Recombinant IgG1 Antibody Research Articles

Modular Semisynthetic Approach to Generate T Cell-Dependent Bispecific Constructs from Recombinant IgG1 Antibodies.

Redirecting T cells to tumor cells by bispecific antibodies is an effective approach to treat cancer, and T cell-dependent bispecific antibodies (TDBAs) are an emerging class of potent immunotherapeutic agents. By simultaneously targeting antigens on tumor cells and T cells, T cells are activated to kill tumor cells. Herein, we report a platform to generate a novel class of 2:1 structure of T cell-dependent bispecific antibody with bivalency for HER2 receptors on tumor cells and monovalency for CD3 receptors on T cells. For this, we use a biogenic inverse electron-demand Diels-Alder (IEDDA) click reaction on genetically encoded tyrosine residues to install one TCO handle on therapeutically approved antibody trastuzumab. Subsequent TCO-tetrazine click with a tetrazine-functionalized CD3-binding Fab yields a 2:1 HER2 × CD3 TDBA that exhibits a tumor-killing capability at picomolar concentrations. Monovalency toward the CD3 receptor on T cells can lower the chances of cytokine release syndrome, which is a common side effect of such agents. Our semisynthetic approach can generate highly potent TDBA constructs in a few chemoenzymatic and synthetic steps.

Conversion of Mouse-Derived Hybridomas to Tasmanian Devil Recombinant IgG Antibodies.

The hybridoma method for production of monoclonal antibodies has been a cornerstone of biomedical research for several decades. Here we convert the monoclonal antibody sequence from mouse-derived hybridomas into a "devilized" recombinant antibody with devil IgG heavy chain and IgK light chain. The chimeric recombinant antibody can be used in functional assays, immunotherapy, and to improve understanding of antibodies and Fc receptors in Tasmanian devils. The process can be readily modified for other species.

BEVACIZUMAB-INDUCED BRONCHIAL PERFORATION

SESSION TITLE: Lung Cancer SESSION TYPE: Fellow Case Report Posters PRESENTED ON: 10/09/2018 01:15 PM - 02:15 PM INTRODUCTION: Bevacizumab is a recombinant IgG1 antibody to VEGF currently approved for use in advanced non-small cell lung cancer. Phase III trials have demonstrated that bevacizumab addition to antineoplastic regimens have improved survival in this patient population. Bevacizumab causes microvascular involution in tumors and suppression of tumor angiogenesis. There are many recorded sinopulmonary toxicities such as pulmonary hemorrhage, venous thromboembolism, and nasal septal perforation. Here we report bronchial perforation in the setting of advanced pulmonary adenocarcinoma. CASE PRESENTATION: Our patient is a 56 year old male with a 30 pack-year smoking history who initially presented to the pulmonary clinic with wheezing, cough and shortness of breath. Imaging at the time showed a right supra hilar mass and right upper lobe collapse. Bronchoscopy with endobronchial ultrasound (EBUS) was performed with subsequent pathology showing poorly differentiated adenocarcinoma (EGFR negative, ALK-EML4 negative, ROS1 negative); left adrenal biopsy confirmed metastasis. His treatment consisted of cisplatin and pemetrexed every three weeks for four total cycles with complete adrenal and partial pulmonary response. Following this treatment, he underwent six weeks of low dose carboplatin and paclitaxel with concurrent thoracic radiation, followed by three weeks of maintenance pemetrexed. He suffered from recurrent post-obstructive pneumonias due to his chronic RUL obstruction and the obstruction unfortunately was not amenable to endobronchial intervention. He also developed pneumonitis after multiple rounds of radiation and a right-sided para malignant pleural effusion. Video-assisted thoracoscopic surgery and indwelling pleural catheter placement by thoracic surgery was performed. Carboplatin and paclitaxel with the addition of bevacizumab every three weeks for six cycles was then started followed by maintenance bevacizumab. He presented with hemoptysis 15 months after starting bevacizumab. Bronchoscopy was performed, revealing loss of the integrity of the bronchial wall of the right bronchus intermedius with tissue protruding through the bronchial wall. It was recommended at that time to discontinue bevacizumab DISCUSSION: To our knowledge, this is the only case in the current literature of bronchial perforation secondary to bevacizumab. Literature review predominantly revealed nasal perforation as the most common sinopulmonary complication from bevacizumab treatment with pulmonary hemorrhage being a close second. In our case, the patient was on bevacizumab 15 mg/kg every 3 weeks for 6 total cycles and then maintenance dose for 37 total cycles (15 total months). His treatment had to be stopped given the extent of the perforation. CONCLUSIONS: Bevacizumab toxicity is well reported in the upper airway. Our case is one of pulmonary involvement most likely due to a vascular mediated effect causing airway necrosis Reference #1: Keating, Gillian M. “Bevacizumab: A Review of Its Use in Advanced Cancer.” Drugs, vol. 74, no. 16, 2014, pp. 1891–1925. Reference #2: Higa, Gerald M, and Jame Abraham. “Biological Mechanisms of Bevacizumab-Associated Adverse Events.” Expert Review of Anticancer Therapy, vol. 9, no. 7, 2009, pp. 999–1007. Reference #3: Vahid, Bobbak, and Paul E. Marik. “Pulmonary Complications of Novel Antineoplastic Agents for Solid Tumors.” Chest, vol. 133, no. 2, 2008, pp. 528–538. DISCLOSURES: No relevant relationships by Chhaya Patel, source=Web Response No relevant relationships by Juan Sanchez, source=Web Response

