APPLICATION NOTE 9

CaptureSelect affinity ligands for antibody detection and characterization

Pim Hermans, Gerwin Grit and Sven Blokland, BAC BV

Introduction Antibody purification BAC’s CaptureSelect affinity ligands provide a unique group Using Protein A, Protein G, and Protein L for antibody purifi- of molecules that can be designed for almost any purification cation can be highly effective. However, there are gaps in the target, with as narrow or as broad specificity as is required. The functionality that they provide, and when a target protein falls fragments are derived from the unique heavy chain antibod- into one of these gaps the process of finding an alternative puri- ies found in Camelidae. Heavy chain antibodies are devoid of fication strategy can be extremely frustrating. The main binding the entire light chain and CH1 domain found in conventional characteristics of Proteins A, G, and L are listed below: antibodies (Figure 1). Without the light chain, antigens are bound only by the variable domain of the heavy chain (VHH), without a PROTEIN A AND G loss in binding affinities compared to conventional antibodies. • Bind to IgG Fc domain (CH2-CH3 interface) The VHH domain is the smallest functional antigen-binding • Bind IgG of many species including bovine and mouse (not fragment known that shows high affinity and stability. The equiv- human specific) alent fragment from conventional antibodies is the Fv domain, • Protein A binds human IgG1, 2 and 4 but not IgG3; Protein G which consists of the heavy chain variable domain and the light binds all sub-types chain variable domain held together by a non-covalent bond. If this bond is broken, all antigen binding functionality will be lost. Because the VHH domain exists as a single polypeptide chain, PROTEIN L it is an extremely stable and easy to produce antibody fragment • Binds to VL domain of an antibody light chain that retains the full antigen binding activity of the parent heavy • Binds human VL-kappa 1, 3 and 4 but not to VL-kappa 2 nor to chain antibody. any VL- lambda During the screening process, VHH molecules that meet very • Binds mouse VL-kappa 1 specific requirements for specificity, affinity, elution profile and cleaning conditions are selected. In this way VHH affinity ligands can be created to match exactly the processes they are lgG Camelid lg intended for. VHH fragments have been used successfully as CH1 affinity ligands in chromatography processes for the purification of many types of protein, including antibodies,1-4, 6, 9 blood factors5 CL and adeno-associated viruses7, 8, 10 among others. For more CH2 CH2 information on CaptureSelect affinity ligands, please visit www. captureselect.com. CH3 CH3

VH domain VL domain VHH

Figure 1: Comparison of conventional IgG structure versus Camelid heavy chain antibody.

1 Since more and more antibody-based therapeutics, (e.g., Fab analytical tools for antibody detection and characterization, fragments) lack a regular Protein A or G binding site, it becomes a set of CaptureSelect affinity ligands has been chemically a challenge to find a suitable equivalent for primary capture of biotinylated for use in capture assay formats incorporating antibody formats. For this reason, BAC has developed a range streptavidin-coated surfaces (as used in ELISA, SPR and BLI). of CaptureSelect affinity ligands directed against a unique panel ForteBio’s Streptavidin (SA) biosensors demonstrated excel- of antibody sub-domains, providing an antibody toolbox for re- lent compatibility with the biotinylated CaptureSelect affinity searchers and manufacturers, facilitating affinity purification and ligands, enabling a whole range of novel selectivities for anti- detection of virtually any antibody-based format. A schematic body analytics on the Octet platform. representation of an IgG antibody and the specific domains Several CaptureSelect biotin conjugates have been studied in that are targeted by various CaptureSelect affinity ligands is detail using an Octet system and the results are described. presented in Figure 2.

