Host cell protein detection gap risk mitigation: quantitative IAC-MS for ELISA antibody reagent coverage determination.

Host cell proteins (HCPs) must be sufficiently cleared from recombinant biopharmaceuticals during the downstream process (DSP) to ensure product quality, purity, and patient safety. For monitoring of HCP clearance, the typical method chosen is an enzyme-linked immunosorbent assay (ELISA) using polyclonal anti-HCP antibodies obtained from an immunization campaign. This polyclonal reagent is a critical factor for functionality and confidence of the ELISA. Therefore, it is important to ensure that the pool of ELISA antibodies covers a broad spectrum of the HCPs that potentially could persist in the final drug substance. Typically, coverage is determined by gel-based approaches. Here, we present a quantitative proteomics approach combined with purification of HCPs by immunoaffinity chromatography (qIAC-MS) for assessment of ELISA coverage. The cell culture fluid (CCF) of a mock fermentation and a recombinant monoclonal antibody product were characterized in detail to investigate whether the HCPs used for immunization of animals accurately represent HCPs that are relevant to the process. Using the qIAC-MS approach, the ELISA antibody coverage was determined for mock fermentation and product CCF, as well as several different DSP intermediates. Here, the use of different controls facilitated the identification and quantification of HCPs present in the polyclonal reagent and those that nonspecifically bound to IAC material. This study successfully demonstrates that the described qIAC-MS approach is not only a suitable orthogonal method to commonly used 2D SDS-PAGE-based analysis for evaluating ELISA antibody coverage, but that it further identifies HCPs covered as well as missed by the ELISA, enabling an improved risk assessment of HCP ELISA.

Mass spectrometric evaluation of upstream and downstream process influences on host cell protein patterns in biopharmaceutical products.

For production of different monoclonal antibodies (mAbs), biopharmaceutical companies often use related upstream and downstream manufacturing processes. Such platforms are typically characterized regarding influence of upstream and downstream process (DSP) parameters on critical quality attributes (CQAs). CQAs must be monitored strictly by an adequate control strategy. One such process-related CQA is the content of host cell protein (HCP) which is typically analyzed by immunoassay methods (e.g., HCP-ELISA). The capacity of the immunoassay to detect a broad range of HCPs, relevant for the individual mAb-production process should be proven by orthogonal proteomic methods such as 2D gel electrophoresis or mass spectrometry (MS). In particular MS has become a valuable tool to identify and quantify HCP in complex mixtures. We evaluate up- and DSP parameters of four different biopharmaceutical products, two different process variants, and one mock fermentation on the HCP pattern by shotgun MS analysis and ELISA. We obtained a similar HCP pattern in different cell culture fluid harvests compared to the starting material from the downstream process. During the downstream purification process of the mAbs, the HCP level and the number of HCP species significantly decreased, accompanied by an increase in diversity of the residual HCP pattern. Based on this knowledge, we suggest a control strategy that combines multi product ELISA for in-process control and release analytics, and MS testing for orthogonal HCP characterization, to attain knowledge on the HCP level, clusters and species. This combination supports a control strategy for HCPs addressing safety and efficacy of biopharmaceutical products.

Haptoglobin-α1, -α2, vitamin D-binding protein and apolipoprotein C-III as predictors of etanercept drug response in rheumatoid arthritis.