Evaluation of different vector design strategies for the expression of recombinant monoclonal antibody in CHO cells

ABSTRACTMonoclonal antibodies (mAbs) have emerged as the most promising category of recombinant proteins due to their high efficiency for the treatment of a wide range of human diseases. The complex nature of mAbs creates a great deal of challenges in both upstream and downstream manufacturing processes. Proportional expression and correct folding and assembly of the light chain and heavy chain are required for efficient production of the mAbs. In this regard, expression vector design has proven to have profound effects on the antibody expression level as well as its stability and quality. Here, we have explored the efficiency of different vector design strategies for the expression of a recombinant IgG1 antibody in Chinese hamster ovary (CHO) cells. The antibody expression level was analyzed in transient expression and stable cell pools followed by expression analysis on single-cell clones. While detectable amounts of antibody were observed in all three systems, dual-promoter single-vector system showed the highest expression level in transient and stable expression as well as the highest productivity among clonal cells. Our results here show the importance of vector design for successful production of whole mAbs in CHO cells.

Alga-Made Anti-Hepatitis B Antibody Binds to Human Fcγ Receptors.

Microalgae are unicellular eukaryotic organisms which represent an emerging alternative to other cell biofactories commonly used to produce monoclonal antibodies. Microalgae display several biotechnological advantages such as their rapid growth rate and their phototrophic lifestyle allowing low production costs as protein expression is solar-fueled. Recently, a fully assembled recombinant IgG antibody directed against Hepatitis B surface antigen is produced and secreted in the culture medium of the diatom Phaeodactylum tricornutum. A biochemical characterization of this recombinant antibody demonstrated that the Asn-297 is N-glycosylated by oligomannosides. In the immune system, antibodies interact with effector molecules and cells through their Fc part and the recognition of Fcγ receptors (FcγR) which are important for inducing phagocytosis of opsonized microbes. Interactions between IgG and FcγR are influenced by the N-glycan structures present on the Asn-297. In this study, the authors characterized the binding capacity of the anti-hepatitis B recombinant IgG produced in P. tricornutum to two human Fcγ receptors (FcγRI and IIIa) using a cellular binding assay and surface plasmon resonance (SPR). This allowed us to demonstrate that the alga-made antibody is able to bind FcγRI with a reduced affinity and engages FcyRIIIa with 3-times higher affinity compared to a control human IgG1.

An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves.

