Potential aggregation prone regions in biotherapeutics

Aggregation of a biotherapeutic is of significant concern and judicious process and formulation development is required to minimize aggregate levels in the final product. Aggregation of a protein in solution is driven by intrinsic and extrinsic factors. In this work we have focused on aggregation as an intrinsic property of the molecule. We have studied the sequences and Fab structures of commercial and non-commercial antibody sequences for their vulnerability towards aggregation by using sequence based computational tools to identify potential aggregation-prone motifs or regions. The mAbs in our dataset contain 2 to 8 aggregation-prone motifs per heavy and light chain pair. Some of these motifs are located in variable domains, primarily in CDRs. Most aggregation-prone motifs are rich in β branched aliphatic and aromatic residues. Hydroxyl-containing Ser/Thr residues are also found in several aggregation-prone motifs while charged residues are rare. The motifs found in light chain CDR3 are glutamine (Q)/asparagine (N) rich. These motifs are similar to the reported aggregation promoting regions found in prion and amyloidogenic proteins that are also rich in Q/N, aliphatic and aromatic residues. The implication is that one possible mechanism for aggregation of mAbs may be through formation of cross-β structures and fibrils. Mapping on the available Fab – receptor/antigen complex structures reveals that these motifs in CDRs might also contribute significantly towards receptor/antigen binding. Our analysis identifies the opportunity and tools for simultaneous optimization of the therapeutic protein sequence for potency and specificity while reducing vulnerability towards aggregation.     

Competing aggregation pathways for monoclonal antibodies

Aggregation is mediated by local unfolding to allow aggregation “hot spot(s)” to become solvent exposed and available to associate with a hot spot on another partially unfolded protein. Historically, the unfolding of either the crystallizable fragment (Fc) or the antigen binding fragment (Fab) regions of a given monoclonal antibody (MAb) has been implicated in aggregation, with differing results across different proteins. The present work focuses on separately quantifying the aggregation kinetics of isolated Fc, isolated Fab, and intact MAb as a function of pH under accelerated (high temperature) conditions. The results show that both Fab and Fc are aggregation prone and compete within the same MAb.     

Aspartate isomerization in the complementarity-determining regions of two closely related monoclonal antibodies

The aspartic acid residues (Asp) present in the complementarity-determining regions (CDRs) of the light chains of two recombinant monoclonal antibodies (MAbs), MAb I and MAb II, are highly susceptible to isomerization due to the presence of glycine residues (Gly) on their C-terminal ends. Asp isomerization in these MAbs leads to formation of the isoaspartate (IsoAsp) and the cyclic imide (Asu) variants of these MAbs. Both MAb I and MAb II, employed in this study, elicit their pharmacological responses through binding human IgE. The formation of the MAb variants as a result of Asp isomerization significantly reduces the binding affinities of these antibodies to IgE, thereby reducing their potencies. Here we report on significant differences in the susceptibility of the MAb I and the MAb II to Asp isomerization. The molecular basis for these differences in rates of Asp isomerization was elucidated. The effect of primary sequence on Asp isomerization was evaluated using pentapeptide models of the MAbs, which included the labile Asp residues and their neighboring amino acid residues. The separation of the parent MAbs and pentapeptides from their isomerization products was achieved using hydrophobic interaction chromatography (HIC) and rp-HPLC, respectively. Structural characterization of the MAbs was performed using differential scanning calorimetry (DSC), circular dichroism (CD), and X-ray crystallography. Our investigations demonstrate that the differences in the Asp isomerization rates between MAb I and MAb II can be attributed to structural factors including the conformational flexibility and the extent of solvent exposure of the labile Asp residue.     

Development of human monoclonal antibodies: A review

This article describes the current status in the development of human monoclonal antibodies. Over the last ten years a lot of information about the human immune system has emerged. Combining these with the many new (bio-)technologies it is plausible that the long awaited breakthrough of this technology is close. This paper focuses on the classical cell-biological methods of achieving stable, antibody-producing human cell lines via cell fusion methods or virus derived transformations of human B-lymphocytes, as well as genetic engineering methods e.g. DNA libraries or phage display technology. The available in vitro immunization methods are critically reviewed and their impact on this topic is discussed. Therapeutic applications for cancer treatment or passive immunization against infectious diseases with antibodies derived by both ways are also reviewed.     

Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins

Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5–6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5–6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development.     

