Novel Multidimensional Liquid Chromatography Workflow with In-Loop Enzymatic Digests of Multiple Heart-Cuts for Fast and Flexible Characterization of Biotherapeutic Protein Variants.

Multidimensional liquid chromatography (mD-LC) is becoming a powerful tool for complete characterization of individual peaks and protein variants through separation methods such as nondenaturing ion exchange (IEC) or size-exclusion chromatography coupled to reversed-phase (RP) chromatography. The flexibility of commercially available and customized mD-LC systems is still limited in terms of enzymatic peak processing between chromatographic dimensions. In this regard, only a few column-immobilized proteases are available for detailed peak characterization by mD-LC coupled to mass spectrometry (mD-LC-MS). Here, we present a purpose-built and automated multiple heart-cutting mD-LC design with a novel analytical workflow involving in-loop enzymatic heart-cut digestion between the first-dimensional column and transfer to the second dimension before MS or MS/MS analyses. The setup facilitates the spike-in of any enzyme to multiple heart-cuts for multilevel analysis, for example, for peptide mapping, fragment generation, or deglycosylation, to reduce heterogeneity and provide maximum flexibility in terms of incubation time for optimal peak characterization. We demonstrate the application of IEC coupled to RP-LC-MS and automated in-loop deglycosylation and on-column reduction of an IgG antibody combined with upper hinge region cleavage for Fab generation. We further employ mD-LC-MS and mD-LC-MS/MS to assess post-translational modifications of a bispecific antibody and to support molecule selection by evaluating the best downstream purification strategy. The novel design and automated workflow of the mD-LC system described here offers enhanced flexibility for in-solution processing and real-time monitoring of multiple heart-cuts enabling streamlined characterization of unknown biotherapeutic charge and size variants.

Inter-laboratory study to evaluate the performance of automated online characterization of antibody charge variants by multi-dimensional LC-MS/MS.

An international study was conducted to evaluate the performance and reliability of an online multi-dimensional (mD)-LC-MS/MS approach for the characterization of antibody charge variants. The characterization of antibody charge variants is traditionally performed by time-consuming, offline isolation of charge variant fractions by ion exchange chromatography (IEC) that are subsequently subjected individually to LC-MS/MS peptide mapping. This newly developed mD-LC-MS/MS approach enables automated and rapid characterization of charge variants using much lower sample requirements. This online workflow includes sample reduction, digestion, peptide mapping, and subsequent mass spectrometric analysis within a single, fully-automated procedure. The benefits of using online mD-LC-MS/MS for variant characterization include fewer handling steps, a more than 10-fold reduction in required sample amount, reduced sample hold time as well as a shortening of the overall turnaround time from weeks to few days compared to standard offline procedures. In this site-to-site comparison study, we evaluated the online peptide mapping data collected from charge variants of trastuzumab (Herceptin®) across three international laboratories. The purpose of this study was to compare the overall performance of the online mD-LC-MS/MS approach for antibody charge variant characterization, with all participating sites employing different mD-LC-MS/MS setups (e.g., instrument vendors, modules, columns, CDS software). The high sequence coverage (95%-97%) obtained in each laboratory, enabled a reproducible generation of tryptic peptides and the comparison of values of the charge variants. Results obtained at all three participating sites were in good agreement, highlighting the reliability and performance of this approach, and correspond with data gained by the standard offline procedure. Overall, our results underscore of the benefit mD-LC-MS/MS technology for therapeutic antibody characterization, confirming its potential to become an important tool in the toolbox of protein characterization scientists.

From proof of concept to the routine use of an automated and robust multi-dimensional liquid chromatography mass spectrometry workflow applied for the charge variant characterization of therapeutic antibodies.