INTRODUCTION: The introduction of tumor necrosis factor-alpha (TNF-α) antagonists has substantially improved patient's clinical outcome in rheumatoid arthritis (RA). However, nearly 20% to 40% of RA patients do not respond to anti-TNF-α treatment strategies. To identify valid predictors of TNF-α antagonist response in RA, serum proteome profiles from responders (R) and non-responders (NR) to etanercept, a soluble recombinant TNF-α receptor/IgG Fc fusion protein receptor, were compared in a prospective cohort study. METHODS: In this clinical study 50 RA patients with inadequate response to conventional DMARDs were included and treated with etanercept. The primary efficacy endpoint was response according to the European League against Rheumatism (EULAR) improvement criteria. Serum samples collected prior to initiation and after six months of etanercept therapy were cleared of the most abundant major proteins by immunoaffinity chromatography. After separation by two-dimensional differential gel electrophoresis (2D-DIGE) and identification by mass spectrometry (MS) data were validated by Western blot analysis. RESULTS: After six months of etanercept treatment 62% (n=31) of RA patients achieved response. Haptoglobin-α1 (Hp-α1) and -α2 (Hp-α2) and vitamin D-binding protein (VDBP) were found to be significantly upregulated in responder sera (P≤0.02) at study entry. In contrast, apolipoprotein C-III (ApoC-III) showed significantly higher levels in non-responders (P=0.0162). At study end ApoA-II, Hp-α1, Hp-α2 and VDBP were identified to be expressed at significantly higher levels (P<0.05) in responder sera. CONCLUSIONS: By application of clinical proteomics in immunodepleted sera we could identify and validate for the first time Hp-α1, -α2, VDBP and ApoC-III as potential biomarkers for prediction of etanercept drug response in RA.

Development of a fluorescence resonance energy transfer peptide library technology for detection of protease contaminants in protein-based raw materials used in diagnostic assays.

Protease impurities in raw materials used in enzyme immunoassays can impair assay performance. This risk may be greatly decreased if incoming protein-based raw materials are controlled for protease impurities or if protease inhibitors are used in the assay formulations. As many different proteases might occur in protein raw materials, it is desirable to have a general test for protease contamination. With the help of a fluorescence resonance energy transfer peptide library containing about 2.5 million peptides, we have succeeded in establishing such a system, with sensitivity in the nanogram range for known proteases. Protease contamination was found to differ between different raw materials and was correlated with assay performance. Protease activity in contaminated raw materials could be suppressed to various degrees with different chemical inhibitors or by thermal treatment. This technology is suited for the control of incoming protein-based raw materials used for enzyme immunoassays, as well as for the optimization of the use of protein inhibitors or thermal treatment of protein-based raw materials for the inactivation of proteases.

Identification of serum proteins involved in pancreatic cancer cachexia.

AIMS: Treatment of cachexia requires pharmacological intervention which, in turn, requires knowledge of the mediators and processes. Cachexia markers that are specifically expressed in pancreatic cancer and secreted into the blood circulation have yet to be identified. The aim of our study was to investigate the serum protein profiles and protein alterations associated with cachexia and to identify potential disease protein biomarkers indicative for this syndrome. MAIN METHODS: Serum samples from cachectic and non-cachectic patients undergoing pancreatic cancer (PaCa) surgery and controls were investigated by Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS). The identity of detected discriminatory markers was determined by a combination of protein fractionation, chromatographic purification steps, gel electrophoresis, and mass spectrometry. KEY FINDINGS: Using Cu-IMAC array and CM-10 array based SELDI-TOF-MS. we identified eleven up- and four down-regulated proteins associated with cachexia. CiphergenExpress analysis revealed four disease-associated protein features (38559Da, 9138Da, 8925Da and 3358Da) that were elevated by a factor of 2.3, 1.7, 1.4 and 1.4, respectively. Zinc-α2-glycoprotein (ZAG), apolipoproteins apo C-II and apo C-III and glucagon-like peptide-1 (GLP-1) were identified as markers for PaCa-associated cachexia syndrome. ZAG levels were additionally evaluated in serum and tissue samples by ELISA and immunohistochemistry and the obtained data confirmed the SELDI-TOF-MS results. SIGNIFICANCE: The identified proteins could be routinely and reliably measured in the serum of patients and provide an elegant non-invasive approach for early diagnosis of cachectic pancreatic cancer patients. Controlling ZAG and GLP-1 activity could be beneficial in the management of cancers and cachexia-induced conditions.

Immuno-MALDI-TOF MS: new perspectives for clinical applications of mass spectrometry.

