Subsequent Polishing Step Research Articles

CH1-specific affinity resins possess the potential of separating heterodimer from homodimers in asymmetric bispecific antibody purification.

CaptureSelect CH1-XL and Praesto 70 CH1 are two affinity media that specifically bind to the CH1 domain of an antibody. In the current work, we first demonstrated that these two CH1-specific affinity media bound to different monoclonal antibodies (mAbs) with varied strengths under identical conditions. We previously had observed the same on a Protein L-conjugated resin and showed that such a property could facilitate homodimer removal in asymmetric bispecific antibody (bsAb) purification. Next, using Praesto 70 CH1, we showed that a small difference in binding between two mAbs could be significantly exaggerated by adding sodium chloride to the mobile phase, further demonstrating this resin can potentially play a role in bsAb purification. Finally, with a concrete bsAb case study, we showed that, like Protein L, Praesto 70 CH1 could separate the target heterodimer from the homodimer by-product. Homodimers are common product-related impurities associated with the recombinant production of asymmetric bsAbs, which can be difficult to remove. Their removal, even a partial one, at the capture stage is a big advantage as it can alleviate the purification burden on subsequent polishing steps and render the overall process more robust. Therefore, Praesto 70 CH1's unique property is highly desirable, and this affinity resin can be a better alternative than Protein A for product capture in asymmetric bsAb purification.

The beneficial impact of kosmotropic salts on the resolution and selectivity of Protein A chromatography

During the manufacturing of therapeutic antibodies, effective Protein A chromatography as initial column step is crucial to simplify the remaining purification effort for subsequent polishing steps. This is particularly relevant for molecules with high impurity content so that desired product purity can be attained. The present study demonstrates beneficial effects on impurity removal when applying kosmotropic salts, e.g., sodium sulfate or sodium chloride, in the elution phase. Initially, a screen using negative linear pH gradient elution evaluated the impact of the kosmotropic salts in comparison to no additive and chaotropic urea using three mAbs and three common resins. Retaining acceptable yield, the kosmotropic salts improved resolution of monomer and impurities and reduced the contents of process-related host cell proteins and DNA as well as of product-related low and high molecular weight forms, despite some resin- and mAb-dependent variations. Moreover, a decrease in hydrolytic activity measured by a new assay for polysorbase activity was observed. In contrast, urea was hardly effective. The findings served to establish optimized step elution conditions with 0.25 M of sodium sulfate for a challenging mAb with complex format (bispecific 2 + 1 CrossMab) displaying high relative hydrophobicity and impurity levels. With yield and purity both in the range of 90 %, the contents of all impurity components were reduced, e.g., low molecular weight forms by two-fold and polysorbase activity by four-fold. The study indicates the potential of kosmotropic salts to establish efficient and comprehensive impurity separation by Protein A for facilitated downstream processing and economic manufacturing of complex antibodies.

Atmospheric Plasma Jet processing for figure error correction of an optical element made from S-BSL7

To meet the increasing market demand for optical components, Plasma Jet Machining (PJM) of Borosilicate Crown Glass (BCG), which can be an alternative to Fused Silica, is presented. Surface figure error correction was performed by applying reactive plasma jet etching, where a fluorine-containing microwave driven plasma jet is employed to reduce the figure error in a deterministic dwell-time controlled dry etching process. However, some of the glass constituents of BCG cause the formation of a residual layer during surface treatment which influences the local material removal. By heating the substrate to about TS = 325 °C to 350 °C during processing, the etching behavior can clearly be improved. Geometric conditions of the optical element nevertheless lead to a characteristic temperature distribution on the substrate surface, which requires an adjustment of the local dwell times in order to obtain the required material removal. Furthermore, the resulting local surface roughness is also influenced by the surface temperature distribution. It is shown that figure error can be significantly reduced by taking the local temperature distribution and resulting local etching rates into account. A subsequent polishing step smoothens roughness features occurring during etching to provide optical surface quality.

Minimization of CaF2 spherical surface deformation caused by annealing

We explore causes and possible techniques to minimize shape deformation of high-precision monocrystalline calcium fluoride optical surface formed during thermal annealing. Due to the anisotropic mechanical properties of calcium fluoride crystals, machining of lens shapes introduces defects into the crystal lattice. The thermal annealing thus leads to activation of the processes of recovery, resulting in the formation of characteristic surface structures causing both shape error and increased microroughness. The surface deformation can be gradually minimized by thermal treatment of the optical element and subsequent polishing steps to the order of units of nanometers, so they do not represent a fundamental problem for optical performance.

