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  • 1.
    Bandmann, Nina
    et al.
    KTH, Superseded Departments, Biotechnology.
    van Alstine, James
    KTH, Superseded Departments, Biotechnology.
    Veide, Andres
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Functional selection of phage displayed peptides for facilitated design of fusion tags improving aqueous two-phase partitioning of recombinant proteins2002In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 93, no 1, p. 1-14Article in journal (Refereed)
    Abstract [en]

    Aqueous two-phase systems allow for the unequal distribution of proteins and other molecules in water-rich solutions containing phase separating polymers or surfactants. One approach to improve the partitioning properties of recombinant proteins is to produce the proteins as fused to certain peptide tags. However, the rational design of such tags has proven difficult since it involves a compromise between multivariate parameters such as partitioning properties, solvent accessibility and production/secretion efficiency. In this work, a novel approach for the identification of suitable peptide tag extensions has been investigated, Using the principles of selection, rather than design, peptide sequences contributing to an improved partitioning have been identified using phage display technology. A 40 million member phagemid library of random nona-peptides, displayed as fusion to the major coat protein pVIII of the filamentous phage M 13, was employed in the selection of top-phase partitioning phage particles in a PEG/sodium phosphate system. After multiple cycles of selection by partitioning, peptides with high frequencies of both tyrosine and proline residues were found to be over represented in selected clones. The identified peptide sequences, or derivatives thereof, were subsequently individually analyzed for their partitioning behavior as displayed on phage, as free synthetic peptides and as genetically fused to a recombinant model target protein. The results showed that novel peptide sequences capable of enhancing top-phase partitioning without interfering with protein production and secretion indeed could be identified for the aqueous two-phase system investigated.

  • 2. Rodrigo, Gustav
    et al.
    Gruvegard, Mats
    Van Alstine, James M.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. JMVA Biotech, Sweden.
    Antibody Fragments and Their Purification by Protein L Affinity Chromatography2015In: ANTIBODIES, ISSN 2073-4468, Vol. 4, no 3, p. 259-277Article, review/survey (Refereed)
    Abstract [en]

    Antibodies and related proteins comprise one of the largest and fastest-growing classes of protein pharmaceuticals. A majority of such molecules are monoclonal antibodies; however, many new entities are antibody fragments. Due to their structural, physiological, and pharmacological properties, antibody fragments offer new biopharmaceutical opportunities. In the case of recombinant full-length antibodies with suitable Fc regions, two or three column purification processes centered around Protein A affinity chromatography have proven to be fast, efficient, robust, cost-effective, and scalable. Most antibody fragments lack Fc and suitable affinity for Protein A. Adapting proven antibody purification processes to antibody fragments demands different affinity chromatography. Such technology must offer the unit operation advantages noted above, and be suitable for most of the many different types of antibody fragments. Protein L affinity chromatography appears to fulfill these criteriasuggesting its consideration as a key unit operation in antibody fragment processing.

  • 3. Soares, R.R.G.
    et al.
    Azevedo, A.M.
    Van Alstine, James
    KTH, School of Biotechnology (BIO). JMVA Biotech, Stockholm, Sweden.
    Raquel Aires-Barros, M.
    Partitioning in aqueous two-phase systems: Analysis of strengths, weaknesses, opportunities and threats2015In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 10, no 8, p. 1158-1169Article in journal (Refereed)
    Abstract [en]

    For half a century aqueous two-phase systems (ATPSs) have been applied for the extraction and purification of biomolecules. In spite of their simplicity, selectivity, and relatively low cost they have not been significantly employed for industrial scale bioprocessing. Recently their ability to be readily scaled and interface easily in single-use, flexible biomanufacturing has led to industrial re-evaluation of ATPSs. The purpose of this review is to perform a SWOT analysis that includes a discussion of: (i) strengths of ATPS partitioning as an effective and simple platform for biomolecule purification; (ii) weaknesses of ATPS partitioning in regard to intrinsic problems and possible solutions; (iii) opportunities related to biotechnological challenges that ATPS partitioning may solve; and (iv) threats related to alternative techniques that may compete with ATPS in performance, economic benefits, scale up and reliability. This approach provides insight into the current status of ATPS as a bioprocessing technique and it can be concluded that most of the perceived weakness towards industrial implementation have now been largely overcome, thus paving the way for opportunities in fermentation feed clarification, integration in multi-stage operations and in single-step purification processes.