The discovery of novel biomarkers by means of advanced detection tools based on proteomic analysis technologies necessitates the development of improved diagnostic methods for application in clinical routine. On the basis of three different application examples, this review presents the limitations of conventional routine diagnostic assays and illustrates the advantages of immunoaffinity enrichment combined with MALDI-TOF MS. Applying this approach increases the specificity of the analysis supporting a better diagnostic recognition, sensitivity, and differentiation of certain diseases. The use of MALDI-TOF MS as detection method facilitates the identification of modified peptides and proteins providing additional information. Further, employing respective internal standard peptides allows for relative and absolute quantitation which is mandatory in the clinical context. Although MALDI-TOF MS is not yet established for clinical routine diagnostics this technology has a high potential for improvement of clinical diagnostics and monitoring therapeutic efficacy.

Neural cell adhesion molecule expression on renal interstitial cells.

BACKGROUND: At early stages of kidney development, the neural cell adhesion molecule (NCAM) is highly expressed on cells of the metanephrogenic mesenchyme. During maturation of the fetal kidney, NCAM gradually disappears. So far, it has been widely accepted that NCAM in the adult kidney is only expressed by nerves, and not by other cell types. METHODS: NCAM expression was analysed in human adult healthy and diseased kidneys by immunohistochemistry and western blot analysis. NCAM+ renal interstitial cells were further characterized by double immunofluorescent staining using antibodies against neurofilaments, alpha smooth muscle actin, vimentin, alpha5beta1 integrin, CD68, CD11c, HLA-DR and the potential progenitor cell markers CD34, CD117, CD133, CD24, nestin and cadherin-11. RESULTS: In adult human kidneys, NCAM expression is restricted to rare interstitial cells with dendritic morphology, which are neurofilament-negative and predominantly localized on the corticomedullary junction. They are also negative for fibroblast cell markers, but co-express the haematopoietic stem cell markers CD34 and CD133. The number of NCAM+ interstitial cells increased in the initial phases of interstitial fibrosis. Western blot analysis of renal tissues with incipient interstitial fibrosis tissues showed the expression of the 140 kDa NCAM isoform. CONCLUSIONS: These data indicate that a rare subpopulation of NCAM+ interstitial cells could represent renal progenitors, and that NCAM+ interstitial cells can participate in the initial phase of interstitial fibrosis.

Whole cell profiling and identification of galectin-1 as a potential marker of renal cell carcinoma.

For most cancers, the patient's prognosis improves dramatically if the disease is detected at an early stage. Although advancements in imaging technology have improved early detection, many cancers remain undetected until it is too late for curative intervention. We have established, for the first time, expression difference mapping analysis of whole cell proteins from renal cell carcinoma (RCC) cell lines using ProteinChip technology. A total of 20 different RCC cell lines were cultured in vitro directly on ProteinChip arrays for 24 h. Direct MS analysis of proteins from the attached cells showed identical protein profiles by all analysed RCC lines. Comparative on-chip analysis of isolated malignant cells from native tumour specimens revealed protein patterns highly similar to those from the continuous RCC lines. However, cultured primary cortex cells showed specific protein differences. Differential protein profiling of isolated cytosolic and enriched membrane fractions from the RCC lines revealed that the protein pattern of the membrane proteins included or were identical to those of the entire cells. Proteomics analysis of the chip-binding membrane fractions allowed the identification of three forms of galectin-1 as potential RCC marker. ProteinChip analysis with a bound-specific antibody certified that galectin-1 could be an RCC marker. Immunostaining methods confirmed the overexpression of galectin-1 in renal carcinoma in comparison to healthy tissue.

T cell epitope definition by differential mass spectrometry: identification of a novel, immunogenic HLA-B8 ligand directly from renal cancer tissue.