The overall quality of recombinant IgG antibodies in plants is dramatically compromised by host endogenous proteases. Different approaches have been developed to reduce the impact of endogenous proteolysis on IgGs, notably involving site-directed mutagenesis to eliminate protease-susceptible sites or the in situ mitigation of host protease activities to minimize antibody processing in the cell secretory pathway. We here characterized the degradation profile of H10, a human tumour-targeting monoclonal IgG, in leaves of Nicotiana benthamiana also expressing the human serine protease inhibitor α1-antichymotrypsin or the cysteine protease inhibitor tomato cystatin SlCYS8. Leaf extracts revealed consistent fragmentation patterns for the recombinant antibody regardless of leaf age and a strong protective effect of SlCYS8 in specific regions of the heavy chain domains. As shown using an antigen-binding ELISA and LC-MS/MS analysis of antibody fragments, SlCYS8 had positive effects on both the amount of fully-assembled antibody purified from leaf tissue and the stability of biologically active antibody fragments containing the heavy chain Fc domain. Our data confirm the potential of Cys protease inhibitors as convenient antibody-stabilizing expression partners to increase the quality of therapeutic antibodies in plant protein biofactories.

Production process reproducibility and product quality consistency of transient gene expression in HEK293 cells with anti-PD1 antibody as the model protein.

Demonstration of reproducibility and consistency of process and product quality is one of the most crucial issues in using transient gene expression (TGE) technology for biopharmaceutical development. In this study, we challenged the production consistency of TGE by expressing nine batches of recombinant IgG antibody in human embryonic kidney 293 cells to evaluate reproducibility including viable cell density, viability, apoptotic status, and antibody yield in cell culture supernatant. Product quality including isoelectric point, binding affinity, secondary structure, and thermal stability was assessed as well. In addition, major glycan forms of antibody from different batches of production were compared to demonstrate glycosylation consistency. Glycan compositions of the antibody harvested at different time periods were also measured to illustrate N-glycan distribution over the culture time. From the results, it has been demonstrated that different TGE batches are reproducible from lot to lot in overall cell growth, product yield, and product qualities including isoelectric point, binding affinity, secondary structure, and thermal stability. Furthermore, major N-glycan compositions are consistent among different TGE batches and conserved during cell culture time.

Abstract 1669: Preclinical efficacy of Sym004, novel anti-EGFR antibody, in esophageal squamous cell carcinoma cell lines

Abstract Background: Epidermal growth factor receptor (EGFR) is a well-validated oncology target molecule for monoclonal antibody therapies. Sym004 is a novel anti-EGFR antibody mixture that consists of two recombinant IgG1 antibodies against domain III of the EGFR. The primary mechanism of action of Sym004 is thought to be EGFR cross-linking, internalization and degradation of the EGFR from the cell surface. EGFR is highly expressed in the overwhelming majority of esophageal squamous cell carcinoma (ESCC). We, therefore, investigated the effect of Sym004 in human ESCC cell lines in the present study. Method: In this preclinical study, approximately 50 ESCC cell lines and 3 kinds of anti-EGFR antibodies (Sym004, cetuximab, and panitumumab) were used. According to protein expression status and gene mutation status analyzed by Western blotting and next generation sequencing, these ESCC cell lines were classified into several groups. High EGFR-expressing cell lines were used for further investigations. Internalization of anti-EGFR antibodies into ESCC cells and inhibition of the EGFR signaling cascade by anti-EGFR antibodies were studied in in vitro experiments. Growth inhibitions by anti-EGFR antibody treatment were investigated in in vitro and in vivo experiments. The present preclinical studies were conducted at the National Cancer Center East Japan as collaborative studies with Merck Serono. Result: Even though protein expression of EGFR was detected in all ESCC cell lines, a variety of the expression levels were seen among the cell lines consistent with the previous data of clinical specimens. In addition, following gene alterations were found: EGFR amplification, Ras mutation, and PIK3CA mutation, and their frequency was 17% (8/48), 6.3% (3/48) and 8.3% (4/48), respectively. More effective internalization of Sym004 was observed than cetuximab and panitumumab in the high EGFR-expression cell lines. In a growth inhibition assay, more potent anti-proliferative activity of Sym004 was observed than those of other anti-EGFR antibodies in some cell lines. No obvious correlation between EGFR expression and the anti-proliferative activity was observed, although cell lines with higher EGFR expression showed a trend toward higher sensitivity to Sym004. In vivo xenograft studies of Sym004 using human ESCC cell lines are currently underway and will be presented in the annual meeting. Conclusions: These studies showed that Sym004 exhibited antitumor activity in some ESCC cell lines in preclinical settings, and are expected to provide a scientific rationale along with a possible clinical indication of ESCC in clinical setting. Citation Format: Shota Fukuoka, Takashi Kojima, Yoshikatsu Koga, Mayumi Yamauchi, Rie Komatsuzaki, Kazuhiko Aoyagi, Hiroki Sasaki, Masahiro Yasunaga, Yasuhiro Matsumura, Toshio Kuronita, Toshihiko Doi, Atsushi Ohtsu. Preclinical efficacy of Sym004, novel anti-EGFR antibody, in esophageal squamous cell carcinoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1669. doi:10.1158/1538-7445.AM2015-1669

