mAb Monomer Research Articles

High Performance Size Exclusion Chromatography and High-Throughput Dynamic Light Scattering as Orthogonal Methods to Screen for Aggregation and Stability of Monoclonal Antibody Drug Products

The presence of aggregates in monoclonal antibody (mAb) drug product (DP) formulations can present product quality challenges. Here we show that use of High Performance Size Exclusion Chromatography (HP-SEC), in conjunction with high-throughput dynamic light scattering (HT-DLS) analyses of mAb DPs can be a useful strategy to determine monomer content and the presence of aggregates under simulated stress conditions. This analytical approach was used to evaluate four commercially available mAb DPs under different conditions i.e.; original formulations, diluted, and thermo-mechanical stressed condition. Due to particle size limitations of HP-SEC columns, resulting in particles accumulating in the column frits prior to reaching the detector for analysis, there is a possibility that large mAb aggregates may not be detected. Both HP-SEC and HT-DLS were able to detect and resolve the mAb monomer (~10–12 nm) of the DPs in their recommended storage conditions. However, the ability to detect large aggregates (>40 nm) by both analytical methods differed, and HT-DLS was able to detect aggregates between 60 nm and 1400 nm under stress conditions. Our data indicates that HP-SEC, in conjunction with HT-DLS, may be beneficial to detect both mAb DP monomer content and multiple aggregate species (1–1000 nm) in the submicron size range.

Cation exchange frontal chromatography for the removal of monoclonal antibody aggregates

A low ligand density cation exchange (CEX) chromatography resin, Eshmuno® CP-FT resin, was investigated for the removal of aggregates from monoclonal antibody (mAb) feeds using a continuous loading process. Removing mAb aggregates with a CEX resin using continuous loading is advantageous relative to a bind/elute loading process, because the resin can use nearly its full capacity to bind the aggregates enabling much higher loadings. The removal of mAb aggregates with Eshmuno® CP-FT resin using a continuous loading process was found to be consistent with a frontal chromatography mechanism where the mAb monomer initially binds to the column and is subsequently displaced by dimers and higher molecular weight aggregates. The removal of mAb aggregates with Eshmuno® CP-FT resin using a continuous loading process was compared with six other commercially available strong CEX chromatography resins and found to correlate with their ionic densities, but not their mAb static binding capacities. The influence of pH, conductivity, residence time, and mAb concentration on the removal of aggregates with Eshmuno® CP-FT resin using a continuous loading process was also investigated. Finally, the percentage of aggregates in a mAb feed was varied to examine the effect on the removal of aggregates with Eshmuno® CP-FT resin using a continuous loading process.

Selective protein quantification for preparative chromatography using variable pathlength UV/Vis spectroscopy and partial least squares regression

In preparative protein chromatography, broad dynamic ranges of protein concentrations as well as co-elution of product and impurities are common. Despite being the standard in biopharmaceutical production, monitoring of preparative chromatography is generally limited to surrogate signals, e.g. UV absorbance at 280 nm. To address this problem, variable pathlength (VP) spectroscopy in conjunction with Partial Least Squares regression (PLS) was used to monitor preparative chromatography. While VP spectroscopy enabled the acquisition of absorbance data for a broad concentration range, PLS modelling allowed for the differentiation between the protein species. The approach was first implemented for monitoring the separation of lysozyme from cytochrome c at an overall loading density of 92 g/l. The same method was then applied to the polishing step of a monoclonal antibody (mAb) at 40 g/l loading density. For PLS model prediction of the mAb monomer and the high molecular weight variants (HMWs), the root mean square error (RMSE) was 1.07 g/l and 0.42 g/l respectively. To demonstrate the usability of the approach for in-line control, pooling decisions for both separation problems were subsequently taken based on the computed concentrations or thereof derived purities. In summary, VP spectroscopy in conjunction with PLS modelling is a promising option for in-line monitoring and control of future chromatography steps at large scale.

Choosing the right protein A affinity chromatography media can remove aggregates efficiently.

Protein A chromatography (PAC) is commonly used as an efficient capture step in monoclonal antibody (mAb) separation processes. Usually dynamic binding capacity is used for choosing the right PAC. However, if aggregates can be efficiently removed during elution, it can make the following polishing steps easier. In this study a method for choosing the right PAC media in terms of mAb aggregate removal is proposed. Linear pH gradient elution experiments of two different mAbs on various PAC columns are carried out, where the elution behavior of aggregates as well as the monomer is measured. Aggregates of one mAb are more strongly retained compared with the mAb monomer. Another mAb showed different elution behavior, where the aggregates are eluted as both the weakly and strongly retained peaks. In order to remove the two types of aggregates by stepwise elution two protocols are tested. The first protocol A consisted of the sample loading, the wash with the equilibration buffer and the low pH elution. The wash stage of the second protocol B included the wash with 1.0 M arginine. No detectable peaks are observed during the wash stage of protocol A whereas significant peaks are monitored during the arginine wash of protocol B. One of the PAC columns showed a smaller peak during the arginine wash. In addition, both aggregate removal and monomer yield are higher with protocol B compared with the other PAC columns. This method is found to be useful for choosing the right PAC column.