The identification and quantification of post-translational modifications (PTMs) is a crucial step required during the development of therapeutic proteins. In particular, the characterization of charge variants separated by cation exchange chromatography (CEX) is a tedious process commonly performed with an off-line manual fraction collection followed by peptide mapping. To improve the efficiency of this time-consuming approach, a generic on-line multi-dimensional LC/MS approach was developed for the characterization of various monoclonal antibody (mAb) isotypes and a bi-specific antibody (BsAb). Fractions collected from 1D CEX analysis were consecutively reduced on a 2D reversed phase liquid chromatography (RPLC) column (polyphenyl), digested within 1-2 min using a 3D immobilized trypsin cartridge, and finally the obtained peptides were separated on another 4D RPLC column (C18), and simultaneously identified with a Q Exactive™ mass spectrometer. 2D RPLC columns and 3D trypsin cartridges from different suppliers were compared, as well as the effects of reducing agents. The effect of 2D and 4D RPLC column temperature, and 2D RPLC column mass load were also systematically studied. Under optimal conditions, the multi-dimensional LC/MS system described in this paper is a robust tool for the on-line digestion of proteins and shows high repeatability. Similar levels of oxidation and deamidation were measured using the off-line and on-line approaches for the same stressed samples. The lower amounts of deamidation and isomerization measured at some asparagine and aspartic acid residues by the on-line approach compared to the manual off-line procedure suggest reduced artifacts using the on-line methodology. The multi-dimensional LC/MS described here enables fast, on-line, automated characterization of therapeutic antibodies without the need for off-line fraction collection and sample pre-treatment (manual approach). The entire workflow can be completed within less than one day, compared to weeks with the manual off-line procedure.

Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine, two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics.

Biotherapeutics may contain a multitude of different post-translational modifications (PTMs) that need to be assessed and possibly monitored and controlled to ensure reproducible product quality. During early development of biotherapeutics, unexpected PTMs might be prevented by in silico identification and characterization together with further molecular engineering. Mass determinations of a human IgG1 (mAb1) and a bispecific IgG-ligand fusion protein (BsAbA) demonstrated the presence of unusual PTMs resulting in major +80 Da, and +16/+32 Da chain variants, respectively. For mAb1, analytical cation exchange chromatography demonstrated the presence of an acidic peak accounting for 20%. A + 79.957 Da modification was localized within the light chain complementarity-determining region-2 and identified as a sulfation based on accurate mass, isotopic distribution, and a complete neutral loss reaction upon collision-induced dissociation. Top-down ultrahigh resolution MALDI-ISD FT-ICR MS of modified and unmodified Fabs allowed the allocation of the sulfation to a specific Tyr residue. An aspartate in amino-terminal position-3 relative to the affected Tyr was found to play a key role in determining the sulfation. For BsAbA, a + 15.995 Da modification was observed and localized to three specific Pro residues explaining the +16 Da chain A, and +16 Da and +32 Da chain B variants. The BsAbA modifications were verified as 4-hydroxyproline and not 3-hydroxyproline in a tryptic peptide map via co-chromatography with synthetic peptides containing the two isomeric forms. Finally, our approach for an alert system based on in-house in silico predictors is presented. This system is designed to prevent these PTMs by molecular design and engineering during early biotherapeutic development.

DuoMab: a novel CrossMab-based IgG-derived antibody format for enhanced antibody-dependent cell-mediated cytotoxicity.

High specificity accompanied with the ability to recruit immune cells has made recombinant therapeutic antibodies an integral part of drug development. Here we present a generic approach to generate two novel IgG-derived antibody formats that are based on a modification of the CrossMab technology. MoAbs harbor two heavy chains (HCs) resulting in one binding entity and one fragment crystallizable region (Fc), whereas DuoMabs are composed of four HCs harboring two binding entities and two Fc regions linked at a disulfide-bridged hinge. The latter bivalent format is characterized by avidity-enhanced target cell binding while simultaneously increasing the 'Fc-load' on the surface. DuoMabs were shown to be producible in high yield and purity and bind to surface cells with affinities comparable to IgGs. The increased Fc load directed at the surface of target cells by DuoMabs modulates their antibody-dependent cell-mediated cytotoxicity competency toward target cells, making them attractive for applications that require or are modulated by FcR interactions.

Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy.

Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti-4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fcγ receptor-mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP-4-1BBL (RG7826) and CD19-4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen-mediated hyperclustering without systemic activation by FcγR binding. We could show targeting of FAP-4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer-bearing rhesus monkey. Combination of FAP-4-1BBL with tumor antigen-targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-γ and granzyme B secretion. Further, combination of FAP- or CD19-4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8+ T cells. FAP- and CD19-4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies.

CD19-/CD3-bispecific antibody of the BiTE class is far superior to tandem diabody with respect to redirected tumor cell lysis.

Many kinds of bispecific antibodies recruiting T cells for cancer therapy have been developed. Side-by-side comparison has shown that CD19-/CD3-bispecific antibodies of the diabody, tandem diabody (Tandab) and quadroma format had similar cytotoxic activity, with Tandab being the most active format. Tandab has also been claimed to be superior to single-chain (sc) Fv-based bispecific constructs although data from a side-by-side comparison are not available. In this study, we compared side-by-side MT103 (bscCD19xCD3), a single-chain bispecific antibody of the BiTE class, with a CD19-/CD3-bispecific representative of the Tandab class. Based on literature data, we have constructed, produced and characterized the LL linker version of Tandab, which was reported to be the most active version of Tandab proteins. A dimeric protein of 114kDa was obtained that showed proper bispecific binding to CD3- and CD19-positive cells and could redirect both pre-stimulated and unstimulated human T cells for lysis of human B lymphoma lines Raji, MEC-1 and Nalm-6. Raji cells were lysed at a half-maximal concentration (EC50) of 10 nM Tandab using pre-stimulated T cells, which closely matched the published activity of LL-Tandab with this particular cell line. MT103 had between 700- and 8000-fold higher efficacy than Tandab for redirected lysis of the three human B lymphoma lines. These data demonstrate that under identical experimental conditions, the BiTE format has far superior activity compared to the Tandab format and is also superior to conventional diabody and quadroma formats. The extraordinary potency of the BiTE class and its representative MT103 may translate into improved anti-tumor activity, lower dosing and lower costs of production compared to other bispecific antibody formats.

A human monoclonal IgG1 potently neutralizing the pro-inflammatory cytokine GM-CSF.

The pro-inflammatory cytokine GM-CSF is aberrantly produced in many autoimmune and chronic inflammatory human diseases. GM-CSF neutralization by antibodies has been shown to have a profound therapeutic effect in animal models of rheumatoid arthritis, inflammatory lung diseases, psoriasis and multiple sclerosis. Moreover, the absence of GM-CSF in null mutant mice ameliorates or prevents certain of these diseases. Here we describe the biophysical and biological properties of a human anti-GM-CSF IgG1 antibody designated MT203, which was derived by phage display guided selection. MT203 bound with picomolar affinity to an epitope on human and macaque GM-CSF involved in high-affinity receptor interaction. As a consequence, the antibody potently prevented both GM-CSF-induced proliferation of TF-1 cells with a sub-nanomolar IC50 value and the production of the chemokine IL-8 by U937 cells. MT203 neutralized equally well recombinant (r) human (h) GM-CSF from Escherichia coli and yeast, and also normally glycosylated GM-CSF secreted by human lung epithelial cells in response to IL-1beta stimulation. Furthermore, MT203 significantly reduced both survival and activation of peripheral human eosinophils as may be required for effective treatment of inflammatory lung diseases. The antibody did not show a detectable loss of neutralizing activity after 5 days in human serum at 37 degrees C. Based on its favorable properties, MT203 has been selected for development as a novel anti-inflammatory human monoclonal antibody with therapeutic potential in a multitude of human autoimmune and inflammatory diseases.

A humanized monoclonal antibody against interleukin-2 that can inactivate the cytokine/receptor complex.

