Real-time product attribute control to manufacture antibodies with defined N-linked glycan levels.

Pressures for cost-effective new therapies and an increased emphasis on emerging markets require technological advancements and a flexible future manufacturing network for the production of biologic medicines. The safety and efficacy of a product is crucial, and consistent product quality is an essential feature of any therapeutic manufacturing process. The active control of product quality in a typical biologic process is challenging because of measurement lags and nonlinearities present in the system. The current study uses nonlinear model predictive control to maintain a critical product quality attribute at a predetermined value during pilot scale manufacturing operations. This approach to product quality control ensures a more consistent product for patients, enables greater manufacturing efficiency, and eliminates the need for extensive process characterization by providing direct measures of critical product quality attributes for real time release of drug product.

The criticality of high-resolution N-linked carbohydrate assays and detailed characterization of antibody effector function in the context of biosimilar development.

Accurate measurement and functional characterization of antibody Fc domain N-linked glycans is critical to successful biosimilar development. Here, we describe the application of methods to accurately quantify and characterize the N-linked glycans of 2 IgG1 biosimilars with effector function activity, and show the potential pitfalls of using assays with insufficient resolution. Accurate glycan assessment was combined with glycan enrichment using lectin chromatography or production with glycosylation inhibitors to produce enriched pools of key glycan species for subsequent assessment in cell-based antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity effector function assays. This work highlights the challenges of developing high-quality biosimilar candidates and the need for modern biotechnology capabilities. These results show that high-quality analytics, combined with sensitive cell-based assays to study in vivo mechanisms of action, is an essential part of biosimilar development.

An optimized approach to the rapid assessment and detection of sequence variants in recombinant protein products.

The development of sensitive techniques to detect sequence variants (SVs), which naturally arise due to DNA mutations and errors in transcription/translation (amino acid misincorporations), has resulted in increased attention to their potential presence in protein-based biologic drugs in recent years. Often, these SVs may be below 0.1%, adding challenges for consistent and accurate detection. Furthermore, the presence of false-positive (FP) signals, a hallmark of SV analysis, requires time-consuming analyst inspection of the data to sort true from erroneous signal. Consequently, gaps in information about the prevalence, type, and impact of SVs in marketed and in-development products are significant. Here, we report the results of a simple, straightforward, and sensitive approach to sequence variant analysis. This strategy employs mixing of two samples of an antibody or protein with the same amino acid sequence in a dilution series followed by subsequent sequence variant analysis. Using automated peptide map analysis software, a quantitative assessment of the levels of SVs in each sample can be made based on the signal derived from the mass spectrometric data. We used this strategy to rapidly detect differences in sequence variants in a monoclonal antibody after a change in process scale, and in a comparison of three mAbs as part of a biosimilar program. This approach is powerful, as true signals can be readily distinguished from FP signal, even at a level well below 0.1%, by using a simple linear regression analysis across the data set with none to minimal inspection of the MS/MS data. Additionally, the data produced from these studies can also be used to make a quantitative assessment of relative levels of product quality attributes. The information provided here extends the published knowledge about SVs and provides context for the discussion around the potential impact of these SVs on product heterogeneity and immunogenicity.

Rapid identification of an antibody DNA construct rearrangement sequence variant by mass spectrometry.

During cell line development for an IgG1 antibody candidate (mAb1), a C-terminal extension was identified in 2 product candidate clones expressed in CHO-K1 cell line. The extension was initially observed as the presence of anomalous new peaks in these clones after analysis by cation exchange chromatography (CEX-HPLC) and reduced capillary electrophoresis (rCE-SDS). Reduced mass analysis of these CHO-K1 clones revealed that a larger than expected mass was present on a sub-population of the heavy chain species, which could not be explained by any known chemical or post-translational modifications. It was suspected that this additional mass on the heavy chain was due to the presence of an additional amino acid sequence. To identify the suspected additional sequence, de novo sequencing in combination with proteomic searching was performed against translated DNA vectors for the heavy chain and light chain. Peptides unique to the clones containing the extension were identified matching short sequences (corresponding to 9 and 35 amino acids, respectively) from 2 non-coding sections of the light chain vector construct. After investigation, this extension was observed to be due to the re-arrangement of the DNA construct, with the addition of amino acids derived from the light chain vector non-translated sequence to the C-terminus of the heavy chain. This observation showed the power of proteomic mass spectrometric techniques to identify an unexpected antibody sequence variant using de novo sequencing combined with database searching, and allowed for rapid identification of the root cause for new peaks in the cation exchange and rCE-SDS assays.

