[23] Large-scale purification of recombinant human leukocyte interferons

Tarnowski, S.Joseph; Roy, Swapan Kumar; Liptak, R.A.; Lee, David K.; Ning, Robert Y.

doi:10.1016/0076-6879(86)19025-2

Cited by 25 publications

(7 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…High purity is required for pharmaceutical proteins, placing a premium on methods that can achieve significant purification in a single step. The high specificity and affinity of antibodies make immunoaffinity separations natural choices for such products, and immunoadsorption has been chosen as the central purification step for a number of pharmaceutical products (Tarnowski et al, 1986).…”

Section: Nonchemical Elution Techniques Temperature Elution Pressure mentioning

confidence: 99%

“…There is considerably more potential for reducing costs by increasing the useful life of the immunoadsorbent, whose binding capacity declines with repeated use (Eveleigh and Levy, 1977). Some anecdotal reports describe immunoadsorption column uses in the hundreds (Tarnowski et al, 1986; Vetterlein and Calton, 19831, but a more systematic study has shown that significant deactivation can typically take place over 40-100 cycles function by modifying the strength of the interaction between antigen and antibody or, more relevant to the elution process, by raising the dissociation rate of the antigen-antibody complex.…”

Section: Nonchemical Elution Techniques Temperature Elution Pressure mentioning

confidence: 99%

See 1 more Smart Citation

Immunoadsorption: Strategies for Antigen Elution and Production of Reusable Adsorbents

Yarmush

Antonsen

Sundaram

et al. 1992

Biotechnology Progress

View full text Add to dashboard Cite

Immunoadsorption is a powerful and generalizable method for protein purification that exploits the fine specificity of antigen-antibody interactions. In spite of its potential utility, the more widespread process scale use of immunoadsorption has been limited by the high cost of the antibody and the lack of gentle elution schemes that completely preserve the activity of both the immunoadsorbent and the eluted product. In this report, we review common chemical elution strategies such as pH, ionic strength, chaotropic salts, denaturants, and organic solvents as well as physical techniques such as pressure, electrokinetics, and temperature. In general, selection of elution strategies has largely been an empirical art, balancing stability of the immunoadsorbent and the eluted product and efficiency. The limitations of the available choices demonstrate that more attention must be placed on the antibody. Techniques which assist in the identification or creation of new antibodies with improved binding properties and resistance to degradation, e.g., screening and/or rational protein engineering, are also discussed.

show abstract

Section: Nonchemical Elution Techniques Temperature Elution Pressure mentioning

confidence: 99%

Section: Nonchemical Elution Techniques Temperature Elution Pressure mentioning

confidence: 99%

Immunoadsorption: Strategies for Antigen Elution and Production of Reusable Adsorbents

Yarmush

Antonsen

Sundaram

et al. 1992

Biotechnology Progress

View full text Add to dashboard Cite

show abstract

“…The SMM form of the molecule contains free sulfhydryl groups and has the potential to form disulfide-bonded oligomers. The FMM that lacks free sulfhydryl groups apparently contains two intramolecular disulfide bonds (7). Figure 4A shows the nonreducing SDS-PAGE analysis of the recombinant IFN-␣ preparation along with several standard proteins that serve as molecular weight markers.…”

Section: Characterization Of Rh-ifn-␣mentioning

confidence: 99%

“…The relative proportions of monomers, dimers, and oligomers in an IFN-␣ preparation can be analyzed by performing SDS-PAGE in the absence of ␤-mercaptoethanol (nonreducing SDS-PAGE). Two different monomeric forms can be distinguished under nonreducing conditions, namely slow-moving monomer (SMM) and fast-moving monomer (FMM) (7). The SMM form of the molecule contains free sulfhydryl groups and has the potential to form disulfide-bonded oligomers.…”

Section: Characterization Of Rh-ifn-␣mentioning

confidence: 99%

“…A recent protocol for the purification of IFN-␣ employs sequential chromatography on metal-chelation, cation-exchange, anion-exchange, and gel-filtration columns (9). Some protocols replace the metal-chelation step and use immunoaffinity chromatography in combination with cation exchange and gel filtration (7). Such multistep procedures are prone to product losses that grow geometrically with each step, resulting in reduced overall yields.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Affinity Purification of Recombinant Interferon-α on a Mimetic Ligand Adsorbent

Swaminathan

Khanna

1999

Protein Expression and Purification

View full text Add to dashboard Cite

Effect of antibody orientation on immunosorbent performance

Subramanian

Velander

1996

J. Mol. Recognit.

View full text Add to dashboard Cite

The impact of antibody orientation on immunosorbent efficiency was quantitatively assessed. A pH-dependent murine monoclonal antibody (Mab) against human protein C (hPC), recombinant hPC (rhPC) and two different immobilization chemistries and matrices were used as model systems. The lysyl groups of the rhPC were covalently modified with an acetic acid ester of N-hydroxysuccinimide and this modified rhPC was used as a Fab masking agent (FMA). The FMA was used to mask the antigen binding regions (Fab) of the Mab prior to and during covalent immobilization. Thereafter, the residual active sites of the support were inactivated and the FMA was removed. Mab was immobilizeed at low bead-averaged densities of about 0.4-1.1 mg Mab/mL matrix to minimize local density effects. Immunosorbents made using masked Mab (oriented coupling) gave antigen binding efficiencies (nAg) of 42-48% compared with 18-22% for those made by random coupling. The amount of (Fab)2 released from pepsin digestion of immunosorbents was about 3-4-fold higher for matrices having been made with FMA-masked Mab relative to unmasked Mab. Thus, the (Fab)2 accessibility to pepsin correlates well with higher functional efficiency (nAg) and serves as a measure of orientation. In summary, at low Mab density and a 2:1 molar rhPC to Mab binding stoichiometry, about 80% or more of the Mab randomly coupled through amino moieties was improperly oriented relative to oriented coupled Mab, which correlated with about 50% of lost Mab functionality upon immobilization.

show abstract

[23] Large-scale purification of recombinant human leukocyte interferons

Cited by 25 publications

References 9 publications

Immunoadsorption: Strategies for Antigen Elution and Production of Reusable Adsorbents

Immunoadsorption: Strategies for Antigen Elution and Production of Reusable Adsorbents

Affinity Purification of Recombinant Interferon-α on a Mimetic Ligand Adsorbent

Effect of antibody orientation on immunosorbent performance

Contact Info

Product

Resources

About