Besides antibody purification at both a research and biopro- BINDING SELECTIVITY OF CAPTURESELECT cess scale, analytical applications such as online monitoring of BIOTIN CONJUGATES antibody expression and/or analyzing product yield and purity during downstream processing, are facilitated by incorpo- The following biotin conjugated affinity ligands were used in rating CaptureSelect affinity ligands onto HPLC-compatible this study: backbones (e.g., POROS). To further broaden the range of Biotin anti-IgG-Fc (Hu) • Binds to CH3 domain of human BAC Cat. No. 710.0822.100 IgG-Fc • Binds all 4 human IgG subclasses Antibody Fc domains CaptureSelect affinity ligands • Human-specific (Hu) Biotin anti-LC-kappa (Hu) • Binds to constant domain of hu- VH BAC Cat. No. 710.0833.100 man kappa light chains (LC) CH-1 Human IgG-Fc • Human-specific (Hu) • Binds all subclasses, human-spe- cific Biotin anti-LC-lambda (Hu) • Binds to constant domain of • Human IgG subclass-specific: IgG1, BAC Cat. No. 710.3080.100 human lambda light chains CH-2 IgG3 and IgG4 • Human-specific (Hu) • Human isotupe specific: IgM, IgA CH-3 Biotin anti-IgG-CH1 • Binds to CH1 domain of human multi-species IgG-Fc (in development) IgG • Binds all 4 human IgG subclasses No binding to bovine, mouse and Antibody light chains (LC) CaptureSelect affinity ligands • rat IgG • Cross-binds with dog and cat IgG VL Human LC-kappa and LC‑lambda In all experiments, the biotin conjugates were loaded onto CL • Human-specific Streptavidin (SA) biosensors using an Octet system. After load- ing, a baseline was established by incubating the biosensors for • Binds all human Ig antibodies 2.5 minutes in PBS buffer prior to antigen incubation. All binding (e.g., IgG, IgM, IgA, IgD, IgE) studies were performed in kinetic mode, allowing further analy- • Binds human Fab fragments sis of kinetic parameters.

In a first experiment, the binding selectivity of the different Antibody IgG CH1 CaptureSelect affinity ligands CaptureSelect affinity ligands was analyzed using a broad panel of monoclonal and polyclonal antibodies including different human antibody isotypes, IgG subclasses and antibody frag- VH CH-1 IgG-CH1 ments. ForteBio’s Protein L biosensors were also included in • Binds all human IgG subclasses this study. • Covers all human IgG Fab frag- As shown in Table 1, the binding selectivity of each Capture- ments Select affinity ligand correlated well with the binding profiles CH-2 CH -2 • Binding independent of type of observed on the Octet system. Biotin anti-IgG-Fc only showed light chain CH-3 binding reactivity towards human IgG antibodies (all subclasses) CH -3 • No binding to free light chains and IgG-Fc but not to any other species tested. No cross-binding towards bovine and mouse IgG was observed, confirming its selectivity for human IgG only. The same pattern was found for Figure 2: Overview of different antibody domains targeted by a variety of Cap- tureSelect affinity ligands. biotin anti-IgG-CH1, except that it showed reactivity for human