Functional assessment of antibody oxidation by native mass spectrometry

Oxidation of methionine (Met) residues is one of several chemical degradation pathways for recombinant IgG1 antibodies. Studies using several methodologies have indicated that Met oxidation in the constant IgG1 domains affects in vitro interaction with human neonatal Fc (huFcRn) receptor, which is important for antibody half-life. Here, a completely new approach to investigating the effect of oxidative stress conditions has been applied. Quantitative ultra-performance liquid chromatography mass spectrometry (MS) peptide mapping, classical surface plasmon resonance and the recently developed FcRn column chromatography were combined with the new fast-growing approach of native MS as a near native state protein complex analysis in solution. Optimized mass spectrometric voltage and pressure conditions were applied to stabilize antibody/huFcRn receptor complexes in the gas phase for subsequent native MS experiments with oxidized IgG1 material. This approach demonstrated a linear correlation between quantitative native MS and IgG-FcRn functional analysis.In our study, oxidation of the heavy chain Met-265 resulted in a stepwise reduction of mAb3/huFcRn receptor complex formation. Remarkably, a quantitative effect of the heavy chain Met-265 oxidation on relative binding capacity was only detected for doubly oxidized IgG1, whereas IgG1 with only one oxidized heavy chain Met-265 was not found to significantly affect IgG1 binding to huFcRn. Thus, mono-oxidized IgG1 heavy chain Met-265 most likely does not represent a critical quality attribute for pharmacokinetics.

IGHV1-69 B cell chronic lymphocytic leukemia antibodies cross-react with HIV-1 and hepatitis C virus antigens as well as intestinal commensal bacteria.

B-cell chronic lymphocytic leukemia (B-CLL) patients expressing unmutated immunoglobulin heavy variable regions (IGHVs) use the IGHV1-69 B cell receptor (BCR) in 25% of cases. Since HIV-1 envelope gp41 antibodies also frequently use IGHV1-69 gene segments, we hypothesized that IGHV1-69 B-CLL precursors may contribute to the gp41 B cell response during HIV-1 infection. To test this hypothesis, we rescued 5 IGHV1-69 unmutated antibodies as heterohybridoma IgM paraproteins and as recombinant IgG1 antibodies from B-CLL patients, determined their antigenic specificities and analyzed BCR sequences. IGHV1-69 B-CLL antibodies were enriched for reactivity with HIV-1 envelope gp41, influenza, hepatitis C virus E2 protein and intestinal commensal bacteria. These IGHV1-69 B-CLL antibodies preferentially used IGHD3 and IGHJ6 gene segments and had long heavy chain complementary determining region 3s (HCDR3s) (≥21 aa). IGHV1-69 B-CLL BCRs exhibited a phenylalanine at position 54 (F54) of the HCDR2 as do rare HIV-1 gp41 and influenza hemagglutinin stem neutralizing antibodies, while IGHV1-69 gp41 antibodies induced by HIV-1 infection predominantly used leucine (L54) allelic variants. These results demonstrate that the B-CLL cell population is an expansion of members of the innate polyreactive B cell repertoire with reactivity to a number of infectious agent antigens including intestinal commensal bacteria. The B-CLL IGHV1-69 B cell usage of F54 allelic variants strongly suggests that IGHV1-69 B-CLL gp41 antibodies derive from a restricted B cell pool that also produces rare HIV-1 gp41 and influenza hemagglutinin stem antibodies.

Abstract 4322: Efficacy of Sym004, a novel anti-EGFR antibody mixture, against EGFRvIII positive glioblastoma xenografts.