The molecular weight and concentration of dextran sulfate affect cell growth and antibody production in CHO cell cultures.

To investigate the effect of dextran sulfate (DS), a widely used anti-aggregation agent, on cell growth and monoclonal antibody (mAb) production including the quality attributes, DS with the three different MWs (4,000 Da, 15,000 Da, and 40,000 Da) at various concentrations (up to 1 g/L) was added to suspension cultures of two different recombinant CHO (rCHO) cell lines producing mAb, SM-0.025 and CS13-1.00. For both cell lines, the addition of DS, regardless of the MW and concentration of DS used, improved cell growth and viability in the decline phase of growth. However, it increased mAb production only in the CS13-1.00 cells. Among the three different MWs, 40,000 Da DS was most effective in attenuating cell aggregation during the cultures of CS13-1.00 cells, and showed the highest maximum mAb concentration. For SM-0.025 cells, it significantly decreased specific mAb productivity, particularly at a high concentration of DS. Overall, DS addition did not negatively affect the quality attributes of mAbs (aggregation, charge variation, and glycosylation), though its efficacy on mAb quality depended on the MW and concentration of DS and cell lines. For both cell lines, the addition of DS did not affect N-glycosylation of mAbs and decreased basic charge variants in mAbs. For CS13-1.00 cells, the mAb monomer increased with the addition of 40,000 Da DS at 0.3-1.0 g/L. Taken together, to maximize the beneficial effect of DS addition on mAb production, the optimal MW and concentration of DS should be determined for each specific rCHO cell line. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1113-1122, 2016.

Gelation of a Monoclonal Antibody at the Silicone Oil–Water Interface and Subsequent Rupture of the Interfacial Gel Results in Aggregation and Particle Formation

The formation of viscoelastic gels by a monoclonal antibody (mAb) at the silicone oil-water interface was studied using interfacial shear rheology. At a concentration of 50 μg/mL, the mAb formed gels in less than 1 h, and the gelation time decreased with increasing protein concentration. To probe the effects of mechanical rupture of the interfacial gel layers, a layer of silicone oil was overlaid on the surface of aqueous solutions of mAb, and the interface was ruptured periodically with a needle. Rupture of the interfacial gel resulted in formation of subvisible particles and substantial losses of mAb monomer, which were detected by microflow imaging and quantified by size-exclusion chromatography, respectively. Resonance mass measurement showed that levels of both protein particles and silicone oil droplets increased as the gel was repeatedly ruptured with a needle. In contrast, in samples wherein the interfacial gels were not ruptured, much lower levels of aggregates and particles were detected. Addition of nonionic surfactants (polysorbate 20 or polysorbate 80) protected against aggregation and protein particle formation, with increased protection seen with increasing surfactant levels, and with the greatest inhibition observed in samples containing polysorbate 80.

Direct analysis of mAb aggregates in mammalian cell culture supernatant.

BackgroundProtein aggregation during monoclonal antibody (mAb) production can occur in upstream and downstream processing (DSP). Current methods to determine aggregate formation during cell culture include size exclusion chromatography (SEC) with a previous affinity chromatography step in order to remove disturbing cell culture components. The pre-purification step itself can already influence protein aggregation and therefore does not necessarily reflect the real aggregate content present in cell culture. To analyze mAb aggregate formation directly in the supernatant of Chinese hamster ovary (CHO) cell culture, we established a protocol, which allows aggregate quantification using SEC, without a falsifying pre-purification step.ResultsThe use of a 3 μm silica SEC column or a SEC column tailored for mAb aggregate analysis allows the separation of mAb monomer and aggregates from disturbing cell culture components, which enables aggregate determination directly in the supernatant. Antibody aggregate analysis of a mAb-producing CHO DG44 cell line demonstrated the feasibility of the method. Astonishingly, the supernatant of the CHO cells consisted of over 75% mAb dimer and larger oligomers, representing a substantially higher aggregate content than reported in literature so far.ConclusionThis study highlights that aggregate quantification directly in the cell culture supernatant using appropriate SEC columns with suitable mAb aggregate standards is feasible without falsification by previous affinity chromatography. Moreover, our results indicate that aggregate formation should be addressed directly in the cell culture and is not only a problem in DSP.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-014-0099-3) contains supplementary material, which is available to authorized users.