Ultrashort pulse laser micro polishing of steel – Investigation of the melt pool depth

For the most demanding applications in mold and tool manufacturing, ultrashort pulsed laser structured surfaces sometimes do not meet the requirements for surface roughness. A subsequent polishing step in the same machine with the same beam source offers a time and cost effective solution. The novel approach of ultrashort pulse laser polishing with a pulse duration of τ=10 ps is investigated experimentally and theoretically in the present study. It is shown that a low micro roughness is achieved with a melt pool depth between 1 and 3 µm in hot-working steel 1.2738, which allows a polishing process for surface structures with small and fine geometry features without influencing the structure itself.

Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation.

In this protocol, we describe a method to purify human calcium-binding protein S100A12 and its ion-induced oligomers from Escherichia coli culture for immune cell stimulations. This protocol is based on a two-step chromatography strategy, which comprises protein pre-purification on an anion-exchange chromatography column and a subsequent polishing step on a hydrophobic-interaction column. This strategy produces S100A12 protein of high purity and yield at manageable costs. For functional assays on immune cells eventual remnant endotoxin contamination requires careful monitoring and further cleaning steps to obtain endotoxin-free protein. The majority of endotoxin contaminations can be excluded by anion-exchange chromatography. To deplete residual contaminations, this protocol describes a removal step with centrifugal filters. Depending on the available ion-strength S100A12 can arrange into different homomultimers. To investigate the relationship between structure and function, this protocol further describes ion-treatment of S100A12 protein followed by chemical crosslinking to stabilize S100A12 oligomers and their subsequent separation by size-exclusion chromatography. Finally, we describe a cell-based assay that confirms the biological activity of the purified protein and confirms LPS-free preparation.

Phosphorus reduction in filters for on-site wastewater treatment

Discharges of phosphorus (P) from on-site wastewater treatment systems generally contribute to eutrophication problems in Swedish freshwaters and, ultimately, in the Baltic Sea. Such concerns have led to a growing interest in improving P removal in treatment facilities. This study investigated the reduction of P in 12 full-scale on-site treatment systems featuring sand filters and alkaline P-filters by sampling and analysing filter influents and effluents. The flow-proportional samples collected were analysed for total and dissolved P, BOD7, total and dissolved organic carbon (TOC and DOC), and pH. Seven of the eight investigated sand filters did not provide satisfactory total P reduction; the likely explanations are filter clogging and wastewater dilution by extraneous water. In addition, effluents from four of the eight sand filters had total P concentrations higher than 3 mg L−1, which is the Swedish effluent limit recommended for common receiving waters, indicating that a subsequent polishing step would be needed. Six of the nine investigated P-filters reduced P adequately, with total P concentrations in the effluent ranging between 0.1 and 1.9 mg L−1. The three underperforming P-filters had effluent pH values below 9; filter age, clogging, and hydraulic overload were identified as probable reasons for their poor performance. A statistically significant correlation was found between total-P reduction and filtrate pH, but no significant correlation was detected between organic load in the influent and P reduction by the P-filters. The P-filter media replacement frequency could not be established, but filtrate pH appeared to be a good estimator.

A membrane-based purification process for cell culture-derived influenza A virus

A simple membrane-based purification process for cell culture-derived influenza virus was established that relies on only two chromatographic unit operations to achieve the contamination limits required according to regulatory authorities. After clarification and concentration, a pseudo-affinity membrane adsorber (sulfated cellulose, SCMA) was applied for virus capture. The subsequent polishing step consisted of a salt-tolerant anion exchange membrane adsorber (STMA) to bind residual DNA. For the presented process neither a buffer exchange step nor a nuclease step for further DNA digestion were required. As a starting point, a two-salt strategy (including a polyvalent ion) was employed to screen STMA conditions in a 96-well plate format. After optimization on chromatographic laboratory scale, the virus recovery was up to 97% with a residual DNA level below 0.82%. In addition, the STMA was characterized regarding its dynamic binding capacity and the impact of flow rate on yields and contamination levels. Overall, the total virus yield for influenza virus A/PR/8/34 (H1/N1) of this two-step membrane process was 75%, while the protein and the DNA contamination level could be reduced to 24% and at least 0.5%, respectively. With 19.8μg protein and 1.2ng DNA per monovalent dose, this purity level complies with the limits of the European Pharmacopeia for cell culture-derived vaccines for human use. Overall, the presented downstream process might serve as a generic and economic platform technology for production of cell culture-derived viruses and viral vectors.