Abstract INTRODUCTION Glioblastoma (GBM) is the most common intracranial cancer in adults but despite recent advances in therapy the overall survival remains about 20 months. The epidermal growth factor receptor variant III (EGFRvIII) is a truncated, constitutively active, and highly oncogenic form of the EGFR expressed on approximately 30% of GBMs. Sym004 is a recombinant IgG1 antibody mixture consisting of two antibodies against domain III of the epidermal growth factor receptor (EGFR). Like anti-EGFR monoclonal antibodies, Sym004 inhibits cancer cell growth and survival by blocking ligand-binding and receptor signaling. However, unlike the monoclonal antibodies, Sym004 induces a rapid and efficient internalization and degradation of the EGFR. Here, we examine whether the more efficient removal of the EGFR and the constitutive active EGFRvIII by Sym004 would translate into increased tumor growth inhibition of EGFRvIII GBM xenografts. METHODS Both subcutaneous (sc) and intracranial (ic) adult brain tumor xenografts were grown in athymic BALB/c mice. After tumor size reached 200-500 mm3 subcutaneously or 3 days after intracranial implantation, groups of 10 mice were randomly treated with either drug vehicle, Sym004 (50 mg/kg) or cetuximab (50 mg/kg) IP twice weekly for 5 weeks. Tumor responses for sc xenografts were assessed by tumor growth delay and regression and for ic xenografts by difference in median survival. Xenograft lines utilized for this study expressed either primarily EGFR wildtype (D-54 MG) or mutant EGFRvIII (D-270 MG and D-317 MG). RESULTS AND DISCUSSION The sc results for D-54 MG did not demonstrate statistically significant growth delays for either Sym004 or cetuximab. In sc EGFRvIII models, however, Sym004 produced statistically significant (p<0.001) growth delays of 7.8 and 76.5 days and outperformed cetuximab by 4.6 and 71.9 days in D-270 MG and D-317 MG, respectively. Intracranially, Sym004 produced statistically significant (p<0.001) increases in survival of 39% and 163% and outperformed cetuximab by 27% and 115% in D-270 MG and D-317 MG, respectively. We speculate that the superior effect of the Sym004 antibody mixture compared to the monoclonal antibody cetuximab in the EGFRvIII models is due to a more efficient internalization and degradation - and thereby shutdown - of the constitutively active EGFRvIII. Histological examination of the receptor status in the tumors following treatment is ongoing. CONCLUSIONS The novel strategy to target EGFR demonstrates the significant anti-tumor activity of Sym004 against adult glioblastoma xenografts that express EGFRvIII. These results warrant further exploration of Sym004 in clinical trials for the treatment of EGFRvIII positive brain tumors. Funding for these studies was provided by Symphogen and the Tisch Preclinical Therapy Screening Program Citation Format: Stephen T. Keir, Martin A. Roskoski, Michael Kragh, Mikkel W. Pederson, Helle J. Jacobsen, Ivan D. Horak, Henry S. Friedman, Darell D. Bigner. Efficacy of Sym004, a novel anti-EGFR antibody mixture, against EGFRvIII positive glioblastoma xenografts. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4322. doi:10.1158/1538-7445.AM2013-4322

Identification of a single base-pair mutation of TAA (Stop codon) → GAA (Glu) that causes light chain extension in a CHO cell derived IgG1

We describe here the identification of a stop codon TAA (Stop) → GAA (Glu) = Stop221E mutation on the light chain of a recombinant IgG1 antibody expressed in a Chinese hamster ovary (CHO) cell line. The extended light chain variants, which were caused by translation beyond the mutated stop codon to the next alternative in-frame stop codon, were observed by mass spectra analysis. The abnormal peptide peaks present in tryptic and chymotryptic LC–MS peptide mapping were confirmed by N-terminal sequencing as C-terminal light chain extension peptides. Furthermore, LC-MS/MS of Glu-C peptide mapping confirmed the stop221E mutation, which is consistent with a single base-pair mutation in TAA (stop codon) to GAA (Glu). The light chain variants were approximately 13.6% of wild type light chain as estimated by RP-HPLC analysis. DNA sequencing techniques determined a single base pair stop codon mutation, instead of a stop codon read-through, as the cause of this light chain extension. To our knowledge, the stop codon mutation has not been reported for IgGs expressed in CHO cells. These results demonstrate orthogonal techniques should be implemented to characterize recombinant proteins and select appropriate cell lines for production of therapeutic proteins because modifications could occur at unexpected locations.