Characterization of antibody charge heterogeneity resolved by preparative immobilized pH gradients.

A capillary isoelectric focusing (cIEF) method has been developed as an alternative to cation exchange chromatography to determine charge heterogeneity for a therapeutic antibody. Characterization of the cIEF profile is important to understand the charged isoform distribution. A variety of preparative IEF methods have been developed over the years but have had various limitations including high levels of contaminating ampholytes and complex fractionation and isolation procedures. More recently, an off-line method that uses pI-based separation on immobilized pH gradients was developed to preparatively isolate material with convenient liquid phase recovery. This method uses the Agilent OFFGEL 3100 Fractionator and was optimized to produce fractions of antibody charge isoforms differing by as little as 0.1 pI units. The isolation of highly resolved fractions then allowed for the identification of N- and C-terminal basic charge modifications including noncyclized glutamine, signal peptide extensions, and various levels of C-terminal lysine processing and high mannose structures. These species could then be correlated to specific peaks in the cIEF profile. This work shows that a preparative IEF method using immobilized pH gradients can be optimized to generate highly resolved, pI-based fractions in solution which can be used for successful cIEF profile characterization. Access to preparative amounts of discrete charged species allows for a better understanding of the underlying covalent modifications responsible for the charge differences and facilitates evaluation of the impact of these modifications on stability and potency of therapeutic antibodies.

Asparagine-linked oligosaccharides present on a non-consensus amino acid sequence in the CH1 domain of human antibodies.

We report that N-linked oligosaccharide structures can be present on an asparagine residue not adhering to the consensus site motif NX(S/T), where X is not proline, described in the literature. We have observed oligosaccharides on a non-consensus asparaginyl residue in the C(H)1 constant domain of IgG1 and IgG2 antibodies. The initial findings were obtained from characterization of charge variant populations evident in a recombinant human antibody of the IgG2 subclass. HPLC-MS results indicated that cation-exchange chromatography acidic variant populations were enriched in antibody with a second glycosylation site, in addition to the well documented canonical glycosylation site located in the C(H)2 domain. Subsequent tryptic and chymotryptic peptide map data indicated that the second glycosylation site was associated with the amino acid sequence TVSWN(162)SGAL in the C(H)1 domain of the antibody. This highly atypical modification is present at levels of 0.5-2.0% on most of the recombinant antibodies that have been tested and has also been observed in IgG1 antibodies derived from human donors. Site-directed mutagenesis of the C(H)1 domain sequence in a recombinant-human IgG1 antibody resulted in an increase in non-consensus glycosylation to 3.15%, a greater than 4-fold increase over the level observed in the wild type, by changing the -1 and +1 amino acids relative to the asparagine residue at position 162. We believe that further understanding of the phenomenon of non-consensus glycosylation can be used to gain fundamental insights into the fidelity of the cellular glycosylation machinery.

O-fucosylation of an antibody light chain: characterization of a modification occurring on an IgG1 molecule.