  • 4. Torto, N.
    et al.
    Ohlrogge, M.
    Gorton, L.
    Van Alstine, James M.
    KTH, Superseded Departments, Chemical Engineering and Technology.
    Laurell, T
    Marko-Varga, G
    In situ poly(ethylene imine) coating of hollow fiber membranes used for microdialysis sampling2004In: Pure and Applied Chemistry, ISSN 0033-4545, E-ISSN 1365-3075, Vol. 76, no 4, p. 879-888Article in journal (Refereed)
    Abstract [en]

    A method for the in situ modification of hollow fiber membranes used as sampling units for microdialysis probes is presented. The method consists of adsorption-coating, high-molecular-weight poly(ethylene imine), PEI, onto membranes, already fitted on microdialysis probes. Modification of membranes was designed to specifically explore the so-called Andrade effects and thus enhance the interaction of membranes with enzyme. The performances of polysulfone, polyethersulfone, and polyamide membranes modified with PEI-enzyrne complex were evaluated based on the membrane extraction fraction for maltose and maltotriose and membrane morphology as examined by scanning electron microscopy. Of the membranes tested, the PEI-enzyme complex least affected the performance of the polysulfone membranes. Conversion of maltoheptaose to maltotriose and maltose was increased reproducibly (within a 5 % relative standard deviation) by 50 % for modified membranes compared to the native hollow fiber membranes. The results demonstrate the potential to effectively modify membranes already fitted on a microdialysis probe. Such a procedure can be modified and employed to either promote or reduce membrane-protein interaction for hollow fibers used as microdialysis sampling units or other similar membrane applications.

  • 5.
    Van Alstine, J. M.
    et al.
    KTH.
    Jagschies, G.
    Łacki, K.M.
    Alternative Separation Methods: Crystallization and Aqueous Polymer Two-Phase Extraction2018In: Biopharmaceutical Processing: Development, Design, and Implementation of Manufacturing Processes, Elsevier , 2018, p. 241-267Chapter in book (Other academic)
    Abstract [en]

    As with many other alternative separations methods, crystallization and aqueous polymer two-phase extraction (ATPE) have been well-studied academically and are amenable to predictive and process modeling. Their solution nature allows them to be optimized using high-throughput microtiter plate-based methodologies. Their liquid nature allows them to be readily scaled and, in comparison with some other separation methods, they do not require overt energy use or capital investment. Crystallization is a well-established industrial method whose application for purifying bio-macromolecules presents interesting challenges. It typically requires reasonably concentrated and quite pure target-containing solutions and its operation can require days. The resulting crystals can be very pure, and in some cases, may be suitable for use as drug product, drug storage, or even as drug-delivery colloids. ATPE can rapidly effect preliminary clarification, target concentration, and some reduction in host cell proteins, toxins, nucleic acids, and other contaminants. As noted in previous chapters, and discussed in more detail in this chapter, it is perhaps best employed upstream of other selective operations that might include chromatography or crystallization.

  • 6.
    Van Alstine, J. M.
    et al.
    KTH.
    Jagschies, G.
    Łacki, K.M.
    Alternative Separation Methods: Flocculation and Precipitation2018In: Biopharmaceutical Processing: Development, Design, and Implementation of Manufacturing Processes, Elsevier , 2018, p. 221-239Chapter in book (Other academic)
    Abstract [en]

    The most common alternative separation methods used in bioprocessing are flocculation and precipitation. They are proven, readily scaled, do not require overt energy input or capital investment, and can offer significant selectivity. They have the ability to handle modern challenges such as high-density feeds that tax the limits of industry "workhorse" methods such as centrifugation, filtration, and chromatography. They appear particularly well suited to be used in the initial stages of bioprocessing, either in target-capture or contaminant-removal. Such methods are compatible with modern high-throughput screening of operating conditions, as well as single-use equipment for use in batch or continuous processes. This chapter provides more insight into a discussion of flocculation and precipitation, initiated in the preceding chapter.

  • 7.
    Van Alstine, J. M.
    et al.
    KTH.
    Jagschies, G.
    Łacki, K.M.
    Overview of Alternative Separation Methods in Relation to Process Challenges2018In: Biopharmaceutical Processing: Development, Design, and Implementation of Manufacturing Processes, Elsevier , 2018, p. 207-220Chapter in book (Other academic)
    Abstract [en]

    This chapter provides an overview of alternative separation methods in order to help the reader decide if such methods are of interest, how they compare with other methods, and what particular approaches may suit specific process challenges. Alternative separation methods are enjoying a resurgence of interest for use in bioprocessing as they have the ability to handle various challenges that tax the limits of industry "workhorse" methods such as centrifugation, filtration, and chromatography. They are proven, readily scaled, and do not require significant energy input or capital investment. Although they typically involve a single separation stage, alternative separation methods can still offer effective separations. They are well-suited to be used in the initial stages of bioprocessing, either in target-capture or contaminant-removal modes. In addition, they are compatible with modern high-throughput screening of operating conditions, as well as single-use equipment for batch or continuous processing. This chapter presents a survey of a wide range of alternative separation methods and key references. The next two chapters provide more details, including some cost of goods estimation references, in regard to the popular methods of flocculation and precipitation (Chapter 11) as well as two methods of growing interest; crystallization, and liquid-liquid aqueous polymer two-phase extraction (Chapter 12).

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