2 IgG-derived Fab fragments and not for Fc. Both anti-LC-kappa were from Jackson Immunochemistry; Human IgD and IgE and anti-LC-lambda conjugates clearly demonstrated explicit from Calbiochem; free human kappa and lambda light chains selectivity for either LC-kappa or LC-lambda containing antibody (Bence Jones) were purchased from BioDesign. formats, respectively. Besides binding to light chains present in intact antibodies, reactivity was observed for free human light VARIANCE IN BINDING REACTIVITY OF BIOTIN chains as well (Bence Jones). The Protein L biosensors also ANTI-LC-KAPPA VERSUS PROTEIN L FOR A RANGE showed the expected binding profiles, however, for one human OF ANTIBODY FORMATS IgG1 kappa monoclonal antibody (Mab) no significant binding was observed. It should be noted that Protein L is restricted to specif- For this purpose, additional human LC-kappa containing anti- ic subclasses of the human VL domain (i.e., VL-kappa 1, 3 and 4) body formats were tested for binding to biotin anti-LC-kappa and and does not bind to the human VL-kappa 2 subclass. The tested Protein L biosensors. As shown in Figure 3, a higher variance human IgG1 Mab may have comprised a VL-kappa 2 domain, in binding reactivity can be observed for Protein L compared to explaining the lack of binding to Protein L. In this respect, both biotin anti-LC-kappa. Although good reactivity was found for iso- anti-LC-kappa and anti-LC-lambda affinity ligands are directed types IgM, A, D and E, only two out of four LC-kappa containing against the constant domain of a human light chain (CL) instead IgG Mabs showed significant binding with Protein L. This again of the variable domain (VL). Since the amino acid sequence could be due to the presence of a VL kappa 2 domain within the of the constant domains is more conserved than for VL, less set of antibodies tested. However, very low binding signals were variance can be expected in the binding of different antibody observed for human IgG Fab fragments as well (both polyclonal formats compared to Protein L. and recombinant). Although a Fab fragment is only one third of the size of an IgG molecule, one would have expected more or The CaptureSelect biotin conjugates were loaded onto less comparable binding signals with the biotin anti-LC-kappa Streptavidin (SA) biosensors at a concentration of 5 μg/mL in biosensors. On the other hand, both the free kappa light chain 200 μL PBS for 10 minutes (biotin anti-IgG-CH1 was loaded samples (Bence Jones and recombinant) did show proper bind- at 10 μg/mL) using a shake speed of 1000 rpm followed by ing with Protein L. In this respect, results obtained with the biotin a washing step in PBS for 2.5 minutes to set a baseline. All anti-LC-kappa biosensors revealed a more consistent binding antibody target samples (10 μg/mL in PBS) were then allowed profile, showing less variance in binding within the broad panel of to bind to the functionalized biosensors (including Protein L LC-kappa containing antibody formats tested (as also observed biosensors) for 10 minutes, followed by a 10-minute dissocia- for biotin anti-LC-lambda). Results indicate that affinity ligands tion step in PBS. All human IgG monoclonal antibodies were that target the constant domain of human light chains provide a purchased from Sigma; Human IgM and IgA, polyclonal human more generic tool for detection and characterization of antibody IgG Fab and Fc fragments, polyclonal bovine- and mouse IgG formats compared to those such as Protein L, which target the variable domain.

CaptureSelect biotin conjugates Biotin anti-IgG- Biotin anti- Biotin anti-LC- Biotin anti-LC- Antibody target Isotype/subclass Fc (Hu) IgG-CH1 kappa (Hu) lambda (Hu) Protein L

Hu IgG-1 kappa ü ü ü − −

Hu IgG-2 lambda ü ü − ü − IgG subclasses Hu IgG-3 kappa ü ü ü − ü

Hu IgG-4 lambda ü ü − ü −

Hu IgM kappa − − ü − ü

Hu IgD kappa − − ü − ü Ab isotypes Hu IgE kappa − − ü − ü

Hu IgA (poly) − − ü ü ü

Hu IgG Fab (poly) − ü ü ü ü

Hu IgG Fc (poly) ü − − − − Ab fragments Free Hu LC kappa − − ü − ü

Free Hu LC lambda − − − ü −

Bovine IgG − − − − − Species Mouse IgG − − − − ü

Table 1: Binding selectivity of CaptureSelect Biotin conjugates.

3 100 Biotin anti-LC-kappa Protein-L

) 80 % (

l a n g i S 60 g n i d n i B

e 40 v i t a l e R 20

0 . ) s ) ) ) ) a a a a a a y a c a y a a y y l e l l l S p d p p d p p e p p p o r n o B p b p p b p p p p p a a a a p a o a p a a po po P m m ( a ( ( ( ( k k k k k J k k k a a p . r 1 l l b A G G 2 3 4 b p E e a B M g D 2 4 a a I g g ‰ G F a g g Ig I I G G G F k I I u g p u I G g g G g 1 e e B I I I G p u u u s g g C H in u I I g G a H H u u u u I L H v k H u u Ig o H H H u u o H H h C B M H h r L

ee u r h f

ee r f Figure 3: Variance in binding reactivity towards a broad panel of kappa light chain containing anti- body formats analyzed on biosensors immobilized with biotin anti-LC-kappa versus Protein L.

