Isolation and Characterization of Monoclonal Antibody Charge Variants by Free Flow Isoelectric Focusing

Capillary isoelectric focusing (cIEF) is widely used in the biopharmaceutical industry to measure the charge distribution of therapeutic proteins. The implementation of this technology has created a new challenge. Capillary volumes are on the order of hundreds of nanoliters and cannot be scaled up for the preparative collection of charge variants. This makes it difficult to identify the charge variants in a cIEF electropherogram. Therefore, preparative IEF methods are needed to fractionate charge variants for characterization. We used free-flow electrophoresis (FFE) to isolate monoclonal antibody charge variants observed in a cIEF electropherogram. The same antibody was also fractionated using the Rotofor and Offgel instruments for comparison. A strategy for purifying the fractionated charge variants and downstream characterization is described. Acidic and basic variants were identified and related back to the analytical cIEF charge profile. This study establishes free-flow isoelectric focusing as a valuable tool for characterizing therapeutic proteins

Phosphorylation of Dzip1 by GSK3β promotes dissociation of Rab8^GDP from GDI2.

<p>(A) The binding of Dzip1 with GDI2 is prevented by inhibition of GSK3. G0-phase NIH 3T3 cells were treated or not treated with the GSK3 inhibitor BIO, and endogenous Dzip1 and GDI2 were analyzed. (B) The binding of Rab8^GDP with GDI2 is increased by inhibition of GSK3. G0-phase HEK 293T cells expressing GFP-Rab8^T22N were treated or not treated with the GSK3 inhibitors BIO or CHIR99021 (CHIR), and endogenous GDI2 was analyzed. The quantified band intensities are labeled. (C) The binding of GDI2 to Dzip1 is decreased, but to Rab8 is increased, by inhibition of GSK3. Endogenous Dzip1 and Rab8 were pulled down from G0-phase NIH 3T3 cells treated with the GSK3 inhibitor or from control (Con) cells by increasing amounts of GST-GDI2. The maximum amounts of the pulled-down proteins were defined as having a binding affinity of 1.0, and the amounts of the indicated proteins at each point were normalized to the maximum amounts. The results represent two independent assays. (D and E) The amount of free Rab8^GDP at the basal body is decreased by inhibition of GSK3. G0-phase NIH 3T3 cells expressing the fusion protein CFP-RBD-YFP were treated with the GSK3 inhibitors BIO or CHIR99021 or not treated (control). The values shown are mean ± SD, from 20 cells for each group (D). The calculated region was manually selected at the cilium base based on strong accumulation of the CFP-RBD-YFP signal. *<i>p</i> < 0.05. Scale bar: 5 μm. Note that the images from all treatment situations showed comparable CFP-RBD-YFP fluorescence intensity but different FE^CFP distributions in quantitative analysis. The FE^CFP was highest around the basal body (labeled by AcTub staining) in the control cell, but was largely eliminated in GSK3-inhibited cells. The rainbow indicator in the heat map shows the FE^CFP, and each colored dot in the image represents the appearance of FE^CFP. The red boxes indicate the areas selected for calibrating background (E). (F) Expression of Dzip1^S520A leads to increased binding of Rab8 to GDI2. The GFP-Dzip1 mutants and endogenous Rab8 were pulled down from G0-phase HEK 293T cells by increasing amounts of GST-GDI2. The maximum amounts of the pulled-down proteins were defined as having a binding affinity of 1.0, and the amounts of the indicated proteins at each point were normalized to the maximum amounts. The results represent two independent assays. (G) Phosphorylation of Dzip1^S520 increases its binding to GDI2. GFP alone or wild-type (WT) GFP-Dzip1 or S520 mutants were co-expressed with Myc-GDI2 in G0-phase HEK 293T cells, followed by IP assay. White asterisks indicate the heavy chain of IgG.</p

GSK3β phosphorylates Dzip1.