Inhibition of the interleukin-2 (IL-2) pathway has potent immunosuppressive activity in humans as is evident from the broad therapeutic utility of cyclosporine, rapamycin, tacrolimus, and monoclonal antibodies blocking the high-affinity subunit of the IL-2 receptor (CD25). Here we describe a humanized antibody, MT204, interfering with IL-2 signaling by a novel mechanism. Although MT204 did not prevent IL-2 from binding to CD25, it potently antagonized downstream signaling events of IL-2 at sub-nanomolar concentrations, such as STAT3 tyrosine phosphorylation, expression of CD124, production of gamma-interferon and cell proliferation. While MT204 and the anti-CD25 mAb daclizumab were equally effective in inhibiting autocrine growth of human CD4(+) T cells, MT204 was far superior in preventing proliferation of NKL lymphoma cells, production of gamma-interferon by natural killer (NK) cells and proliferation of primary NK cells. MT204 has potential as a novel immunosuppressive and anti-proliferative therapy with an apparently broader spectrum of activities than anti-CD25 antibodies.

Variable heavy-variable light domain and Fab-arm CrossMabs with charged residue exchanges to enforce correct light chain assembly.

Technologies for the production of bispecific antibodies need to overcome two major challenges. The first one is correct heavy chain assembly, which was solved by knobs-into-holes technology or charge interactions in the CH3 domains. The second challenge is correct light chain assembly. This can be solved by engineering the Fab-arm interfaces or applying the immunoglobulin domain crossover approach. There are three different crossovers possible, namely Fab-arm, constant domain and variable domain crossovers. The CrossMabCH1-CL exchange does not lead to the formation of unexpected side products, whereas the CrossMabFab and the CrossMabVH-VL formats result in the formation of typical side products. Thus, CrossMabCH1-CL was initially favored for therapeutic antibody development. Here, we report a novel improved CrossMab design principle making use of site-specific positional exchanges of charged amino acid pairs in the constant domain of these CrossMabs to enable the correct light chain assembly in the CrossMabVH-VL and improvements for the CrossMabFab design.

GingisKHAN™ protease cleavage allows a high-throughput antibody to Fab conversion enabling direct functional assessment during lead identification of human monoclonal and bispecific IgG1 antibodies.

The determination of the binding strength of immunoglobulins (IgGs) to targets can be influenced by avidity when the targets are soluble di- or multimeric proteins, or associated to cell surfaces, including surfaces introduced from heterogeneous assays. However, for the understanding of the contribution of a second drug-to-target binding site in molecular design, or for ranking of monovalent binders during lead identification, affinity-based assessment of the binding strength is required. Typically, monovalent binders like antigen-binding fragments (Fabs) are generated by proteolytic cleavage with papain, which often results in a combination of under- and over-digestion, and requires specific optimization and chromatographic purification of the desired Fabs. Alternatively, the Fabs are produced by recombinant approaches. Here, we report a lean approach for the functional assessment of human IgG1s during lead identification based on an in-solution digestion with the GingisKHAN™ protease, generating a homogenous pool of intact Fabs and Fcs and enabling direct assaying of the Fab in the digestion mixture. The digest with GingisKHAN™ is highly specific and quantitative, does not require much optimization, and the protease does not interfere with methods typically applied for lead identification, such as surface plasmon resonance or cell-based assays. GingisKHAN™ is highly suited to differentiate between affinity and avidity driven binding of human IgG1 monoclonal and bispecific antibodies during lead identification.

Detection of a phosphorylated glycine-serine linker in an IgG-based fusion protein.

Molecular mass determination by electrospray ionization mass spectrometry of a recombinant IgG-based fusion protein (mAb1-F) produced in human embryonic kidney (HEK) cells demonstrated the presence of a dominant +79 Da product variant. Using LC-MS tryptic peptide mapping analysis and collision-induced dissociation (CID) and electron-transfer/higher-energy collision dissociation fragmentations, the modification was localized to the C-terminal serine residue of a glycine-serine linker [(G4S)2] of a fused heavy chain containing in total 2 (G4S)2-linkers. The modification was identified as a phosphorylation (+79.97 Da) by the presence of a 98 Da neutral loss reaction with CID, by spiking a synthetic phosphoserine peptide, and by dephosphorylation with alkaline phosphatase. A thermolysin digest combined with higher-energy collision dissociation (HCD) positioned the phosphoserine to one specific glycine-serine linker of the fused heavy chain, and the relative level of phosphorylated linker was determined to be 11.3% and 0.4% by LC-MS when the fusion protein was transiently expressed in HEK or in stably transformed Chinese hamster ovary cells, respectively. This observation demonstrates that fusions with glycine-serine linker sequences should be carefully evaluated during drug development to prevent the introduction of a phosphorylation site in therapeutic fusion proteins.