Recombinant Human IgG1 Kappa Antibody: Whole Molecule (150 kDa) Recombinant Human IgG1 Kappa Antibody: Fab Fragment (50 kDa)

CaptureSelect: Biotin anti-LC-kappa (Hu) CaptureSelect: Biotin anti-LC-kappa (Hu)

2.40 1.60 2.00

1.60 1.20

1.20 ka = 9.61E+04 0.80 k = 4.29E+04 kd = 7.29E-06 a Fitting View Fitting View K =75.9 pM kd = 1.17E-04 0.80 D KD = 2.73 nM 0.40 0.40

0 0 0 100 200 300 400 500 600 700 800 900 0 200 400 600 800 1,000 1,200 Time (sec) Time (sec)

CaptureSelect: Biotin anti-IgG-CH1 CaptureSelect: Biotin anti-IgG-CH1 0.50 1.00 ka = 2.31E+04 kd = 8.05E-04 K =34.9 nM 0.40 D 0.80

ka = 8.22E+04 0.30 0.60 kd = 2.64E-05

KD = 0.32 nM 0.40 0.20 Fitting View Fitting View

0.20 0.10

0 0 0 200 400 600 800 1,000 1,200 0 200 400 600 800 1,000 1,200 Time (sec) Time (sec)

CaptureSelect: Biotin anti-IgG-Fc (Hu)

1.60 Figure 4: Dose response curves of a recombinant human IgG1 kappa antibody and its corresponding Fab fragment on different antibody capturing biosensors.

1.20 ka = 1.84E+05 The CaptureSelect biotin conjugates were loaded onto Streptavidin (SA) bio- kd = 5.01E-04 sensors as previously described. A dilution series of a recombinant human IgG1 K = 2.73 nM D kappa monoclonal antibody (whole molecule and Fab fragments thereof) were 0.80 allowed to bind for 10 minutes, followed by a 10-minute dissociation step (kinetic

Fitting View mode). Binding curves are displayed for each of the biosensors tested. Using the Octet data analysis software, kinetic data were calculated. 0.40

0 0 100 200 300 400 500 600 700 800 900 Time (sec)

4 Binding analysis was performed as previously described. The capture biosensors is key. When capturing human IgG antibod- residual binding signals after 10 minutes of dissociation were ies, the previous results show that a stable complex can be determined for the whole panel of antibody samples tested. For achieved using biotin anti-LC-kappa and anti-IgG-CH1 biosensors both biotin anti-LC-kappa (Hu) and Protein L the highest binding (based on bivalent binding). For biotin anti-LC-kappa for instance, signals were observed for the human IgG3 kappa antibody (i.e., hardly any loss of bound IgG could be observed after 10 minutes