<p>(A and B) Dzip1 interacts with GSK3β. Endogenous GSK3β was immunoprecipitated by Dzip1 but not IgG (A), and endogenous Dzip1 was immunoprecipitated with GFP-GSK3β in HEK 293T cells (B). (C and D) Dzip1 is co-localized with GSK3β at the basal body. G0-phase NIH 3T3 cells expressing GFP-GSK3β were immunostained for Dzip1 and AcTub (C), or cells expressing BFP-Centrin2 were immunostained with GSK3β and Dzip1 (D). Scale bar: 5 μm. (E) GSK3β binds Dzip1 in a kinase-substrate interaction manner. Wild-type (WT) GFP-GSK3β and the mutants S9A, K85R, and R96A were each co-expressed with Myc-Dzip1 in G0-phase HEK 293T cells, and treated with the CK1 inhibitor D4476 or the CK2 inhibitor CX4945. Note that treatment with CX4945 but not D4476 led to a significant decrease in the extent of the up-shifted Dzip1 bands, although the binding of Dzip1 to the GFP-GSK3β variants showed no difference. The extent of the up-shifting of the Dzip1 bands was decreased in K85R-expressing cells. (F) Phosphorylation of Dzip1 is coordinated with GSK3β activation. The kinase activity of GSK3β was negatively correlated with serum stimulation in NIH 3T3 cells. Note that the up-shifted bands (arrowheads) of Dzip1 became evident after serum depletion for 24–48 h, and disappeared after serum restimulation. γ-Tubulin was set as a loading control. (G) GSK3β phosphorylates Dzip1 in vivo. In resting mouse embryo fibroblast (MEFs) treated versus not treated with GSK3 and CK2 inhibitors, the Dzip1 bands were up-shifted less in GSK3- and CK2-inhibited cells. The protein levels of total β-Catenin and GSK3β were steady, but the phosphorylated (S33/37/T41) β-Catenin specifically disappeared from GSK3-inhibited cells. α-Tubulin was set as a loading control. (H) GSK3β phosphorylates Dzip1 in vitro. Auto-phosphorylation of GSK3β (55 kD), and the phosphorylated bands of the middle (28 kD), C-terminus (36 kD), and N- terminus (50 kD) of Dzip1 are shown (left panel). Coomassie blue staining of the gel shows the loaded amounts of Dzip1 fragments (right panel). Note that the S520A mutation resulted in decreased phosphorylation of Dzip1 by GSK3β. (I) Inhibition of GSK3 by BIO causes loss of phospho-S520 in Dzip1.</p

Dysfunction of GSK3β disrupts ciliogenesis.

<p>(A) Schematic model of the acquisition of synchronized mitosis–G0 phase NIH 3T3 cells, treated with double thymidine block, nocodazole (Noc), and the indicated GSK3 inhibitors. (B and C) Inhibition of GSK3 impairs ciliogenesis. Representative images of ciliated cells during the mitosis–G0 phase transition without (Con) or with BIO or CHIR99021 treatment. Cells were immunostained for γ-Tubulin and AcTub. The DNA was stained with DAPI. Ciliation ratios for (B) are shown in (C). (D and E) Knockout (KO) of GSK3β, but not GSK3α, abolishes ciliogenesis. Wild-type (WT), GSK3α^−/−, and GSK3β^−/− MEFs during the mitosis–G0 phase transition were immunostained with γ-Tubulin and AcTub. The DNA was stained with DAPI. Ciliation percentage ratios for (D) are shown in (E). (F and G) The kinase activity of GSK3β is required for ciliogenesis. GSK3β^−/− MEFs were transfected with wild-type GFP-GSK3β or mutant GFP-GSK3β S9A, K85R, or R96A and immunostained with AcTub. The DNA was stained with DAPI. Representative images are shown in (F), and the percentage ciliation ratios are shown in (G). Note that the wild-type and S9A fully, and R96A partially, rescued the ciliogenesis defect, whereas K85R did not. (H and I) A phosphorylation-mimicking mutant of Dzip1 rescues the ciliogenesis defect in GSK3β^−/− cells. GSK3β^−/− MEFs were transfected with wild-type Myc-Dzip1, mutant S520A, or S520D, and immunostained with AcTub. The DNA was stained with DAPI. Representative images are shown in (H), and the percentage ciliation ratios are shown in (I). The values in (C), (E), (G), and (I) are mean ± SD; 50 pairs of daughter cells were counted in each of three independent experiments. ***<i>p</i> < 0.001. Scale bars in (B), (D), (F), and (H): 5 μm.</p

Dzip1 promotes the release of Rab8^GDP from GDI2 at the cilium base.