Combined Protein A and size exclusion high performance liquid chromatography for the single-step measurement of mAb, aggregates and host cell proteins.

Quantification of monoclonal antibody (mAb) monomer, mAb aggregates, and host cell proteins (HCPs) is critical for the optimization of the mAb production process. The present work describes a single high throughput analytical tool capable of tracking the concentration of mAb, mAb aggregate and HCPs in a growing cell culture batch. By combining two analytical HPLC methods, Protein A affinity and size-exclusion chromatography (SEC), it is possible to detect a relative increase or decrease in the concentration of all three entities simultaneously. A comparison of the combined Protein A-SEC assay to SEC alone was performed, demonstrating that it can be useful tool for the quantification of mAb monomer along with trending data for mAb aggregate and HCP. Furthermore, the study shows that the Protein A-SEC method is at least as accurate as other commonly used analytical methods such as ELISA and Bradford.

A practical strategy for using miniature chromatography columns in a standardized high‐throughput workflow for purification development of monoclonal antibodies

The emergence of monoclonal antibody (mAb) therapies has created a need for faster and more efficient bioprocess development strategies in order to meet timeline and material demands. In this work, a high-throughput process development (HTPD) strategy implementing several high-throughput chromatography purification techniques is described. Namely, batch incubations are used to scout feasible operating conditions, miniature columns are then used to determine separation of impurities, and, finally, a limited number of lab scale columns are tested to confirm the conditions identified using high-throughput techniques and to provide a path toward large scale processing. This multistep approach builds upon previous HTPD work by combining, in a unique sequential fashion, the flexibility and throughput of batch incubations with the increased separation characteristics for the packed bed format of miniature columns. Additionally, in order to assess the applicability of using miniature columns in this workflow, transport considerations were compared with traditional lab scale columns, and performances were mapped for the two techniques. The high-throughput strategy was utilized to determine optimal operating conditions with two different types of resins for a difficult separation of a mAb monomer from aggregates. Other more detailed prediction models are cited, but the intent of this work was to use high-throughput strategies as a general guide for scaling and assessing operating space rather than as a precise model to exactly predict performance.

Investigating Monoclonal Antibody Aggregation Using a Combination of H/DX-MS and Other Biophysical Measurements

To determine how structural changes in antibodies are connected with aggregation, the structural areas of an antibody prone to and/or impacted by aggregation must be identified. In this work, the higher-order structure and biophysical properties of two different monoclonal antibody (mAb) monomers were compared with their simplest aggregated form, that is, dimers that naturally occurred during normal production and storage conditions. A combination of hydrogen/deuterium exchange mass spectrometry and other biophysical measurements was used to make the comparison. The results show that the dimerization process for one of the mAb monomers (mAb1) displayed no differences in its deuterium uptake between monomer and dimer forms. However, the other mAb monomer (mAb2) showed subtle changes in hydrogen/deuterium exchange as compared with its dimer form. In this case, differences observed were located in specific functional regions of the CH 2 domain and the hinge region between CH 1 and CH 2 domains. The importance and the implications of these changes on the antibody structure and mechanism of aggregation are discussed.

Analytical characterization of a monoclonal antibody therapeutic reveals a three-light chain species that is efficiently removed using hydrophobic interaction chromatography

Size exclusion high performance liquid chromatography analysis of a human monoclonal antibody (mAb) showed the presence of a new species that eluted with a retention time between the dimeric and monomeric species of the antibody. Extensive characterization of this species, referred to as “shoulder,” indicated that it was a mAb containing an extra light chain and had a molecular weight of approximately 175 kDa. The extra light chain was found to be non-covalently associated with the Fab portion of the protein. The relative amount of shoulder (typically 1−3% of the total mAb present) varied with the Chinese hamster ovary cell line producing the mAb and was not influenced by the growth conditions. Our three-step mAb purification platform using protein A, anion exchange, and cation exchange process steps was successful at removing dimer and higher and lower molecular weight species, but not the shoulder impurity. It was found that hydrophobic interaction chromatography could be used in place of cation exchange to exploit the subtle differences in hydrophobicity between monomer and shoulder. We developed an antibody polishing process using Butyl Sepharose HP resin that is capable of removing the majority of high and low molecular weight impurities yielding 99% pure mAb monomer, virtually devoid of the shoulder species, with a step recovery of about 80%.