Development of a novel affinity chromatography resin for platform purification of lambda fabs

Eifler, Nora; Medaglia, Giovanni; Anderka, Oliver; Laurin, Linus; Hermans, Pim

doi:10.1002/btpr.1958

Cited by 29 publications

(16 citation statements)

References 31 publications

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“…Approximately 60% of mammalian IgG light chains are kappa chains, with the remaining 40% being lambda chains that lack binding sites for Protein L [73,98]. For Ab-fragments containing the lambda light chain, Camelidprotein-based [79] affinity resins may be employed [19].…”

Section: Protein L and Its Use In Antibody Fragment Bioprocessingmentioning

confidence: 99%

“…In 2010 those three products had total sales of over $3.5 billion (USD) [7]. Many more Ab-fragments are under development [3,[8][9][10][11][12][13][14][15][16][17][18][19][20].…”

Section: Introductionmentioning

confidence: 99%

“…Yet, while robust methods to purify mAbs at relatively large (many Kg) scale are well established [16], there are still challenges associated with the production and purification of antibody fragments [8,[17][18][19][20]. Downstream processing presently constitutes a roadblock to delivering clinically efficacious Ab-fragments at effective unit costs.…”

Section: Introductionmentioning

confidence: 99%

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Antibody Fragments and Their Purification by Protein L Affinity Chromatography

Rodrigo¹,

Gruvegard²,

Alstine

2015

Antibodies

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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 criteria-suggesting its consideration as a key unit operation in antibody fragment processing.

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Section: Protein L and Its Use In Antibody Fragment Bioprocessingmentioning

confidence: 99%

“…In 2010 those three products had total sales of over $3.5 billion (USD) [7]. Many more Ab-fragments are under development [3,[8][9][10][11][12][13][14][15][16][17][18][19][20].…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Antibody Fragments and Their Purification by Protein L Affinity Chromatography

Rodrigo¹,

Gruvegard²,

Alstine

2015

Antibodies

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“…Affinity matrices such as LambdaFabSelect (LFS) and KappaSelect can then be used to selectively capture asymmetric bispecific antibodies that contain at least one light chain within the target subfamily. 26,27 …”

Section: Introductionmentioning

confidence: 99%

A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture

Wang¹,

Vemulapalli²,

Cao³

et al. 2018

mAbs

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Immunoglobulin G–like bispecific antibodies with asymmetric architecture are among the most widely used bispecific antibody formats for diagnostic and therapeutic applications. The primary technical challenge for this format is how to achieve correctly paired assembly of four unique polypeptide chains. Advances in protein engineering and process development are being used to overcome these challenges and are driving a corresponding demand for sensitive analytical tools to monitor and control mispaired species. Here, we report a systematic approach for analysis and characterization of mispairing in asymmetric bispecific antibodies. This approach consists of three orthogonal components, the first of which is a liquid chromatography (LC)-mass spectrometry (MS)–based method to measure the mass of intact antibodies. This method is used for fast analysis of mispairing and requires minimal method development, which makes it an ideal choice for early-stage development. The second component is a hydrophobic interaction chromatography (HIC)–based mispairing method that is suitable for lot release testing. The HIC method is robust and quality control friendly, and offers great linearity, precision, and accuracy. The third component is a two-dimensional LC-MS method for on-line chromatographic peak identification, which not only expedites this task but also reduces the risk of undesirable modifications during conventional fraction collection. These three methods dovetail to form the foundation of a complementary toolbox for analysis and characterization of mispairing in asymmetric bispecific antibodies and provide guidance and support for process development throughout the drug development life cycle.

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“…Thus, the physical and thermal stability, or better, the capacity of VHHs to refold completely after denaturation enables the apparent infinite reusability of the columns15. These characteristics explained that these binding domains have already been successfully applied as affinity ligands in chromatography processes covering a variety of biotherapeutics16171819, can serve as target-specific capture reagents20, and can replace the conventional and expensive protein A purification step2122.…”

mentioning

confidence: 99%

Dual anti-idiotypic purification of a novel, native-format biparatopic anti-MET antibody with improved in vitro and in vivo efficacy

Godar

Morello

Sadi³

et al. 2016

Sci Rep

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Bispecific antibodies are of great interest due to their ability to simultaneously bind and engage different antigens or epitopes. Nevertheless, it remains a challenge to assemble, produce and/or purify them. Here we present an innovative dual anti-idiotypic purification process, which provides pure bispecific antibodies with native immunoglobulin format. Using this approach, a biparatopic IgG1 antibody targeting two distinct, HGF-competing, non-overlapping epitopes on the extracellular region of the MET receptor, was purified with camelid single-domain antibody fragments that bind specifically to the correct heavy chain/light chain pairings of each arm. The purity and functionality of the anti-MET biparatopic antibody was then confirmed by mass spectrometry and binding experiments, demonstrating its ability to simultaneously target the two epitopes recognized by the parental monoclonal antibodies. The improved MET-inhibitory activity of the biparatopic antibody compared to the parental monoclonal antibodies, was finally corroborated in cell-based assays and more importantly in a tumor xenograft mouse model. In conclusion, this approach is fast and specific, broadly applicable and results in the isolation of a pure, novel and native-format anti-MET biparatopic antibody that shows superior biological activity over the parental monospecific antibodies both in vitro and in vivo.

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Development of a novel affinity chromatography resin for platform purification of lambda fabs

Cited by 29 publications

References 31 publications

Antibody Fragments and Their Purification by Protein L Affinity Chromatography

Antibody Fragments and Their Purification by Protein L Affinity Chromatography

A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture

Dual anti-idiotypic purification of a novel, native-format biparatopic anti-MET antibody with improved in vitro and in vivo efficacy

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