<p>(A) The binding of Myc-GDI2 to GFP-Rab8^T22N is abolished by expression of Myc-Dzip1. GFP-Rab8^T22N and Myc-GDI2 were co-expressed in HEK 293T cells with or without Myc-Dzip1 expression. White asterisks indicate the heavy chain of IgG. (B) His-Dzip1 aa 430–600 promotes the dissociation of endogenous Rab8 and GST-GDI2 in vitro. Addition of the His-Dzip1 truncate released Rab8 from GDI2 in a dose-dependent manner. The pulled-down proteins were stained with Fast Green. (C and D) Endogenous Dzip1 and GDI2 form a complex, and are co-localized at the centrosome and the PCM. Scale bar: 5 μm. (E and F) The amount of free Rab8^GDP at the cilium base is decreased in Dzip1-knockdown cells. CFP-RBD-YFP was introduced into G0-phase RNAi control (Con) and Dzip1-knockdown cells. The rainbow indicator shows the fluorescence emission intensity of CFP (FE^CFP), and each colored dot in the image represents the location of FE^CFP. The red boxes are regions selected for calibrating the background. For values in (E), 20 cells were examined. **<i>p</i> < 0.01. Note that the representative images show comparable CFP-RBD-YFP fluorescence intensity and YFP bleaching efficiency in both cell lines, but quantitative analysis of the acceptor-bleaching fluorescence resonance energy transfer (AB-FRET) efficiency showed a significant difference. The FE^CFP occurred intensively around, but not at, the basal body (labeled by AcTub) in control but not Dzip1-knockdown cells. Scale bar: 5 μm.</p

Dzip1 is localized to the periciliary diffusion barrier and concentrated at the mother centriole in ciliated cells.

<p>(A) Examination of the Dzip1 antibody. The antibody Mid2 targeting aa 594–610 of full-length human Dzip1 recognized endogenous Dzip1 at ~110 kD both in non-ciliated human (HeLa) and ciliated mouse (NIH 3T3) cells. GAPDH was set as a loading control. (B) Super-resolution microscopy showing that a fraction of Dzip1 is concentrated at the mother centriole and the PCM. G0-phase NIH 3T3 cells were immunostained with Dzip1 and acetylated α-tubulin (AcTub). DC, daughter centriole; MC, mother centriole; TZ, transition zone. Scale bar: 1 μm. (C) Super-resolution microscopy showing that Dzip1 is asymmetrically concentrated at the mother centriole. NIH 3T3 cells transfected with GFP-Cep120 were immunostained for Dzip1. Note that the brighter immunofluorescence signal of Dzip1 is partially merged with the darker signal of GFP-Cep120, and vice versa. The boxed area in the main image is magnified below it. Scale bar: 5 μm. (D) Super-resolution microscopy showing that GFP-Dzip1 is localized to the cilium base and the PCM. NIH 3T3 cells transfected with GFP-Dzip1 were immunostained for PCM1 and IFT88. Note that in addition to the co-localization of GFP-Dzip1 with PCM1 at the PCM, a portion of Dzip1 was also localized to the cilium base, labeled by IFT88. The boxed area in the main image is magnified below it. Scale bar: 5 μm. (E and F) Super-resolution microscopy showing that pericentriolar Dzip1 resides in the PDB. NIH 3T3 cells expressing the PDB marker YFP-GL-GPI were immunostained for AcTub and Dzip1. A model of localization of the indicated proteins is shown in (F). MC, mother centriole; PDB, periciliary diffusion barrier; TZ, transition zone. Scale bar: 5 μm. (G) The PCM localization of Dzip1 is dynamic in the cell cycle. NIH 3T3 cells were immunostained for Dzip1 and AcTub in the cell cycle. Note that Dzip1 was fuzzy at the centrosome in mitosis, but became evident and partially co-localized with the grandmother centriole marker GFP-Cep164 (arrow) in daughter cells in early G1 phase, during which Dzip1 also showed midbody-localization. Also note that Dzip1 was absent from one of the two centrosomes in late G2 phase and in telophase (arrowhead). Boxes labeled “1” are magnified on the right, showing the centrosomal localization of Dzip1. Scale bar: 5 μm.</p

Dzip1 promotes ciliary entry of Rab8 and preferentially binds to Rab8^GDP.