Evaluation of high‐capacity cation exchange chromatography for direct capture of monoclonal antibodies from high‐titer cell culture processes

Advances in molecular biology and cell culture technology have led to monoclonal antibody titers in excess of 10 g/L. Such an increase can pose concern to traditional antibody purification processes due to limitations in column hardware and binding capacity of Protein A resins. Recent development of high capacity cation exchangers can make cation exchange chromatography (CEX) a promising and economic alternative to Protein A capture. This work investigates the feasibility of using CEX for direct capture of monoclonal antibodies from high titer cell culture fluids. Two resin candidates were selected from seven newer generation cation exchangers for their higher binding capacity and selectivity. Two monoclonal antibodies with widely differing pI values were used to evaluate the capability of CEX as a platform capture step. Screening of loading pH and conductivity showed both resins to be capable of directly capturing both antibodies from undiluted cell culture fluid. At appropriate acidic pH range, product loading of over 65 g/L resin was achieved for both antibodies. A systematic design of experiment (DOE) approach was used to optimize the elution conditions for the CEX step. Elution pH showed the most significant impact on clearance of host cell proteins (HCPs). Under optimal conditions, HCP reduction factors in the range of 9-44 were achieved on the CEX step based on the pI of the antibody. Apart from comparing CEX directly to Protein A as the capture method, material from either modality was also processed through the subsequent polishing steps to compare product quality at the drug substance level. Process performance and product quality was found to be acceptable using the non-affinity based process scheme. The results shown here present a cheaper and higher capacity generic capture method for high-titer antibody processes.

Novel peptide ligand with high binding capacity for antibody purification

Small synthetic ligands for protein purification have become increasingly interesting with the growing need for cheap chromatographic materials for protein purification and especially for the purification of monoclonal antibodies (mAbs). Today, Protein A-based chromatographic resins are the most commonly used capture step in mAb down stream processing; however, the use of Protein A chromatography is less attractive due to toxic ligand leakage as well as high cost. Whether used as an alternative to the Protein A chromatographic media or as a subsequent polishing step, small synthetic peptide ligands have an advantage over biological ligands; they are cheaper to produce, ligand leakage by enzymatic degradation is either eliminated or significantly reduced, and they can in general better withstand cleaning in place (CIP) conditions such as 0.1M NaOH. Here, we present a novel synthetic peptide ligand for purification of human IgG. Immobilized on WorkBeads, an agarose-based base matrix from Bio-Works, the ligand has a dynamic binding capacity of up to 48mg/mL and purifies IgG from harvest cell culture fluid with purities and recovery of >93%. The binding affinity is ∼105M−1 and the interaction is favorable and entropy-driven with an enthalpy penalty. Our results show that the binding of the Fc fragment of IgG is mediated by hydrophobic interactions and that elution at low pH is most likely due to electrostatic repulsion. Furthermore, we have separated aggregated IgG from non-aggregated IgG, indicating that the ligand could be used both as a primary purification step of IgG as well as a subsequent polishing step.

Development of a Laser Based Process Chain for Manufacturing Freeform Optics

The current state of the development of a laser based process chain for manufacturing fused silica optics is presented. In a first step fused silica is ablated with laser radiation to produce the geometry of the optics. A subsequent polishing step reduces the surface roughness and a third step uses micro ablation to remove the last remaining redundant material. Although the process chain is still under development, the ablation of fused silica already reaches ablation rates above 20 mm3/s with a resulting surface roughness of Ra < 5 μm and the polishing process is able to significantly reduce this roughness.

Diamond components with integrated abrasion sensor for tribological applications

Tribological components made from CVD diamond are commonly used for protection against abrasion in rough environments. Such components can be used e.g. in textile industry as thread guiding devices provided that surface roughness and resultant friction are low. In this work we report on the fabrication of CVD diamond components of non-planar shape. These devices were fabricated in a negative replication approach on mechanically structured substrates. Using this technique cylindrically shaped diamond devices with smooth surfaces were produced by using the nucleation side of the diamond layers as the exposed surface, thus making subsequent polishing steps unnecessary. Applying an improved two-step nucleation method further reduced surface roughness. Sensing elements in the form of resistors made from boron-doped CVD diamond were integrated into the device surface. Device performance was characterized with respect to the temperature dependence of the resistors and the suitability as abrasion sensor.