2.103 nm and 5.475 nm for anti-LC-kappa (Hu) and Protein L, of dissociation. The kd value as calculated for biotin anti-IgG-CH1 respectively). To visualize the variance in binding reactivity with- also serves well for generating a stable captured IgG surface. in this broad panel of LC-kappa containing antibody formats, When capturing Fab fragments, higher dissociation rates were all binding signals were compared to that produced by human observed for both affinity ligands, which preferably should be IgG3 kappa , which was set as 100%. in the 10-5 s1 or lower range instead of 10-4 s1. However, since a human IgG Fab fragment possesses one constant domain on DOSE RESPONSE CURVES the light chain and one CH1 domain on the heavy chain, simul- A fully human recombinant IgG1 kappa monoclonal antibody taneous binding of these domains could again facilitate bivalent and its corresponding Fab fragment were tested at concentra- binding to a biosensor surface. This would then also result in a tion series ranging from 0.5 μg/mL up to 50 μg/mL. Figure 4 stable Fab surface with low dissociation. As proof of concept, displays the kinetic data from the corresponding curve fits. one Streptavidin (SA) Biosensor was loaded with a mixture of biotin anti-LC-kappa (at 5 μg/mL) and biotin-anti-IgG-CH1 Human IgG1 samples on biotin anti-LC-kappa and anti-IgG-CH1 (at 10 μg/mL) and compared for Fab binding with biosensors biosensors demonstrated proper association and dissociation immobilized with each of the biotin conjugates separately. The curves for the concentration range of IgG tested. Due to biva- resulting data shown in Figure 5 indicate a decrease in dissocia- lent binding that occurs through either the CH1 domain or the tion rate for the mixed biosensor compared to (e.g., the mono- constant domain on the kappa light chain that is present twice specific anti‑LC‑kappa biosensor), demonstrating that bivalent in an IgG format, very low dissociation rate constant (k ) values d binding can occur through binding of two separate domains on were obtained, as depicted in Figure 4 (i.e., 2.64 x 10-5 s-1 and a human Fab fragment. Since both ligands bind to the opposite 7.29 x 10-6 s-1 for anti-IgG-CH1 and anti-LC-kappa, respectively). site of the actual antigen binding site of a Fab (Fv domain), one Proper binding curves for human IgG1 on biotin anti-IgG-Fc bio- can expect that antigen binding characteristics of the captured sensors were also generated; however, since this affinity ligand Fab are not altered. A further decrease in dissociation rates may interacts with the Fc domain in a monovalent manner, a higher be achieved by further optimizing ligand mixtures for immo- dissociation rate was found compared to anti-LC-kappa and an- bilization. Note that this principle can also be used for human ti-IgG-CH1 (i.e., 5.01 x 10-4 s-1). Nevertheless, it does show that Fab fragments that possess a lambda light chain by combining this set of CaptureSelect biosensors can find their use in quanti- biotin anti-LC-lambda with biotin anti-IgG-CH1. tation of human IgG antibodies on the Octet platform. Note that none of these affinity ligands cross-bind to bovine IgG and as such can be used for human IgG detection in the presence of EFFECT OF FREE LIGHT CHAINS ON MEASURING FCS containing media. HUMAN FAB FRAGMENTS IN THE OCTET SYSTEM When producing recombinant monoclonal antibodies and Fab Biotin anti-LC-kappa and anti-IgG-CH1 biosensors displayed fragments, one often has to deal with over-expression of free proper dose response curves for human Fab fragments as well. light chains. For quantitation of IgG antibodies, this problem can However, since Fab only contains one light chain and one CH1 easily be circumvented by using affinity ligands that target the domain, no bivalent binding can occur with either of these affinity IgG Fc domain and do not show any cross-binding towards anti- ligands. Therefore, the calculated k values for Fab were signifi- d body light chains (e.g., biotin anti-IgG-Fc or Protein A). However, cantly higher compared to those for human IgG. These ligands for detection and quantitation of human Fab fragments, the can still be valuable for Fab quantitation purposes, although final majority of currently available binding agents target epitopes detection limits will likely be higher than for measuring whole IgG that reside on the antibody light chain (e.g., VL-kappa by Pro- antibodies. Fab binding was also observed for Protein L, although tein L or light chain constant domains by biotin anti-LC-kappa at a significantly lower binding affinity compared to biotin an- and biotin anti-LC- lambda). Quantitation of Fab fragments then ti-LC-kappa (data not shown). becomes problematic since cross-binding to free light chain contaminants will also occur, resulting in unreliable outcomes. IGG AND FAB CAPTURE FOR SCREENING However, by using an affinity ligand that targets the CH1 domain PURPOSES on the heavy chain of a Fab fragment, this problem can be elim- For numerous applications, it is advantageous to have high-af- inated. To demonstrate this concept, two different biosensors finity binding agents showing very low dissociation rate values, (biotin anti-IgG-CH1 and Protein L) were used to determine the resulting in a stable complex between the binding agent and its effect of free light chains on Fab quantitation. For this purpose antigen. Especially for screening antibodies against antigen to a human IgG1 Fab sample (at 10 μg/mL) was spiked with levels determine binding characteristics, the availability of high-affinity of free kappa light chains (recombinant) ranging from 0.5 μg/mL