Comparative in vitro and experimental in vivo studies of the anti–epidermal growth factor receptor antibody nimotuzumab and its aglycosylated form produced in transgenic tobacco plants

A broad variety of foreign genes can be expressed in transgenic plants, which offer the opportunity for large-scale production of pharmaceutical proteins, such as therapeutic antibodies. Nimotuzumab is a humanized anti-epidermal growth factor receptor (EGFR) recombinant IgG1 antibody approved in different countries for the treatment of head and neck squamous cell carcinoma, paediatric and adult glioma, and nasopharyngeal and oesophageal cancers. Because the antitumour mechanism of nimotuzumab is mainly attributed to its ability to interrupt the signal transduction cascade triggered by EGF/EGFR interaction, we have hypothesized that an aglycosylated form of this antibody, produced by mutating the N(297) position in the IgG(1) Fc region gene, would have similar biochemical and biological properties as the mammalian-cell-produced glycosylated counterpart. In this paper, we report the production and characterization of an aglycosylated form of nimotuzumab in transgenic tobacco plants. The comparison of the plantibody and nimotuzumab in terms of recognition of human EGFR, effect on tyrosine phosphorylation and proliferation in cells in response to EGF, competition with radiolabelled EGF for EGFR, affinity measurements of Fab fragments, pharmacokinetic and biodistribution behaviours in rats and antitumour effects in nude mice bearing human A431 tumours showed that both antibody forms have very similar in vitro and in vivo properties. Our results support the idea that the production of aglycosylated forms of some therapeutic antibodies in transgenic plants is a feasible approach when facing scaling strategies for anticancer immunoglobulins.

Pharmacokinetics and Biodistribution of a Human Monoclonal Antibody to Oxidized LDL in Cynomolgus Monkey Using PET Imaging

PurposeOxidized low-density lipoprotein (LDL) plays an essential role in the pathogenesis of atherosclerosis. The purpose of this study was to characterize the pharmacokinetics (PK) of a human recombinant IgG1 antibody to oxidized LDL (anti-oxLDL) in cynomolgus monkey. The tissue biodistribution of anti-oxLDL was also investigated using positron emission tomography (PET) imaging.MethodsAnti-oxLDL was conjugated with the N-hydroxysuccinimide ester of DOTA (1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraacetic acid) and radiolabeled by chelation of radioactive copper-64 (64Cu) for detection by PET. Anti-oxLDL was administered as a single intravenous (IV) dose of 10 mg/kg (as a mixture of radiolabeled and non-labeled material) to two male and two female cynomolgus monkeys. Serum samples were collected over 29 days. Two ELISA methods were used to measure serum concentrations of anti-oxLDL; Assay A was a ligand binding assay that measured free anti-oxLDL (unbound and partially bound forms) and Assay B measured total anti-oxLDL. The biodistribution was observed over a 48-hour period following dose administration using PET imaging.ResultsAnti-oxLDL serum concentration-time profiles showed a biphasic elimination pattern that could be best described by a two-compartment elimination model. The serum concentrations obtained using the two ELISA methods were comparable. Clearance values ranged from 8 to 17 ml/day/kg, while beta half-life ranged from 8 to12 days. The initial volume of distribution and volume of distribution at steady state were approximately 55 mL/kg and 150 mL/kg, respectively. PET imaging showed distribution predominantly to the blood pool, visible as the heart and great vessels in the trunk and limbs, plus diffuse signals in the liver, kidney, spleen, and bone marrow.ConclusionsThe clearance of anti-oxLDL is slightly higher than typical IgG1 antibodies in cynomolgus monkeys. The biodistribution pattern appears to be consistent with an antibody that has no large, rapid antigen sink outside the blood space.

