Novel affinity resins enabling the purification of next generation antibody fragments: KANEKA KanCap™ G and KanCap™ L


A guest article by Yuji Okubo, Director, Pharmaceutical Unit, Kaneka US Innovation Center and Harkewal Singh, Manager- Pharmaceutical Unit, Kaneka US Innovation Center

Ever since the licensing of first monoclonal antibody (mAb) drug, biologics have seen unprecedented growth as drugs to treat a variety of malignancies1. The modular structure of mAbs have allowed protein engineers to create smaller, nimbler, and multispecific next-generation antibody therapeutics. Some of these modalities can be easily produced using microbial expression systems, offering higher yields and excellent process economy2.

Smaller sized next-generation modalities can have easy access to difficult targets and often elicit reduced immunogenicity. Additionally, they have been shown to lack bystander effect and makes it possible to target multiple epitopes3. Fabs, diabodies (dAb), single chain Fragment variable (scFv), bispecific scFv (Bis-scFv), ScFv-Fab, Fc modified full IgG, Dual-affinity Re-targeting Antibody (DART) are some popular examples of next-generation antibodies3. However, the purification of the next generation modalities that lack or have modified Fc domain is not straightforward since it precludes efficient binding to most existing protein A resins.

To address this need, Kaneka has developed two cellulose-based affinity resins: KANEKA KanCap G (KanCap G) and KANEKA KanCap L (KanCap L). KanCap G is developed using modified Protein G ligand and KanCap L consists of our proprietary Protein L ligand.

Specifically, KanCap G binds to the CH1 domain of Fabs and thus an excellent resource to purify all types of human Fab (k and l type). Whereas, KanCap L binds to the VL domain (only for k types) of the antibody and therefore geared towards the purification of Fab, scFv, and diabody like modalities. Figure 1, provides a general description of antibody fragments that can be purified by KanCap G and KanCap L.

Structure of a full-length Immunoglobulin (IgG) showing various domains. KANEKA KanCap™ G
Figure 1. Structure of a full-length Immunoglobulin (IgG) showing various domains. KANEKA KanCap™ G targets CH1 domain while KanCap™ L focuses on VL domain of IgGs. Examples of target purification modalities by KanCap™ G and KanCap™ L are shown in respective boxes.

KANEKA KanCap G

KanCap G is a newly developed cellulose-based affinity resin for the purification of Fab, F(ab’)2, Bis-Fab, Fab-scFv etc. Its proprietary Protein G ligand exhibits an increased binding affinity to the CH1 domain, thereby making KanCap G a powerful tool for efficient capture and purification of antibody formats containing CH1 domain. It can also be used as an alternative tool for full length antibody purification.

Performance

To test the comparative performance of KanCap G, three Fab molecules (k and l type) were purified using KanCap G and existing products in the market. Our data shows that KanCap G offers best in class dynamic binding capacity and outperforms existing protein G resins (Figure 2). It is noteworthy that purification of different Fab modalities using KanCap G also results in high level of protein purity as adjudged by SDS-PAGE (Figure 3)

Dynamic Binding Capacity of KanCapTM G as compared to other protein G ligand resins (SpG agarose (A), (B) and CH1 specific agarose resin
Figure 2. Dynamic Binding Capacity of KanCap™ G as compared to other protein G ligand resins (SpG agarose (A), (B) and CH1 specific agarose resin

 

SDS-PAGE analysis of Fab purification samples using KanCapTM G under non-reduced and reduced conditions
Figure 3. SDS-PAGE analysis of Fab purification samples using KanCap™ G under non-reduced and reduced conditions. Lane M, Marker; Lane 1, Fab in E. coli lysate*1 (left), Yeast Supernatant*2 (right); Lane 2, Flow through; Lane 3, Wash; Lane 4, Elution *1 Mixture of purified polyclonal Fab (containing λ light chain) and E. coli lysate (cytoplasmic fraction) *2 Yeast Supernatant including expressed monoclonal Fab (containing κ light chain)

KANEKA KanCap L

KANEKA KanCap™ L is a novel cellulose-based Protein L resin that exhibits an increased affinity to VL region of antibody κ light chain. This resin is designed for capture and purification of antibody fragments such as Fab, scFv, diabody as well as an alternative tool to purify full-length mAbs containing a κ light chain that poorly binds to Protein A resins.