In this study, we describe a differential mass spectrometric technique for the immuno-proteomic analysis of the major histocompatibility complex (MHC) peptides of a renal cell carcinoma (RCC) biopsy compared with the healthy kidney tissue of the same patient after nephrectomy. Using a stable isotope labeling approach, we could directly compare and relatively quantify 43 MHC-peptide pairs, most of which were present in similar proportions on both normal kidney and tumor. Significantly, two dominant peptides of monoisotopic masses ([M+H](+)) 973.43 u and 967.59 u, respectively, were found exclusively in the tumor sample. One of these was identified as originating from heme oxygenase-1 (HO-1), a protein involved in induction of apoptosis resistance, immuno-suppression and neoangiogenesis and reported to be up-regulated in various cancer types. Moreover, the corresponding synthetic HO-1-derived peptide was shown to be immunogenic in vitro by generation of CD8+ T cell lines with peptide-specific cytolytic activity. Thus, this peptide is an example of a differentially identified T cell epitope that could be considered as a target for immunotherapy.

Cathepsin D is present in human eccrine sweat and involved in the postsecretory processing of the antimicrobial peptide DCD-1L.

The protein pattern of healthy human eccrine sweat was investigated and 10 major proteins were detected from which apolipoprotein D, lipophilin B, and cathepsin D (CatD) were identified for the first time in human eccrine sweat. We focused our studies on the function of the aspartate protease CatD in sweat. In vitro digestion experiments using a specific fluorescent CatD substrate showed that CatD is enzymatically active in human sweat. To identify potential substrates of CatD in human eccrine sweat LL-37 and DCD-1L, two antimicrobial peptides present in sweat, were digested in vitro with purified CatD. LL-37 was not significantly digested by CatD, whereas DCD-1L was cleaved between Leu(44) and Asp(45) and between Leu(29) and Glu(30) almost completely. The DCD-1L-derived peptides generated in vitro by CatD were also found in vivo in human sweat as determined by surface-enhanced laser desorption/ionization (SELDI) mass spectrometry. Furthermore, besides the CatD-processed peptides we identified additionally DCD-1L-derived peptides that are generated upon cleavage with a 1,10-phenanthroline-sensitive carboxypeptidase and an endoprotease. Taken together, proteolytic processing generates 12 DCD-1L-derived peptides. To elucidate the functional significance of postsecretory processing the antimicrobial activity of three CatD-processed DCD-1L peptides was tested. Whereas two of these peptides showed no activity against Gram-positive and Gram-negative bacteria, one DCD-1L-derived peptide showed an even higher activity against Escherichia coli than DCD-1L. Functional analysis indicated that proteolytic processing of DCD-1L by CatD in human sweat modulates the innate immune defense of human skin.

Differential detection of S100A8 in transitional cell carcinoma of the bladder by pair wise tissue proteomic and immunohistochemical analysis.

The search for novel molecular markers of tumor invasion is vital if strategies are to become more effective in the diagnostic and prognostic management of transitional cell carcinoma of the bladder. Up to 50% of tumors detected at stage 1 (pT1) progress to a higher grade even after endoscopic surgical resection, and there are currently no protein markers of this aggressive, invasive phenotype. We have combined SELDI-TOF-MS, ClinProt magnetic bead enrichment, Nano-LC-ESI-ion trap tandem mass spectrometry and immunohistochemical analysis to the study of 12 invasive bladder cancer tissue biopsies paired with normal bladder tissue samples obtained from the same patients for the definition and identification of proteins up-regulated in the tumors. We report the inflammation-associated calcium binding protein S100A8 (MRP-8, calgranulin A) to be highly expressed in tumor cells in contrast to normal urothelium in 50% of the samples, as well as two unidentified protein markers at 5.75 and 6.89 kDa that were differentially detected in 9/12 and 10/12 tumor samples, respectively. These new markers, when fully characterized, may contribute to new target proteins for the prediction of aggressive, invasive bladder tumors.

Expansion of peripheral CD8+ CD28- T cells in response to Epstein-Barr virus in patients with rheumatoid arthritis.