5 Biotin anti-LC-kappa (Hu)

ka = 4.41E+04

kd = 1.56E-04

2.00 KD = 3.54 nM

Figure 5: Introducing bivalency to capture human 1.60 IgG Fab kappa fragments by loading a mixture of biotin anti-IgG-CH1 and biotin anti-LC-kappa (Hu) on Biotin anti-LC-kappa + one Streptavidin (SA) Biosensor. The CaptureSelect Biotin anti-IgG-CH1 biotin conjugates anti-LC-kappa and anti-IgG-CH1 ka = 5.34E+04 1.20 kd = 1.77E-05 were immobilized onto Streptavidin (SA) biosensors K = 0.33 nM D at 5 and 10 μg/mL, respectively. One biosensor was

Fitting View immobilized with a mixture of both biotin conjugates using above concentrations for each affinity ligand 0.80 (15 μg/mL in total). Binding analysis was performed using a human IgG1 Fab fragment at a concentration of 10 μg/mL. The Octet Data Analysis software was used Biotin anti-IgG-CH1 to calculate the kinetic values for each biosensor. 0.40 ka = 4.74E+04 Results are displayed in the graph next to a schematic k = 7.59E-04 d representation of a “bi-specific” biosensor enabling K = 16 nM D bivalent capture of a human Fab fragment.

0 0 200 400 600 800 1,000 1,200 Time (sec)

CaptureSelect: Anti-IgG-CH1 Protein L

1.20 4.80

1.00 4.00

0.80 3.20 Free LC-kappa spiked at: 50 μg/mL 2 μg/mL 25 μg/mL 1 μg/mL 10 μg/mL 0.5 μg/mL Binding (nm) Binding 0.60 2.40 5 μg/mL 0 μg/mL Binding (nm)

0.40 1.60

Free LC-kappa spiked at: 50 μg/mL 2 μg/mL 0.20 25 μg/mL 1 μg/mL 0.80 10 μg/mL 0.5 μg/mL 5 μg/mL 0 μg/mL 0 0 0 200 400 600 800 1,000 1,200 0 200 400 600 800 1,000 1,200 Time (sec) Time (sec)

6.0 Binding of 10 μg/mL Fab Anti-IgG-CH1 Protein L without free LC-kappa 5.0 ► Anti-IgG-CH1: 0.826 nm Intact Fab fragments Protein L: 0.136 nm ► Anti-IgG-CH1 4.0 Free kappa light chains Protein L - monomer LC - dimer LC2 3.0 Binding (nm) 2.0

1.0

0 0.1 1 10 100 Free LC-kappa spiked (µg/mL)

Figure 6: Effect of free light chains on quantitation of human Fab fragments using biotin anti-IgG-CH1 versus Protein L. Top: A human Fab fragment derived from a recombinant human IgG1 kappa antibody was analyzed on different antibody capturing biosensors (biotin anti-IgG-CH1 and Protein L) at a concentration of 10 μg/mL in PBS. In order to determine the effect of free kappa light chains on the quantitation of intact Fab fragments, different levels of a recombinant human LC-kappa domain (free light chains) ranging from 0.5 μg/mL up to 50 μg/mL were spiked into the Fab sample (10 μg/mL). Binding analysis was performed as previously described. Bottom right: Graphical representation of binding to anti-IgG-CHI and Protein L. Bottom left: Effect of free light chains on quantitation of human Fab fragments using biotin anti- IgG-CH1 versus Protein L. Bottom right: The corresponding association and dissociation curves. Binding signals (after 10 minutes association) of all spiked samples are displayed in the lower figure for both biotin anti-IgG-CH1 and Protein L.