Performance

KanCap L’s performance was compared to existing protein L resin by purifying two Fab modalities and one diabody. KanCap L also offers best in class binding capacity and outperforms competitor’s agarose-based protein L resin (PpL) (Figure 4 & 5).

Dynamic Binding Capacity of KanCapTM L as compared to PpL agarose resin
Figure 4. Dynamic Binding Capacity of KanCap™ L as compared to PpL agarose resin

 

SDS-PAGE analysis of Fab purification from E. coli lysate* using KanCapTM L under non-reduced and reduced conditions
Figure 5. SDS-PAGE analysis of Fab purification from E. coli lysate* using KanCap™ L under non-reduced and reduced conditions. Lane M, Marker; Lane 1, Fab in E. coli lysate-Fab (1); Lane 2, Flow through; Lane 3, Wash; Lane 4, Elution. * Mixture of purified monoclonal Fab (containing k light chain) and E. coli lysate (cytoplasmic fraction).

Summary

The rapid development of mAbs have benefited from the existence of protein A based platform resins. However, the development and purification of next-generation antibodies lacking Fc domain is complex. Kaneka’s KanCap G and KanCap L affinity resins address this challenge. As shown above, KanCap G exhibits higher binding capacity and aids in the recovery of all types of human Fabs (k and l type). Whereas KanCap L offers wide binding spectra and excellent binding capacity for k light chain containing antibody modalities

In summary, to address the downstream processing challenges, KANEKA has developed a robust toolset of cellulose-based affinity resins. As shown below, our protein A resins (KanCapA and KanCapA 3G) are designed to bind molecules containing Fc domain. The unique features of this class of resins include high dynamic binding capacity, milder pH elution, and excellent ability to resolve impurity contents (aggregates, HCP, DNA etc). These resins can be used to purify full-length mAbs and modalities that have Fc domain.

Our KanCap G and KanCap L fulfill a critical gap in addressing the purification challenges associated with the discovery and development of next-generation antibody modalities of significant medical importance.

Affinity Chromatography Toolbox from KANEKA
Affinity Chromatography Toolbox from KANEKA

References

  • Ecker, D.M.; Jones, S.D.; Levine, H.L. MAbs 2015, 7
  • Fernandes, J.C. Drug Discovery. Today 2018, 23, 1996–2002
  • Bates A, Power CA. Antibodies (Basel). 2019 Apr 9;8(2). pii: E28. doi: 10.3390/antib80200

About Kaneka: Kaneka is an innovation-driven chemical company responding to customer needs with world-class science and technology. Kaneka’s business activities span a broad spectrum and include Chemicals, Functional Plastics, Expandable Products, Foodstuffs Products, Electronic Products, Synthetic Fibers, and Life Science Products (Food Supplements, Medical Devices, Pharmaceutical Intermediates, Protein A, G and L resins).

Kaneka Corporation is headquartered in Osaka-Japan, with subsidiaries in the United States, Belgium, Singapore, Malaysia, China, Australia, Vietnam, India, South Korea, and Taiwan, and employs more than 9300 people worldwide.

For more information, please visit: http://www.bioseparation.kaneka.com/


Authors

Yuji OkuboYuji Okubo,
Director, Pharmaceutical Unit, Kaneka US Innovation Center
He received a Master degree in 1986 (Biochemistry) from Kyoto University. Later he joined Kaneka and worked on broad range of Kaneka’s biotechnology  field include affinity chromatography.  He transferred to US in 2016  and since then he is working in marketing, alliance and open innovation activities.


Harkewal SinghHarkewal Singh
Manager- Pharmaceutical Unit, Kaneka US Innovation Center
Harkewal received his PhD in 2011 from University of Missouri-Columbia in the field of Structural Biology and Protein Biochemistry. Prior to joining Kaneka, Harkewal worked as Senior Scientist with MilliporeSigma in the field of Protein Technologies and later with Beckman Coulter’s life science research.

He joined Kaneka in March 2018 where he manages affinity chromatography products and leads technology commercialization efforts for the life science division.


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