A highly stable polyethylene glycol-conjugated human single-chain antibody neutralizing granulocyte-macrophage colony stimulating factor at low nanomolar concentration.

GM-CSF (granulocyte-macrophage colony stimulating factor) plays a central role in inflammatory processes. Treatment with antibodies neutralizing murine GM-CSF showed significant therapeutic effects in mouse models of inflammatory diseases. We constructed by phage display technology a human scFv, which could potently neutralize human GM-CSF. At first, a human V(L) repertoire was combined with the V(H) domain of a parental GM-CSF-neutralizing rat antibody. One dominant rat/human scFv clone was selected, neutralizing human GM-CSF with an IC50 of 7.3 nM. The human V(L) of this clone was then combined with a human V(H) repertoire. The latter preserved the CDR 3 of the parental rat V(H) domain to retain binding specificity. Several human scFvs were selected, which neutralized human GM-CSF at low nanomolar concentrations (IC50 > or = 2.6 nM). To increase serum half-life, a branched 40 kDa PEG-polymer was coupled to the most potent GM-CSF-neutralizing scFv (3077) via an additional C-terminal cysteine. PEG conjugation had a negligible effect on the in vitro neutralizing potential of the scFv, although it caused a significant drop in binding affinity owing to a reduced on-rate. It also significantly increased the stability of the scFv at elevated temperatures. In mouse experiments, the PEGylated scFv 3077 showed a significantly prolonged elimination half-life of 59 h as compared with 2 h for the unconjugated scFv version. PEGylated scFv 3077 is a potential candidate for development of a novel antibody therapy to treat pro-inflammatory human diseases.

Applying mini-bore HPAEC-MS/MS for the characterization and quantification of Fc N-glycans from heterogeneously glycosylated IgGs.

High-performance anion-exchange chromatography (HPAEC) coupled to pulsed amperometric detection (PAD) is a highly sensitive method for the analysis of oligosaccharides without the need for prior derivatization. However, the method suffers from the lack of chemical information with peak assignments based on the retention times of authentic standards or known peaks of reference materials. Here we applied HPAEC coupled on-line with electrospray ion trap mass spectrometry (HPAEC-MS) using a prototype mini-bore (1mm I.D.) CarboPac PA200 column and challenged the analytical separation based method for the structural assignment of heterogeneous mixtures of N-glycans derived from immunoglobulin G from human plasma, glyco-engineered CHO cells, and Sp2/0 mouse myeloma cells. Compared to an analytical scale 3mm I.D. column, the mini-bore column demonstrated a superior performance with up to 8-fold improved limit of detection for specific N-glycans determined by PAD. Quantitative evaluation by extracted ion current chromatograms revealed detection limits in the 50-100 femtomole range using ion trap MS operated in positive ionization mode. In our hands HPAEC-MS/MS allowed the detection and quantification of even low abundant glycan species including biantennary complex-type, high mannose, hybrid and hybrid bisected structures. In comparison to the detection of N-glycans as lithiated or sodiated adducts, we obtained a 65-fold improved signal-to-noise ratio with protonated ions only. Relative quantitative evaluation by single ion current chromatograms was successfully applied and demonstrated an excellent performance with respect to selectivity in the relative quantification of heterogeneous samples of N-glycans compared to HPAEC-PAD and HILIC-UPLC of 2-AB labelled N-glycans.

Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry.

