator. CyDTA treatment was essential to the production of sHMM. Then such myosin was digested with chymotrypsin in the presence of divalent cations at high ionic strength. Crude sHMM was separated from double-headed HMM by affinity chromatography using an ADP-column. Contaminating S1 was removed by gel filtration. Heavy chain of sHMM obtained by the present method had no nick. Purified sHMM showed normal EDTA-ATPase and Ca-ATPase. It interacted with thin filament and its ATPase was activated by actin normally.     

Monoclonal antibody therapy of cancer

The most significant recent advances in the application of monoclonal antibodies (mAbs) to oncology have been the introduction and approval of bevacizumab (Avastin), an anti-vascular endothelial growth factor antibody, and of cetuximab (Erbitux), an anti-epidermal growth factor antibody. In combination with standard chemotherapy regimens, bevacizumab significantly prolongs the survival of patients with metastatic cancers of the colorectum, breast and lung. Cetuximab, used alone or with salvage chemotherapy, produces clinically meaningful anti-tumor responses in patients with chemotherapy-refractory cancers of the colon and rectum. In addition, the anti-HER2/neu antibody trastuzumab (Herceptin), in combination with standard adjuvant chemotherapy, has been shown to reduce relapses and prolong disease-free and overall survival in high-risk patients after definitive local therapy for breast cancer. These exciting recent results provide optimism for the development of mAbs that bind novel targets, exploit novel mechanisms of action or possess improved tumor targeting. Progress in the clinical use of radioimmunoconjugates remains hindered by complexity of administration, toxicity concerns and insufficiently selective tumor targeting.     

Monoclonal antibody purification by affinity chromatography with ligands derived from the screening of peptide combinatory libraries

The peptide, Ala-Pro-Ala-Arg (APAR), was selected from the screening of a tetrapeptide combinatorial synthetic library as the ligand for affinity purification of an anti-Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) monoclonal antibody (Mab) developed in mouse ascitis. The affinity chromatographic matrix obtained by attachment of APAR to agarose, having a peptide density of 0.5 mol ml^–1, showed a maximum capacity of 9.1 mg Mab ml^–1 and a dynamic capacity of 3.9 mg Mab ml^–1. A 95% yield of electrophoretically pure anti-GM-CSF was obtained in a single step.     

Monoclonal antibody against the catalytic subunit of bovine heart cAMP-dependent protein kinase and the application of enzyme purification

A monoclonal antibody against the catalytic subunit of bovine heart cAMP-dependent protein kinase has been prepared. The antibody, M73/PK1, belongs to the heavy-chain subclass IgG3 and has a titer of 20,480. Purified M73/PK1 was obtained by ammonium sulfate precipitation and Protein A-agarose affinity chromatography and was coupled to CNBr-activated Sepharose 6MB. The catalytic subunit was purified by this affinity column. A 75% yield and 4746-fold purification were obtained. These results show that this is a suitable and effective method for the purification of the catalytic subunit of bovine heart cAMP-dependent protein kinase.     

A new method for purification of anti-glycosphingolipid antibody. Avian anti-hematoside (NeuGc) antibody

A new method for purification of anti-glycosphingolipid antibodies had been developed. N-Glycolylneuraminyl(alpha 2-3)lactosylceramide [hematoside (NeuGc)] could be hydrophobically bound on octyl-Sepharose 4B in the presence of 0.1 M KCl. The Sepharose gel coated with hematoside (NeuGc) was used as immunoadsorbent for affinity column chromatography to purify avian anti-hematoside (NeuGc) antibody. The procedure is very simple, reproducible and applicable to purification of almost all anti-glycosphingolipid antibodies. The glycosphingolipid used for the affinity chromatography could be recovered without any destruction by successive extraction of the gel with methanol and methanol/chloroform (1:2, v/v).     

Monoclonal antibody technology: applications in veterinary science

Monoclonal antibodies have revolutionised the study of animals and their diseases. The author looks at the detection of antigen in samples using a range of techniques from indirect fluorescence, through in-situ hybridization to enzyme linked immunosorbent assays. Examples are given of how Salmonella species, mastitis antigens, viral antigens, chlamydial organisms and E. coli toxins can be detected using specific monoclonal antibodies. The recognition of antigen in tissues by monoclonal antibodies is also discussed using as examples; the vitamin biotin, the chicken anemia virus, the growth promoter clenbuterol and the bovine lymphokine, gamma interferon. The ability of monoclonal antibodies to measure specific antibody is also discussed, with particular reference to chicken anemia agent. The review concludes with a discussion of the ability of monoclonal antibody based ELISAs to discriminate between pigs naturally infected with Aujeszky's disease and those vaccinated against the condition.