Hyperbranched Polylysine: A Versatile, Biodegradable Transfection Agent for the Production of Recombinant Proteins by Transient Gene Expression and the Transfection of Primary Cells

The feasibility of a new transfection agent, HBPL, for the production of recombinant IgG antibody via TGE as well as for the transfection of primary cells is studied. Under the conditions investigated, transfection of CHO-DG44 cells using HBPL results in IgG yields that are comparable to those obtained with PEI. In experiments with CHO-K1 cells and MEFs the use of HPBL allows to achieve transfection efficiencies comparable to or better than those obtained with PEI or Fugene®. HBPL-mediated transfection does not require complex pre-formation, works well in serum-containing media and is biodegradable, which may prevent cumulative cytotoxicity and facilitates downstream processing.

Glycan variability on a recombinant IgG antibody transiently produced in HEK-293E cells

In this study, a recombinant monoclonal IgG antibody was produced by transient gene expression (TGE) in suspension-adapted HEK-293E cells. The objective of the study was to determine the variation in recombinant IgG yield and glycosylation in ten independent transfections. In a ten-day batch process, the variation in transient IgG yield in the ten batches was less than 30% with the specific productivity averaging 20.2 ± 2.6 pg/cell/day. We characterized the N-glycosylation profile of each batch of affinity-purified IgG by intact protein and bottom-up mass spectrometry. Four major glycans were identified at Asn(297) in the ten batches with the maximum relative deviation for a single glycoform being 2.5%. In addition, within any single transfection there was little variation in glycoforms over the ten-day culture. Our experimental data indicate that with TGE, the production of recombinant IgG with little batch-to-batch variation in volumetric yield and protein glycosylation is feasible, even in a non-instrumented cultivation system as described here.

Use of a Human Recombinant Immunoglobulin G1 CH3 Domain as a Probe for Detecting Alternatively Folded Human IgG in Intravenous Ig Products

It has been previously reported that intravenous immunoglobulin (IVIg) contains alternatively folded (aggregation-prone) monomeric immunoglobulin (Ig) G molecules. These alternatively folded IgG molecules may act as precursors for Fc-Fc-mediated dimerization and/or aggregation in IVIg. To study this phenomenon, we set up a fluid-phase binding assay using an acid-shocked (pH 2.5) recombinant human IgG1 CH3 domain as a probe in combination with size-exclusion chromatography. Three IVIg products and a recombinant IgG1 antibody were analyzed. Besides CH3 probe binding to monomeric IgG derived from all IVIg products, the CH3 probe also bound to IgG4 half-molecules. This IgG4 binding could be distinguished from binding to IgG molecules on the basis of molecular weight. In contrast, no CH3 probe binding to IgG from the recombinant IgG1 antibody was observed. After acid-induced aggregation of either IVIg or a recombinant IgG1 antibody, CH3 probe binding to oligomeric complexes was observed, but no longer to monomeric IgG, demonstrating that the alternatively folded monomeric IgG molecules had oligomerized. Our results indicate that the tested IVIg products contain traces of alternatively folded IgG molecules within the "normal" monomeric IgG fraction. Furthermore, we conclude that the fluid-phase binding assay is sensitive to detect these alternatively folded IgG molecules in IVIg.

Simultaneous Assessment of Asp Isomerization and Asn Deamidation in Recombinant Antibodies by LC-MS following Incubation at Elevated Temperatures

The degradation of proteins by asparagine deamidation and aspartate isomerization is one of several chemical degradation pathways for recombinant antibodies. In this study, we have identified two solvent accessible degradation sites (light chain aspartate-56 and heavy chain aspartate-99/101) in the complementary-determining regions of a recombinant IgG1 antibody susceptible to isomerization under elevated temperature conditions. For both hot-spots, the degree of isomerization was found to be significantly higher than the deamidation of asparagine-(387, 392, 393) in the conserved CH3 region, which has been identified as being solvent accessible and sensitive to chemical degradation in previous studies. In order to reduce the time for simultaneous identification and functional evaluation of potential asparagine deamidation and aspartate isomerization sites, a test system employing accelerated temperature conditions and proteolytic peptide mapping combined with quantitative UPLC-MS was developed. This method occupies the formulation buffer system histidine/HCl (20 mM; pH 6.0) for denaturation/reduction/digestion and eliminates the alkylation step. The achieved degree of asparagine deamidation and aspartate isomerization was adequate to identify the functional consequence by binding studies. In summary, the here presented approach greatly facilitates the evaluation of fermentation, purification, formulation, and storage conditions on antibody asparagine deamidation and aspartate isomerization by monitoring susceptible marker peptides located in the complementary-determining regions of recombinant antibodies.