OBJECTIVE: To investigate control of Epstein-Barr virus (EBV) infection in rheumatoid arthritis (RA) by comparing the frequency phenotypes and function of peripheral CD8+ EBV-peptide antigen-specific T cells in patients with RA and healthy longterm carriers of EBV. METHODS: The frequency of interferon-g (IFN-g)-producing HLA-A2 or HLA-B8-restricted EBV-reactive CD8+ T cells in peripheral blood mononuclear cells (PBMC) from 49 RA patients and 26 healthy EBV carriers was evaluated in Elispot assays with 12 lytic/latent peptide epitopes. Direct staining with HLA-peptide tetramers containing 3 of these peptides was performed for comparison. The phenotype and function of these T cells was determined by FACS and cytotoxicity testing. RESULTS: IFN-g production patterns in Elispot assays revealed that EBV-specific CD8+ T cells were directed predominantly against the lytic epitopes A2/GLC and B8/RAK and to a minor extent to all the other lytic and latent epitopes tested, with no significant differences of the frequencies in patients and controls. However, although similar frequencies of CD8+ T cells stained with A2/GLC or B8/RAK tetramers in both groups, the fraction of A2/GLC or B8/RAK-reactive T cells producing IFN-g in response to specific peptide antigen was significantly lower in RA patients than controls. The A2/GLC or B8/RAK tetramer-positive T cells were also substantially enriched in CD28-CD27- T cells of a late-differentiated phenotype in RA patients but not in controls. CONCLUSION: RA patients show clonal expansion of dysfunctional, terminally differentiated CD8+ EBV-specific T cells in their T cell responses to immunodominant lytic peptide EBV epitopes, which could be a sign of specific impairment of virus-host interactions in RA.

Serum protein profiling by SELDI mass spectrometry: detection of multiple variants of serum amyloid alpha in renal cancer patients.

The molecular analysis of serum is an important field for the definition of potential diagnostic markers or disease-related protein alterations. Novel proteomic technologies such as the mass spectrometric-based surface-enhanced laser desorption/ionization (SELDI) ProteinChip technique facilitate a rapid and reproducible analysis of such protein mixtures and affords the researcher a new dimension in the search for biomarkers of disease. Here, we have applied this technology to the study of a cohort of serum samples from well-characterized renal cell carcinoma patients for the identification of such proteins by comparison to healthy controls. We detected and characterized haptoglobin 1 alpha and serum amyloid alpha-1 (SAA-1) as disease related, in addition to an as-yet-unidentified marker of 10.84 kDa. Of particular note is the detection of multiple variants of SAA-1 in multiplex that have not been described in the sera of cancer patients. SAA-1 is detected as full-length protein, des-Arginine and des-Arginine/des-Serine variants at the N terminus by SELDI. In addition, we could also detect a low-abundant variant minus the first five N-terminal amino acids. Such variants may impact the function of the protein. We conclude the technique to be a reproducible, fast and simple mode for the discovery and analysis of marker proteins of disease in serum.

Development of an MHC-class I peptide selection assay combining nanoparticle technology and matrix-assisted laser desorption/ionisation mass spectrometry.

Human leukocyte antigen (HLA)-bound peptides are central for recognition of infected/transformed cells by T cells, and have formed the basis for many immunotherapy strategies. Epitopes from a given protein sequence (e.g. from viral proteins or oncoproteins) can be predicted by algorithms, as individual HLA receptors bind peptides through defined binding motifs. Peptides with the highest predicted binding score are then normally tested for their binding ability in binding assays. However, with the assays already established, only one peptide can be tested for binding per assay. This is certainly not a reflection of the in vivo situation, where several peptides generated via the major histocompatability complex (MHC)-class I processing pathway compete for HLA-receptor binding. Here, we describe the development of a method that can mimic the competition between multiple peptides for binding to a single HLA receptor molecule. We used silica nanoparticles with immobilised HLA-A2 complexes to screen HLA-A2 binder-peptides out of a known peptide mixture. The washed beads were analysed for selectively bound peptides by matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry. The advantage of the system is that the bound peptides can be unambiguously identified without any prior modification (e.g. radioactive or fluorescence labelling), even from complex peptide mixtures.

Detection of dermcidin-derived peptides in sweat by ProteinChip technology.