Identification and quantification of protecting groups remaining in commercial oligonucleotide products using monoclonal antibodies

Quality control is paramount to reproducibly achieving oligonucleotide therapeutics and diagnostics of superior value. However, incomplete deprotection of nucleoside reactive groups after the automated chemical synthesis of oligonucleotides would result in diminished antisense activity and in erroneous array analysis of gene expression. Mass spectrometry and capillary electrophoresis are used to detect aborted sequences of oligonucleotides, but not to identify and quantify incompletely deprotected oligonucleotides. To address this problem, monoclonal antibodies (MAbs), ELISA, and dot-blot assays were developed for the specific identification and quantification of the commonly used nucleic acid base- and sugar-protecting groups: benzoyl, isobutyryl, isopropylphenoxyacetyl, and dimethoxytrityl. Each MAb was capable of reproducibly detecting 8-32 pmol of the respectively protected nucleoside in an intact DNA or RNA sample composed of 320-640 nmol of the deprotected nucleoside. In a direct comparison, HPLC nucleoside composition analysis of enzyme-hydrolyzed DNA was limited to the detection of 2-5 nmol of protected nucleoside. Using the MAb dot-blot assay, 5 of 16 commercial DNA products obtained from eight different companies were found to have 1.0-5.2% of the benzoyl and isopropylphenoxyacetyl protecting groups remaining. Thus, MAbs selectively identify and quantify picomoles of remaining protecting groups on antisense therapeutics and oligonucleotide diagnostics.     

Effect of monoclonal antibodies to defined regions of tropoelastin on elastogenesis in vitro.

Primary cultures of chick embryo aorta cells were grown for one week in the presence of mouse monoclonal antibodies directed against defined regions of chick tropoelastin. This treatment did not significantly alter cell proliferation, cell viability and incorporation of labeled amino acids into total protein or tropoelastin compared with control cultures in which antibodies were either omitted or substituted with an unrelated monoclonal antibody. Tropoelastin-reactive material in the cell layer was revealed by immunologic staining with rabbit antibodies against the chick protein both at the optical and ultrastructural level. Immunofluorescence of control cultures showed that tropoelastin was incorporated into thin and straight fibrils which were sometimes associated with spot-like elements. In the electron microscope tropoelastin-reactive sites were found mainly on the amorphous core of typical, small elastic fibers. The morphological picture of tropoelastin deposits in cultures exposed to anti-tropoelastin monoclonal antibodies depended on the molecular form (whole antibody or Fab fragments) and the binding specificity of the antibody used. Although alterations common to different antibodies were observed, the main structural features were peculiar for each antibody. Two antibodies which bound epitopes present in two regions of tropoelastin grossly altered the formation of amorphous elastin. Moreover, two antibodies directed against the region of tropoelastin containing the polypentapeptide-repeat (VPGVG)n stimulated the deposition of the protein into the amorphous core of normal-looking elastic fibers and disorganized the compact bundles of parallel microfibrils seen in controls. Finally, one antibody which recognized a unique epitope close to the carboxy-terminal end of tropoelastin and Fab fragments from all antibodies apparently inhibited the formation of the amorphous nuclei of elastic fibers, but not the association of tropoelastin with microfibrils. The data suggest that the association of tropoelastin molecules during fiber assembly is not random, but follows an ordered alignment process which the antibodies alter by imposing a different molecular packing.     

A novel platform to produce human monoclonal antibodies: The next generation of therapeutic human monoclonal antibodies discovery

A new technology has been developed by immunologix that allows human antibodies to be quickly generated against virtually any antigen. Using a novel process, naïve human B cells are isolated from tonsil tissue and transformed with efficiency up to 85%, thus utilizing a large portion of the human VDJ/VJ repertoire. Through ex vivo stimulation, the B cells class switch and may undergo somatic hypermutation, thus producing a human “library” of different IgG antibodies that can then be screened against any antigen. Since diversity is generated ex vivo, sampling immunized or previously exposed individuals is not necessary. Cells producing the antibody of interest can be isolated through limiting dilution cloning and the human antibody from the cells can be tested for biological activity. No humanization is necessary because the antibodies are produced from human B cells. By eliminating immunization and humanization steps and screening a broadly diverse library, this platform should reduce both the cost and time involved in producing therapeutic monoclonal antibody candidates.Key words: human, antibody, monoclonal, novel platform, naïve, B cell, therapeutic     

Forced degradation of recombinant monoclonal antibodies: A practical guide

Forced degradation studies have become integral to the development of recombinant monoclonal antibody therapeutics by serving a variety of objectives from early stage manufacturability evaluation to supporting comparability assessments both pre- and post- marketing approval. This review summarizes the regulatory guidance scattered throughout different documents to highlight the expectations from various agencies such as the Food and Drug Administration and European Medicines Agency. The various purposes for forced degradation studies, commonly used conditions and the major degradation pathways under each condition are also discussed.     