We describe the characterization of an O-fucosyl modification to a serine residue on the light chain of a recombinant, human IgG1 molecule expressed in Chinese hamster ovary (CHO) cells. Cation exchange chromatography (CEX) and hydrophobic interaction chromatography (HIC) were used to isolate a Fab population which was 146 Da heavier than the expected mass. Isolated Fab fragments were treated with a reducing agent to facilitate mass spectrometric analysis of the reduced light chain (LC) and fragment difficult (Fd). An antibody light chain with a net addition of 146 Da was detected by mass spectrometric analysis of the modified Fab. A light chain tryptic peptide in complementarity determining region-1 (CDR-1) was subsequently identified with a net addition of 146 Da by a peptide map. Results from a nanospray infusion of the modified peptide into a linear ion trap mass spectrometer with electron transfer dissociation (ETD) functionality indicated that the modified residue was a serine at position 30 in the light chain. Acid hydrolysis of the modified tryptic peptide followed by fluorescent labeling with 2-aminoanthranilic acid (2AA) and HPLC comparison with monosaccharide standards confirmed the presence of fucose on the light chain peptide. The presence of O-fucose on an antibody has not been previously reported. Currently, O-fucose has been described as occurring on mammalian proteins with amino acid sequence motifs associated with epidermal growth factor (EGF)-like repeats or thrombospondin type 1 repeats (TSRs). The amino acid sequence around the modified Ser in the IgG1 molecule does not conform to any known O-fucosylation sequence motif and thus is the first description of this type of modification on a nonconsensus sequence in a mammalian protein.

Molecular mass analysis of antibodies by on-line SEC-MS.

Mass analysis of recombinant protein therapeutics is an important assay for product characterization. Intact mass analysis is used to provide confirmation of proper translation of the DNA sequence and to detect the presence of post-translational modifications such as amino acid processing and glycosylation. We present here a method for the rapid mass analysis of antibodies using a polyhydroxyethyl aspartamide column operated in size-exclusion mode and coupled with ESI-MS. This method allows extremely efficient desalting of proteins under acidic conditions that are optimal for subsequent mass analysis using standard ESI conditions. Furthermore, this technique is significantly faster and more sensitive than rpHPLC methods, typically considered the standard chromatography approach for mass analysis of proteins. This method is flexible and robust, and should prove useful for applications where a combination of speed and sensitivity are required.

Characterization of nonenzymatic glycation on a monoclonal antibody.

We present here an improved analytical method for the analysis of glycation events in proteins. Nonenzymatic glycation of an IgG2 monoclonal antibody was studied using affinity chromatography, mass spectrometry, and chemical derivatization. Analysis of both forced-degraded and bulk-drug substance (BDS) samples showed the presence of glycated protein. A new peptide mapping procedure, incorporating derivatization using sodium borohydride, allowed the development of a sensitive method for detecting and identifying the sites of modification. When combined with tandem mass spectrometry, peptides glycated by glucose showed dramatically improved MS/MS spectra as compared to underivatized controls. Using these methods we were able to map a number of glycation sites in both forced-degraded and BDS samples that were distributed across both light and heavy chain subdomains. The combination of affinity chromatography, high-resolution mass spectrometry, and a simple derivatization procedure should allow the facile analysis of glycation for other antibody and protein samples.

Fluorescent derivatization method of proteins for characterization by capillary electrophoresis-sodium dodecyl sulfate with laser-induced fluorescence detection.

A fast and improved sample preparation scheme was developed for protein analysis using capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) with laser-induced fluorescence detection. This CE-SDS method was developed as a purity assay for recombinant monoclonal antibodies (rMAbs). In this assay, rMAbs are derivatized with the fluorogenic reagent 3-(2-furoyl)-quinoline-2-carboxaldehyde (FQ) in the presence of a nucleophile (CN-), which fluoresces only upon covalent binding to the protein. Purification after labeling is therefore not necessary to remove unreacted reagents. Proteins are incubated at 75 degrees C for 5 min to facilitate denaturation and labeling. For nonreduced preparation, rMAbs are labeled at pH 6.5 with a dye-to-protein (D/P) molar ratio of 50:1, which forms conjugates having 6 +/- 4 FQ labels. For reduced preparation, rMAbs are labeled at pH 9.3 with a D/P molar ratio of 10:1, which generates light chain conjugates incorporated with 3 +/- 2 FQ labels. Labeling artifacts such as fragmentation or aggregation are absent with use of alkylation reagents. This efficient labeling scheme generates detection limits for FQ-labeled rMAbs as low as 10 ng/mL. In comparison to other labeling strategies, labeling proteins with FQ has the advantage of speed, ease of use, and robust quantification.