Recently, a novel antimicrobial peptide DCD-1, derived from the Dermcidin (DCD) gene and secreted by sweat glands, has been described by Schittek et al. [Nat. Immunol. 2 (2001) 1133.]. Here we describe the application of the surface-enhanced laser desorption/ionisation (SELDI) technology for the detection of DCD-1 and other dermcidin-derived peptides directly from microlitre amounts of human sweat. The advantages of the technique are as follows: (a) it can be carried out with ease and rapidity; (b) multiple samples can be processed simultaneously; (c) prior purification is not required; and (d) only a limited sample volume is necessary for both protein profiling and semiquantitation. Profiling of human sweat from various donors revealed that in addition to DCD-1, other DCD-derived peptide species were also present in significant quantities. Four of five identified peptides were DCD-1 related, while the fifth corresponded to a portion of the DCD protein outside the DCD-1 core. This provides clues as to how the novel protein is processed to its active form, though further work remains to elucidate this fully. Thus, we have demonstrated the applicability of such technology to the detection of DCD-1 and for the protein profiling of sweat in general. Such studies could reveal valuable new biomarkers for diagnosis and treatment of skin and sweat gland disorders.

Cathepsin S activity is detectable in human keratinocytes and is selectively upregulated upon stimulation with interferon-gamma.

Keratinocytes are an integral component of the skin immune system and function as nonprofessional antigen-presenting cells in pathophysiologic conditions when they express major histocompatibility complex class II molecules, e.g., in psoriasis. In order to analyze further this function we investigated the activity of cathepsin S in comparison with cathepsins B and L. These enzymes were suggested to be involved in antigen presentation. Specific catalytic activities of these cathepsins were determined fluorometrically by hydrolysis of a synthetic substrate (Z-Phe-Arg-7-amido-4-methylcoumarin) in subcellular fractions of human keratinocytes. It was found that the human keratinocyte cell line HaCaT exhibits activities of all three cathepsins investigated. Endosomal/lysosomal compartments show highest cathepsin activities. Normal human keratinocytes in primary culture show a comparable pattern of cathepsin activities. In contrast to this, in syngeneic Epstein-Barr virus-transformed B cells the level of cathepsin B activity was found to be 10% of that in the corresponding keratinocytes, whereas the activities for cathepsins L and S were in a similar range. Interferon-gamma stimulation of primary keratinocytes and HaCaT cells resulted in a selective upregulation of the cathepsin S activity, the extent of which was very similar. The mechanism of this upregulation was demonstrated as induction at the mRNA and protein levels. This report documents that cathepsin S in human keratinocytes is selectively upregulated, in parallel to major histocompatibility complex class II molecules, in response to a pro-inflammatory cytokine. Our observations support the concept of keratinocytes functioning as nonprofessional antigen-presenting cells in states of inflammation.

Human alpha-defensins HNPs-1, -2, and -3 in renal cell carcinoma: influences on tumor cell proliferation.

The alpha-defensins human neutrophil peptides (HNPs)-1, -2, and -3 have been described as cytotoxic peptides with restricted expression in neutrophils and in some lymphocytes. In this study we report that HNPs-1, -2, and -3 are also expressed in renal cell carcinomas (RCCs). Several RCC lines were found to express mRNA as well as the specific peptides of HNP-1, -2, and -3 demonstrated by reverse transcriptase-polymerase chain reaction, mass spectrometric, and flow cytometric analyses. At physiological concentrations HNPs-1, -2, and -3 stimulated cell proliferation of selected RCC lines in vitro but at high concentrations were cytotoxic for all RCC lines tested. As in RCC lines, alpha-defensins were also detected in vivo in malignant epithelial cells of 31 RCC tissues in addition to their expected presence in neutrophils. In most RCC cases randomly, patchy immunostaining of alpha-defensins on epithelial cells surrounding neutrophils was seen, but in six tumors of higher grade malignancy all tumor cells were diffusely stained. Cellular necrosis observed in RCC tissues in association with extensive patches of HNP-1, -2, and -3, seemed to be related to high concentrations of alpha-defensins. The in vitro and in vivo findings suggest that alpha-defensins are frequent peptide constituents of malignant epithelial cells in RCC with a possible direct influence on tumor proliferation.