A molecular and structural analysis of the VH and VK regions of monoclonal antibodies bearing the A48 regulatory idiotype

The results presented in this paper explore the molecular basis for expression of the A48 regulatory Id (RI). A48 RI+ mAb derived from idiotypically manipulated mice molecularly resembled the A48 and UPC 10 prototypes of this system by utilizing a VHX24-Vk10 combination. Id expression by these antibodies was not restricted by a particular D region sequence, JH, or JK segment, but quantitative differences in Id expression were associated with utilization of different members of the VK10 germ-line gene families. The VL sequences of these A48 RI+ mAb has identified amino acid residues lying in four different idiotope-determining regions which may contribute to the structural correlate of this Id. A comparative sequence analysis of the VH regions of these VHX24 utilizing A48 RI+ mAb with several A48 RI+ mAb utilizing VHJ558 or VH7183 VH genes as well as a hybrid transfectoma antibody derived from two A48 RI-, VHJ558 utilizing hybridomas, all suggested that four nonconsecutive positions which lie outside the idiotope-determining regions may contribute structural elements toward expression of this Id. The VH and VL regions of the A48RI+, VHX24-Vk 10+ mAb showed low to moderate levels of somatic mutation which showed different patterns of distribution between the complementary determining region (CDR) and framework regions in the H and L chains. Although the VK sequences contained 50% of the replacement mutations in the CDR, with a replacement/silent mutation ratio of 10, the CDR of the VH sequences contained only 31% of the replacement mutations with a replacement/silent mutation ratio of 0.69.     

Sequences of the VH and VL regions of murine monoclonal antibodies against 3-fucosyllactosamine

Many mAb that bind the carbohydrate antigenic determinant 3-fucosyl-lactosamine (3-FL), Gal beta 1-4[Fuc alpha-3]GlcNAc-R have been raised in BALB/c mice, and we are studying the structure and regulation of these antibodies. In this report, we present the first information about their amino acid sequences and the Ig gene segments used to encode them. V regions of the H and L chains of three anti-3-FL antibodies, PMN6, PMN29, and PM81, were sequenced by a combination of mRNA and amino acid sequencing. The L chain sequences of PMN6 and PM81 antibodies indicate that their VK and JK regions are encoded by VK24B and JK1 germ-line genes, respectively. The nucleotide and amino acid sequences of the H chains suggest that the three anti-3-FL antibodies are encoded by the VH441 gene segment of the X24 VH family, and this conclusion was supported by Southern filter hybridization with VH441 and JH3-JH4 probes. PMN29 has at least 11 amino acid substitutions, which is an unusually large amount of somatic mutation for an IgM antibody. Previous analyses of BALB/c genomic libraries with VHX24 and VH441 probes make it unlikely that this VH family contains additional germ-line genes, but this possibility cannot be excluded. All three antibodies use the DQ52 and JH4 gene segments. The single VH and VL gene segments used to encode the anti-3-FL antibodies is in contrast to the multiple VH and VL segments used by antibodies against other carbohydrate Ag such as alpha 1-6 dextran and group A streptococcal carbohydrate. VH441 also encodes the VH regions of antibodies against galactan and levan (beta 2-6 fructosan). The similarities among VH segments of antibodies against 3-FL, levan, and galactan, and the striking differences in their CDR3 sequences, suggest that CDR3 plays an important role in the formation of the Ag binding site. The use of a single VH segment from the smallest VH gene family by antibodies against at least three different carbohydrate determinants is noteworthy. It raises the possibility that the amino acid sequence encoded by VH441 has some general structural features that make it particularly well adapted for binding to carbohydrate sequences.     

Epitope analysis and primary structures of variable regions of anti-human FcepsilonRI monoclonal antibodies, and expression of the chimeric antibodies fused with human constant regions

The structural analysis of monoclonal antibodies (mAbs) against the alpha subunit of the high affinity IgE receptor (FcepsilonRIalpha) is an alternative approach to obtaining information for the design of inhibitors that will block complementary interaction between IgE and FcepsilonRIalpha and to analyzing the various biological effects induced by anti-FcepsilonRIalpha autoantibodies in chronic urticaria. In this study, epitopes for mouse anti-human FcepsilonRIalpha mAbs and primary structures of variable regions of the mAbs were analyzed. Three mAbs inhibitory for IgE-binding reacted to the deletion mutants of FcepsilonRIalpha containing the whole second immunoglobulin-like domain as well as IgE did. On the other hand, two uninhibitory mAbs reacted to those containing the whole first immunoglobulin-like domain. The cDNAs for variable regions of the five mAbs were cloned and sequenced. Two inhibitory mouse/human chimeric antibodies were expressed in COS7 cells and bound to Chinese hamster ovary transfectant cells expressing FcepsilonRI (CHO/alphabetagamma), and these inhibited the binding of IgE to CHO/alphabetagamma cells.     