6 Sample loaded on anti-IgG-CH1 resin:

Human IgG Fab Load Flow-through Elution 1 Load M Flow-through Elution • 100 μg in 200 μL PBS 1 2 3 4 5 6 1 2 3

50 kDa

25 kDa

2 Human LC-kappa Load Flow-through Elution Load M Flow-through Elution • 100 μg in 200 μL PBS 1 2 3 4 5 6 1 2 3

50 kDa

25 kDa

3 Human IgG Fab and Load Flow-through Elution Load M Flow-through ßElution Human LC-kappa 1 2 3 4 5 6 1 2 3 • 2x 100 μg in 200 μL PBS

50 kDa

25 kDa

Human LC-kappa standard curve: Quantitation of free human LC-kappa in non-bound fractions Biotin anti-LC-kappa (Hu) biosensor after sample treatment with CaptureSelect anti-IgG-CH1 resin

400 0.240 Hu lgG Fab (Exp 1) LC-kappa (Exp 2) 0.200 Fab/LC mix (Exp 3) 300

0.160 Standard Fitting curve Unknown & control 0.120 200

Binding Rate Binding 0.080 100 0.040 LC-kappa concentration (µg/mL)

0 0 0 10 20 30 40 50 60 Flow-through Fractions Concentration (μg/mL)

Figure 7: Separating intact human IgG Fab fragments from free human kappa light chains using CaptureSelect anti-IgG-CH1 affinity resin. Top: Samples containing poly- clonal human IgG Fab fragments (Jackson Immunochemistry) and/or human kappa light chains (Bence Jones, BioDesign) were prepared in 200 μL PBS and mixed with 100 μL CaptureSelect anti-IgG-CH1 resin (spin columns, Mobitec). The spin columns were tumbled for 1 hour at room temperature. The non-bound fraction (flow-through 1) was collected by spinning for 1 minute at 150 x g followed by 5 washing steps with 400 μL PBS. Bound Fab fragments were eluted using 200 μL 0.1 M glycine pH 2 (1 minute at 1000 x g). Load, flow-through and elution fractions were analyzed by SDS page (CBB stained). Bottom: Levels of free human kappa light chains in the non-bound fractions (flow-through fractions 1) were determined in the Octet system using an anti-LC-kappa standard curve obtained by a biotin anti-LC-kappa (Hu) biosensor.

