Continuous purification of antibodies from cell culture supernatant with aqueous two-phase systems: from concept to process.

An aqueous two-phase extraction (ATPE) process based on a PEG/phosphate system was developed for the capture of human immunoglobulin G and successfully applied to a Chinese hamster ovary and a PER.C6® cell supernatant. A continuous ATPE process incorporating three different steps (extraction, back-extraction, and washing) was set up and validated in a pump mixer-settler battery. Most of the higher molecular weight cell supernatant impurities were removed during the extraction step, while most of the lower molecular weight impurities were removed during the subsequent steps. A global recovery yield of 80% and a final protein purity of more than 99% were obtained for the IgG purification from a CHO cell supernatant, representing a 155-fold reduction in the protein/IgG ratio. For the purification of IgG from a PER.C6® cell supernatant, a global recovery yield of 100%, and a host cell protein purity were attained, representing a 22-fold reduction in the host cell protein/IgG ratio. These results, thus, open promising perspectives for the application of the developed ATPE process as a platform for the capture of antibodies. In fact, this new process has shown the ability to successfully recover and purify different antibodies from distinct cell culture supernatants. This technology can also overcome some of the limitations encountered using the typical chromatographic processes, besides inherent advantages of scalability, process integration, capability of continuous operation, and economic feasibility.

Continuous aqueous two-phase extraction of human antibodies using a packed column.

The performance of a pilot scale packed differential contactor was evaluated for the continuous counter-current aqueous two-phase extraction (ATPE) of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cells supernatant (CS) enriched with pure protein. Preliminary studies have been firstly performed in order to select the dispersed phase (phosphate-rich or polyethylene glycol 3350 Da (PEG)-rich phase) and the column packing material. The PEG-rich phase has been selected as the dispersed phase and the stainless steel as the preferred material for the column packing bed since it was not wetted preferentially by the selected dispersed phase. Hydrodynamic studies have been also performed, and the experimental results were successfully adjusted to the Richardson-Zaki and Mísek equations, typically used for the conventional organic-aqueous two-phase systems. An experimental set-up combining the packed column with a pump mixer-settler stage showed to have the best performance and to be advantageous when compared to the IgG batch extraction. An IgG recovery yield of 85% could be obtained with about 50% of total contaminants and more than 85% of contaminant proteins removal. Mass transfer studies have revealed that the mass transfer was controlled by the PEG-rich phase. A higher efficiency could be obtained when using an extra pump mixer-settler stage and higher flow rates.

Aqueous two-phase extraction as a platform in the biomanufacturing industry: economical and environmental sustainability.

The biotech industry is, nowadays, facing unparalleled challenges due to the enhanced demand for biotechnology-based human therapeutic products, such as monoclonal antibodies (mAbs). This has led companies to improve substantially their upstream processes, with the yield of monoclonals increasing to titers never seen before. The downstream processes have, however, been overlooked, leading to a production bottleneck. Although chromatography remains the workhorse of most purification processes, several limitations, such as low capacity, scale-related packing problems, low chemical and proteolytic stability and resins' high cost, have arisen. Aqueous two-phase extraction (ATPE) has been successfully revisited as a valuable alternative for the capture of antibodies. One of the important remaining questions for this technology to be adopted by the biotech industries is, now, how it compares to the currently established platforms in terms of costs and environmental impact. In this report, the economical and environmental sustainability of the aqueous two-phase extraction process is evaluated and compared to the currently established protein A affinity chromatography. Accordingly, the ATPE process was shown to be considerably advantageous in terms of process economics, especially when processing high titer cell culture supernatants. This alternative process is able to purify continuously the same amount of mAbs reducing the annual operating costs from 14.4 to 8.5 million (US$/kg) when cell culture supernatants with mAb titers higher than 2.5 g/L are processed.

Downstream processing of antibodies: single-stage versus multi-stage aqueous two-phase extraction.

Single-stage and multi-stage strategies have been evaluated and compared for the purification of human antibodies using liquid-liquid extraction in aqueous two-phase systems (ATPSs) composed of polyethylene glycol 3350 (PEG 3350), dextran, and triethylene glycol diglutaric acid (TEG-COOH). The performance of single-stage extraction systems was firstly investigated by studying the effect of pH, TEG-COOH concentration and volume ratio on the partitioning of the different components of a Chinese hamster ovary (CHO) cells supernatant. It was observed that lower pH values and high TEG-COOH concentrations favoured the selective extraction of human immunoglobulin G (IgG) to the PEG-rich phase. Higher recovery yields, purities and percentage of contaminants removal were always achieved in the presence of the ligand, TEG-COOH. The extraction of IgG could be enhanced using higher volume ratios, however with a significant decrease in both purity and percentage of contaminants removal. The best single-stage extraction conditions were achieved for an ATPS containing 1.3% (w/w) TEG-COOH with a volume ratio of 2.2, which allowed the recovery of 96% of IgG in the PEG-rich phase with a final IgG concentration of 0.21mg/mL, a protein purity of 87% and a total purity of 43%. In order to enhance simultaneously both recovery yield and purity, a four stage cross-current operation was simulated and the corresponding liquid-liquid equilibrium (LLE) data determined. A predicted optimised scheme of a counter-current multi-stage aqueous two-phase extraction was hence described. IgG can be purified in the PEG-rich top phase with a final recovery yield of 95%, a final concentration of 1.04mg/mL and a protein purity of 93%, if a PEG/dextran ATPS containing 1.3% (w/w) TEG-COOH, 5 stages and volume ratio of 0.4 are used. Moreover, according to the LLE data of all CHO cells supernatant components, it was possible to observe that most of the cells supernatant contaminants can be removed during this extraction step leading to a final total purity of about 85%.