Transient gene expression with CHO cells in conditioned medium: a study using TubeSpin®bioreactors

Background Transient gene expression (TGE) allows rapid protein production in mammalian cells and has become an important tool in the pharmaceutical product development pipeline [1]. Polyethylenimine (PEI)-mediated, high-density transfection allowed to express recombinant proteins at yields exceeding 1 g/L in only a few weeks [2]. Although highly efficient protocols are available, volumetric scale-up of TGE is still a challenge. A major issue is the need to perform the transfection in fresh medium rather than in conditioned (spent) medium. This implies a medium exchange step just before transfection. In CHO-DG44 [3] cells we observed up to a 100-fold decrease in volumetric protein production if transfections were performed in conditioned medium, compared to fresh medium. The reasons for such a negative effect of conditioned medium on transfectability and/or protein production expression are not yet known. To study this problem we transfected CHO cells at small-scale in TubeSpin bioreactor 50 tubes using 41 different commercially available serum-free media formulations in combination with different transfection parameters and culture conditions. By comparing the transient production of a recombinant IgG antibody among the different media, we observed variation of up to 400-fold when transfecting in fresh media and up to 20-fold when using conditioned medium. The optimization of the PEI:DNA ratio allowed a significant improvement in yields of transfection in conditioned medium. Methods Suspension-adapted CHO-DG44 cells [3] were routinely cultivated in TubeSpin bioreactor 50 tubes (TPP, Trasadingen, Switzerland) in ProCHO5 medium (Lonza, Vervier, Belgium) supplemented with 13.6 mg/L hypoxanthine, 3.9 mg/L thymidine, and 4 mM glutamine. The 38 media samples for transfection were provided by Excellgene SA. Conditioned medium is defined as a cell culture medium where cells have been growing for more than two days up to a density between 4-5 million cells/mL. Cell growth was accessed with the Packed Cell Volume method. The dual expression vector pXLGCHO-A3, containing the cDNAs coding for human anti-Rhesus D IgG1 heavy and light chain cloned in separate expression cassettes in a head-to-head orientation, was kindly provided by Excellgene SA [4]. Transfections are performed at a cell density of 5.5 million cells/mL at a volume of 5 mL by the direct addition of 15 μg of pDNA and 76 μg of linear 25 kDa Polyethylenimine (PEI, Polysciences, Eppenheim, Germany)

Identification of Potential Sites for Tryptophan Oxidation in Recombinant Antibodies Using tert-Butylhydroperoxide and Quantitative LC-MS

Amino acid oxidation is known to affect the structure, activity, and rate of degradation of proteins. Methionine oxidation is one of the several chemical degradation pathways for recombinant antibodies. In this study, we have identified for the first time a solvent accessible tryptophan residue (Trp-32) in the complementary-determining region (CDR) of a recombinant IgG1 antibody susceptible to oxidation under real-time storage and elevated temperature conditions. The degree of light chain Trp-32 oxidation was found to be higher than the oxidation level of the conserved heavy chain Met-429 and the heavy chain Met-107 of the recombinant IgG1 antibody HER2, which have already been identified as being solvent accessible and sensitive to chemical oxidation. In order to reduce the time for simultaneous identification and functional evaluation of potential methionine and tryptophan oxidation sites, a test system employing tert-butylhydroperoxide (TBHP) and quantitative LC-MS was developed. The optimized oxidizing conditions allowed us to specifically oxidize the solvent accessible methionine and tryptophan residues that displayed significant oxidation in the real-time stability and elevated temperature study. The achieved degree of tryptophan oxidation was adequate to identify the functional consequence of the tryptophan oxidation by binding studies. In summary, the here presented approach of employing TBHP as oxidizing reagent combined with quantitative LC-MS and binding studies greatly facilitates the efficient identification and functional evaluation of methionine and tryptophan oxidation sites in the CDR of recombinant antibodies.