The quadroma antibody represents the first attempt to produce a bispecific heterodimeric IgG antibody by somatic fusion of 2 hybridoma cells each expressing monoclonal antibodies with distinctive specificities. However, because of random heavy and light chain pairing, the desired functional bispecific antibody represents only a small fraction of the protein produced. Subsequently, the knobs-into-holes (KiH) approach was developed to enforce correct heavy chain heterodimerization. Assuming equimolar expression of 4 unmodified chains comprising 2 heavy and 2 light chains, the statistical distribution of all paired combinations can be calculated. With equimolar expression as the goal, we transfected HEK cells with 1:1:1:1 plasmid ratios and analyzed the protein A affinity-purified antibodies from the quadroma and KiH approaches qualitatively and quantitatively with regard to the estimated relative amounts of the products using electrospray quadrupole time-of-flight mass spectrometry. Our results show that all expected species are formed, and that, within the methodological limits, the species distribution in the mixtures corresponds approximately to the statistical distribution.

Identification and characterization of a UDP-D-glucuronate 4-epimerase in Arabidopsis.

One of the major sugars present in the plant cell wall is d-galacturonate, the dominant monosaccharide in pectic polysaccharides. Previous work indicated that one of the activated precursors necessary for the synthesis of pectins is UDP-d-galacturonate, which is synthesized from UDP-d-glucuronate by a UDP-d-glucuronate 4-epimerase (GAE). Here, we report the identification, cloning and characterization of a GAE6 from Arabidopsis thaliana. Functional analysis revealed that this enzyme converts UDP-d-glucuronate to UDP-d-galacturonate in vitro. An expression analysis of this epimerase and its five homologs in the Arabidopsis genome by quantitative RT-PCR and promoter::GUS fusions indicated differential expression of the family members in plant tissues and expression of all isoforms in the developing pollen of A. thaliana.

Structure-based prediction of asparagine and aspartate degradation sites in antibody variable regions.

Monoclonal antibodies (mAbs) and proteins containing antibody domains are the most prevalent class of biotherapeutics in diverse indication areas. Today, established techniques such as immunization or phage display allow for an efficient generation of new mAbs. Besides functional properties, the stability of future therapeutic mAbs is a key selection criterion which is essential for the development of a drug candidate into a marketed product. Therapeutic proteins may degrade via asparagine (Asn) deamidation and aspartate (Asp) isomerization, but the factors responsible for such degradation remain poorly understood. We studied the structural properties of a large, uniform dataset of Asn and Asp residues in the variable domains of antibodies. Their structural parameters were correlated with the degradation propensities measured by mass spectrometry. We show that degradation hotspots can be characterized by their conformational flexibility, the size of the C-terminally flanking amino acid residue, and secondary structural parameters. From these results we derive an accurate in silico prediction method for the degradation propensity of both Asn and Asp residues in the complementarity-determining regions (CDRs) of mAbs.

Assessment of chemical modifications of sites in the CDRs of recombinant antibodies: Susceptibility vs. functionality of critical quality attributes.

Modifications like asparagine deamidation, aspartate isomerization, methionine oxidation, and lysine glycation are typical degradations for recombinant antibodies. For the identification and functional evaluation of antibody critical quality attributes (CQAs) derived from chemical modifications in the complementary-determining regions (CDRs) and the conserved regions, an approach employing specific stress conditions, elevated temperatures, pH, oxidizing agents, and forced glycation with glucose incubation, was applied. The application of the specific stress conditions combined with ion exchange chromatography, proteolytic peptide mapping, quantitative liquid chromatography mass spectrometry, and functional evaluation by surface plasmon resonance analysis was adequate to identify and functionally assess chemical modification sites in the CDRs of a recombinant IgG1. LC-Met-4, LC-Asn-30/31, LC-Asn-92, HC-Met-100c, and HC Lys-33 were identified as potential CQAs. However, none of the assessed degradation products led to a complete loss of functionality if only one light or heavy chain of the native antibody was affected.

Identification and monitoring of host cell proteins by mass spectrometry combined with high performance immunochemistry testing.

Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS). However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP) was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day) manner.