Application of aqueous two-phase systems to antibody purification: a multi-stage approach.

The single-stage equilibrium aqueous two-phase extraction (ATPE) of human antibodies from a Chinese hamster ovary (CHO) cells supernatant was investigated. The performance of polyethylene glycol 3350 (PEG 3350)/phosphate aqueous two-phase systems (ATPS) was evaluated by studying several experimental conditions, such as pH, ionic strength, volume ratio and initial antibody concentration in the feed stock. The conditions that favoured the extraction of human immunoglobulin G (IgG) were low pH values, high NaCl concentration and high volume ratios. The percentage of contaminants removal did not depend on the pH value and NaCl concentration, increasing, however, as the volume ratio decreased. About 66% of total contaminants were removed when a volume ratio of 0.4 was used. The multi-stage equilibrium ATPE was also investigated by simulating a four stages cross-current operation in test tubes. According to the IgG equilibrium curves and respective McCabe Thiele diagrams, a predicted optimised scheme of a counter-current multi-stage ATPE was described. An IgG recovery yield of 89% and a protein purity of 75% can be achieved using a PEG/phosphate ATPS containing 10% (w/w) NaCl, five stages and a volume ratio of 0.4. This represents significant improvements in the recovery yield and purity when compared to a single-stage trial performed at the same experimental conditions, where a 61% recovery yield and 55% protein purity were attained. Based on the CHO cells supernatant components equilibrium curves, it was observed that IgG can be completely purified from the higher molecular weight (MW) components and partially purified from the lower MW components.

[Quality of life of patients with malignant facial and skin tumors--development of a questionnaire and initial findings of a study]. / Lebensqualität von Patienten mit bösartigen Gesichts- und Hauttumoren--Entwicklung eines Fragebogens und erste Ergebnisse einer Studie.

Assisted by Deutsche Krebshilfe, the German cancer aid fund, a questionnaire for assessment of quality of life in patients with life-threatening skin tumours and disfiguring tumour sequels was developed at Fachklinik Hornheide between 1986 and 1988. A first step toward determining quality of life and for enhancing treatment-integrated rehabilitative work is the assessment of the subjective distress and problems experienced due to the illness and its treatment. This is the objective of the Hornheider Fragebogen, a questionnaire developed in the framework of the study on the basis of extensive literature search, structured interviews with patients and treating staff. This self-assessment instrument was answered by 131 patients with malignant tumours of the skin, twice during in-patient treatment and a third time some 6 months after discharge. Statistical analysis of the data collected proved the questionnaire to be valid, reliable and practicable. Additionally, it has been possible to draw up a number of significant results of immediate relevancy. It turned out that persons who are unemployed, divorced, or married but separated, as well as persons without school leaving certificate, experienced greater distress and more problems in many dimensions than the other respondents. The questionnaire was viewed as meaningful and as helpful by the patients in order to voice unspecific anxiety and questions. Also, the study entailed positive implications for our rehabilitative work already in this initial testing phase, by enabling us to give more specific support to the patients. The Hornheider Fragebogen, after review and abridgment, consists of 27 problem-oriented questions relating to eight dimensions of life.

[Experience of their environment and disease by facially disfigured patients--recognition of the handicap as a prerequisite for rehabilitation]. / Umwelterfahrung und Krankheitserleben gesichtsversehrter Patienten--Erkennen der Behinderung als Voraussetzung zur Rehabilitation.

In an investigation of psychosocial strain in persons having a facial or skin impairment, a total of 25 patients of Fachklinik Hornheide (a special clinic for the treatment of tumors, tuberculosis, and for facial and skin reconstruction) had participated in in-depth interviewing. It was found with unexpected clarity that these clients are exposed to a high measure of negative environmental reaction. In addition to having to cope with stressful disease consequences, often entailing a "loss of face" with the major problems of self-worth this involves, these negative experiences in many instances lead to behavioural problems, withdrawal reaction, occupational repercussions, or severe emotional crisis in those concerned. The degree to which these patients can overcome their problems is to a large extent dependent on how parents, spouse, colleagues, and important others respond. Early enough involvement of significant others, possibly already at the time of medical information, as well as support during emotional crisis are capable of considerably alleviating the coping process. The psychosocial problems confronting persons with disfiguring facial conditions have to be taken into account during therapy in order to successfully enhance the purpose of integration into the social environment.