Screening of monoclonal antibodies to scarce and labile enzymes: A functional immunoassay for isolating monoclonal antibodies to NADPH:nitrate reductase

A functional immunoassay, that has proved very useful, is described for screening and identifying monoclonal antibodies (McAbs) against scarce and labile enzymes. This method does not require purified enzyme or antigen and it has been successfully applied to isolate three hybridomas secreting McAbs to NADPH:nitrate reductase from the chloronema cells of the mossFunaria hygrometrica. Briefly, the protocol involves: adsorption of murine antibodies from hybridoma supernatants by rabbit antimouse IgG antibody pre-adsorbed toStaphylococcus aureus cells (SAC), reaction with crude extract for 15 min, sedimentation of the SAC complex by centrifugation and measurement of residual enzymatic activity in the supernatant. A depletion indicates the presence of antibodies that bind to the active enzyme. The method is rapid, sensitive and versatile enough to be used to isolate McAbs with exquisite specificities. The three isolated McAbs recognized nitrate reductase protein in a conformation-independent and/or a conformation-dependent manner.     

Conformational stability and aggregation of therapeutic monoclonal antibodies studied with ANS and Thioflavin T binding

Characterization of aggregation profiles of monoclonal antibodies (mAb) is gaining importance because an increasing number of mAb-based therapeutics are entering clinical studies and gaining marketing approval. To develop a successful formulation, it is imperative to identify the critical biochemical properties of each potential mAb drug candidate. We investigated the conformational change and aggregation of a human IgG1 using external dye-binding experiments with fluorescence spectroscopy and compared the aggregation profiles obtained to the results of size-exclusion chromatography. We show that using an appropriate dye at selected mAb concentration, unfolding or aggregation can be studied. In addition, dye-binding experiments may be used as conventional assays to study therapeutic mAb stability.     

Conformational stability and aggregation of therapeutic monoclonal antibodies studied with ANS and thioflavin T binding

Characterization of aggregation profiles of monoclonal antibodies (mAb) is gaining importance because an increasing number of mAb-based therapeutics are entering clinical studies and gaining marketing approval. To develop a successful formulation, it is imperative to identify the critical biochemical properties of each potential mAb drug candidate. We investigated the conformational change and aggregation of a human IgG1 using external dye-binding experiments with fluorescence spectroscopy and compared the aggregation profiles obtained to the results of size-exclusion chromatography. We show that using an appropriate dye at selected mAb concentration, unfolding or aggregation can be studied. In addition, dye-binding experiments may be used as conventional assays to study therapeutic mAb stability.Key words: therapeutic monoclonal antibody, protein aggregation, conformational change, stability and shelf-life prediction, accelerated studiesMonoclonal antibodies (mAbs) have emerged as a novel class of protein drugs and are utilized for a variety of mostly incurable and debilitating diseases such as cancer and rheumatoid arthritis.^1–4 For treatment of chronic diseases, it is desirable for these drugs to be administered subcutaneously, in which case high protein concentrations (>100 mg/mL) are generally needed.^5,6 Protein-based drugs containing mAbs must contain minimum amounts of aggregation and fragmentation and conserve their structural integrity during storage because degraded or aggregated protein may induce immunogenicity or reduce efficacy. Currently, size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) is the most commonly used method to characterize mAb aggregation profiles;⁷ however it is time consuming, expensive and requires expertise. SEC-HPLC cannot be used to obtain accurate biophysical profiles of mAbs at high concentrations because dilution during the experiment might lead to reversible aggregation. Furthermore, the potential interaction of aggregates with surfaces, e.g., needle, tubing, column, will lead to the loss of sample and thus an inaccurate analysis.^8,9 Additional drawbacks of the technique are that different conformations such as partially unfolded monomers also cannot be distinguished by SEC-HPLC and large aggregates may be totally excluded during the injection into the column.External dye binding assays have been used to characterize protein stability and aggregation,^10–12 and studies involving biopharmaceuticals have been reported recently, e.g., for thermostability screening¹⁰ and detection of aggregation.^11–14 These methods are not limited by protein quantity and are more sensitive because they are fluorescence-based. We studied the accelerated unfolding of an IgG1 mAb with the hydrophobic dye 1-anilino-8-naphthale-nesulfonate (ANS), and its accelerated aggregation with aggregate specific Thioflavin T (ThT). We have also conducted accelerated aggregation studies with SEC-HPLC7 and compared the findings to the ThT binding results. We hypothesize that key structures formed during mAb aggregation can be probed selectively by the appropriate dyes (Fig. 1) with specific mAb concentrations.Open in a separate windowFigure 1Key structures of the mAb probed by fluorescent dyes. N and U are native and unfolded monomers, respectively. “n” reactive monomers form aggregates.