7 up to 50 μg/mL. As shown in Figure 6, no significant difference Octet system (Figure 7). Results from the second experiment in Fab binding signals was observed for the biotin anti-IgG-CH1 confirmed selectivity of the resin for intact Fab, leaving all free biosensors with addition of free light chains up to a level of 10 light chains in the non-bound fraction. In the last experiment a μg/mL. When spiking higher concentrations of free light chains mixture of both was applied, resulting in excellent separation (> 25 μg/mL) some non-specific binding was observed, probably of free light chains (non-bound) from the intact Fab material via caused by the stickiness of free light chains at these high con- elution. In this setup, small spin columns were used for deplet- centrations (possibly due to the unpaired cysteine residues pres- ing 100 μg Fab with 100 μL resin. Since the binding capacity ent on monomeric LC). Unlike anti-IgG-CH1, however, the Pro- of the resin for Fab exceeds 5 mg/mL, higher amounts of Fab tein L biosensors clearly indicated co-binding of free light chains, could be depleted. Furthermore, converting this assay into a in fact at a higher binding rate than for the tested Fab fragment. 96-well plate format could facilitate sample analysis on free light It should be noted that the spiked free kappa light chains were chains in a high throughput mode. different from the light chain present on the IgG1 Fab fragment tested. Since Protein L demonstrated variances in binding affinity Conclusion for different types of light chains, this may have boosted its effect in this experimental setup. Nevertheless, co-binding of free light CaptureSelect biotin conjugates demonstrated excellent com- chains does occur and will clearly have an effect on Fab quanti- patibility with Streptavidin (SA) biosensors and can serve as a tation assays, when using Protein L (or biotin anti-LC-kappa and valuable tool for detection, quantitation and characterization anti-LC-lambda). Therefore, introducing biotin anti-IgG-CH1 for of any antibody format on ForteBio’s Octet platform. Octet sys- specific detection of intact Fab fragments may circumvent the tems quantify protein concentrations and monitor protein-pro- need for currently applied sandwich-based assays. tein and other biomolecular interaction kinetics (ka, kd and KD), enabling informed research and development decisions earlier in the process, while their high throughput enables accelerated SAMPLE TREATMENT USING CAPTURESELECT ANTI- TM timelines. The microfluidics-free Dip and Read format enables IGG-CH1 RESIN use of crude media and a simple and easy user interaction. As described in the previous section, the use of affinity ligands directed against epitopes that are not present on antibody light References chains can be advantageous for detection of intact antibodies 1 Liu J, et al. Comparison of Camelid antibody ligand to Protein A for mono- (and in particular Fab fragments) in samples contaminated with clonal antibody purification, BioPharm International September: pp 35–43, free light chains. An even bigger challenge can be developing 2009. assays that enable specific detection and quantitation of free 2 Detmers F, Hermans P and ten Haaft M. Affinity Chromatography based upon light chain contaminants. Currently available affinity ligands unique single chain antibodies, The Peak September: pp 7–14, 2007. directed against light chains do not discriminate between free 3 Beyer T. Serum-free production and purification of chimeric IgA antibodies, Journal of Immunological Methods 346: pp 26–27, 2009. light chains and light chains present in intact antibody formats. 4 Klooster R, et al. Improved anti-IgG and HSA affinity ligands: Clinical applica- However, as shown in Figure 7, this problem can be solved by tion of VHH antibody technology, Journal of Immunological Methods 324: pp implementing a simple sample pretreatment step using BAC’s 1–12, 2007. CaptureSelect anti-IgG-CH1 resin (cat. no.191.3120.10). Like biotin 5 McCue JT, Selvitelli K and Walker J. Application of a novel affinity adsorbent for the capture and purification of recombinant factor VIII compound, J. anti-IgG-CH1, this resin specifically captures intact human IgG Chrom. A. 1216: pp 7824–7830, 2009. and Fab fragments thereof without cross-binding to free light 6 Kuroiwa Y, et al. Antigen-specific human polyclonal antibodies from hyperim- chains. By incubating a sample with this resin, all intact human munized cattle, Nature Biotechnology 27 (2): pp 173–181, 2009. IgG or IgG Fab fragments will be depleted, leaving all free light 7 Hellström M, et al. Cellular tropism and transduction properties of seven ade- no-associated viral vector serotypes in adult retina after intravitreal injection, chains in the non-bound fraction. This non-bound fraction can Gene Therapy 16: pp 521–532, 2008. then be collected for quantitation of light chains using a standard 8 Smith RH, Levy JR, Kotin RM. A simplified baculovirus-AAV expression vector curve obtained by biotin anti-LC-kappa biosensors, for example. system coupled with one-step affinity purification yields high-titer rAAV stocks from insect cells, Gene Therapy (16 June) doi:10.1038/mt.2009.128, In a first experiment, intact human IgG Fab fragments were 2009. incubated with the anti‑IgG-CH1 resin, resulting in no detect- 9 Low D, O’Leary R and Pujar NS. Future of antibody purification, J. Chrom. B 848: pp 48–63, 2007. able Fab fragments in the non-bound fraction as determined 10 Kotin RM. Large-scale recombinant adeno-associated virus production, Hu- by SDS-PAGE and biotin anti‑LC-kappa biosensors in the man Molecular Genetics doi:10.1093/hmg/ddr141, Vol. 20, Review Issue 1, 2011.

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