<p>(A and B) Knockdown of Dzip1 impairs ciliary localization of Rab8. RNAi control (Con) and Dzip1 stable-knockdown 1308–3 cells were arrested at G0 phase and immunostained for IFT88 and Rab8. Note that the basal body localization of Rab8 was unaffected. Scale bars: 5 μm. The values in (B) are mean ± standard deviation (SD); 100 cells were counted in each of three independent experiments. ***<i>p</i> < 0.001. (C and D) Rab8^GTP is not localized to the cilium in Dzip1-knockdown cells. The active-mimicking mutant GFP-Rab8^Q67L was introduced into RNAi control and RNAi 1308–3 cells, which were then immunostained for AcTub. Scale bars: 5 μm. The values in (D) are mean ± SD; 100 cells were counted in each of three independent experiments. ***<i>p</i> < 0.001. (E and F) Smo-YFP is not transported to the cilium in Dzip1-knockdown cells. The cells transfected with Smo-YFP were immunostained for AcTub, and the DNA was stained with DAPI. Note that, although the ciliary lengths are similar in the representative cells, ciliary Smo-YFP was much less abundant in Dzip1-knockdown cells than in control cells. Scale bars: 5 μm. The values in (F) are mean ± SD; 100 cells were counted in each of three independent experiments. ***<i>p</i> < 0.001. (G) Dzip1 interacts with Rab8a and Rab10. Myc-Dzip1 was co-expressed with GFP-Rab proteins or GFP alone in G0-phase cells. Immunoprecipitation (IP) was carried out using an antibody against GFP, and the immunoprecipitates were probed for the Myc tag. (H) Dzip1 interacts with Rab8. Rab8 was immunoprecipitated by Dzip1 but not by IgG in G0-phase NIH 3T3 cells (left). Dzip1 was immunoprecipitated by monoclonal antibody against Rab8 but not by GFP (right). (I) Dzip1 and Rab8 are co-localized at the cilium base. The localization of GFP-Dzip1 and RFP-Rab8 in living NIH 3T3 cells arrested in G0 phase was modeled by 3-D reconstruction (bottom). Scale bar: 5 μm. (J and K) Dzip1 preferentially binds Rab8^GDP. The Flag-Rab8 variants were each co-expressed with GFP-Dzip1 in HEK 293T cells. Note that ~3.5-fold more Rab8^T22N than Rab8^Q67L was co-immunoprecipitated with Dzip1 (J), and ~5.0-fold more Dzip1 was co-immunoprecipitated with GFP-Rab8^T22N than with wild-type (WT) Rab8 (K).</p

Roles of p300 binding in regulating RKIP promoter activityin A375 and HeLa cells.

<p>In A375 (A) and HeLa (B) nuclear extracts, EMSA of the ³²P-labeled oligonucleotide derived from the +108/+121 putative p300 binding site in the presence or absence of cold WT or mutation oligonucleotide. NE, nuclear extract. Shifted ³²P-probe bands sensitive to competition by the WT cold probe are indicated. (C) Relative luciferase activity of A375 and HeLa cells transfected with the indicated reporter constructs. (D) A375 cells were co-transfected with negative control or p300-specific siRNA and the reporter construct pGL3-Basic RKIP (−56/+261), then cultured for an additional 48 hr. Left panel: RT-PCR. Right panel: relative luciferase activity. (E) A375 cells were transfected with the indicated siRNAs and the mRNA level of RKIP was examined by RT-PCR. (F) Relative luciferase activity of A375 cells co-transfected as indicated and cultured for an additional 48 hr. (G) A375 cells were co-transfected with the reporter construct pGL3-Basic RKIP (−56/+261) and the indicated plasmids and then cultured for an additional 48 hr. Relative luciferase activity was assayed. *P<0.05, **P<0.01.</p

Role of CREB binding in regulating RKIP promoter activity in A375 and HeLa cells.

<p>In A375 (A) and HeLa (B) nuclear extracts, EMSA of the ³²P-labeled oligonucleotide derived from the −28/−17 putative CREB binding site in the presence or absence of cold WT or mutation oligonucleotide. NE, nuclear extract. Shifted ³²P-probe bands sensitive to competition by the WT cold probe are indicated. (C) Relative luciferase activity of cells transfected with each of the indicated reporter constructs. (D) Cells were co-transfected with a negative control or a CREB-specific shRNA and the reporter construct pGL3-Basic RKIP (−56/+261), then cultured for an additional 24 hr. Left panel: immunoblots of cell lysates. Right panel: relative luciferase activity. (E) Cells were transfected with the indicated siRNAs and then the mRNA level of RKIP was examined by RT-PCR. (F) Cells were co-transfected with the vector (pcDNA 3.1) or the CREB expression plasmid and the reporter construct, then cultured for an additional 24 hr. Left panel: immunoblots of cell lysates. Right panel: relative luciferase activity. *P<0.05, **P<0.01.</p

Oligonucleotides of Sp1 binding sites recognized by factors in A375 and HeLa cells.

<p>Shifted ³²P-probe bands sensitive to competition by the WT cold probe are indicated. In A375 (A) and HeLa (B) nuclear extracts, EMSA of the ³²P-labeled oligonucleotide derived from the −17/−6 putative Sp1 binding site in the presence or absence of cold WT or mutation oligonucleotide. In A375 (C) and HeLa (D) nuclear extracts, EMSA of the ³²P-labeled oligonucleotide derived from the −5/+5 putative Sp1 binding site in the presence or absence of cold WT or mutation oligonucleotide. MU, mutated; NE, nuclear extract.</p