biotinylated human c met his  (ACROBiosystems)

Journal: mAbs

Article Title: Reduction of therapeutic antibody self-association using yeast-display selections and machine learning

doi: 10.1080/19420862.2022.2146629

Figure Lengend Snippet: Design and sorting of a bococizumab Fab library to generate enriched library samples with high and low levels of antigen and QD-lenzilumab binding. (a) Ten sites in the heavy chain CDRs of bococizumab that were predicted to mediate high self-association in a physiological solution condition (pH 7.4, PBS) were mutated using degenerate codons. (b) Degenerate codons were used to sample five mutations and the wild-type amino acid at each selected CDR position. (c) Structural model of the bococizumab Fv region with sites selected for mutagenesis highlighted according to wild-type residue (blue for positively charged residues and green for hydrophobic residues). (d-e) Bococizumab library displayed on the surface of yeast was enriched for high and low levels of (d) QD-lenzilumab binding (4 sorts) and (e) antigen binding (PCSK9, 2 sorts) using fluorescence-activated cell sorting. The enriched library samples boxed in red were collected and deep sequenced.

Article Snippet: For single-point binding measurements, reagents [1 nM biotinylated-PCSK9 (Sino Biological 10692-H27H-B), 0.1 mg/mL SMP, 0.1 mg/mL ovalbumin) were incubated with the washed beads.

Techniques: Binding Assay, Mutagenesis, Fluorescence, FACS

Journal: mAbs

Article Title: Reduction of therapeutic antibody self-association using yeast-display selections and machine learning

doi: 10.1080/19420862.2022.2146629

Figure Lengend Snippet: Bococizumab variants enriched for high affinity and low self-association display dissimilar isoelectric points. (a-b) V H isoelectric points for deep sequencing datasets corresponding to high (positive) and low (negative) levels of Fab binding to (a) QD-lenzilumab conjugates and (b) antigen (PCSK9). In (a) and (b), each dataset contains 30,000 bococizumab mutants. Violin plots are kernel density estimations. Inner boxplots extend from the first to third quartiles with the center white datapoints depicting the median values.

Article Snippet: For single-point binding measurements, reagents [1 nM biotinylated-PCSK9 (Sino Biological 10692-H27H-B), 0.1 mg/mL SMP, 0.1 mg/mL ovalbumin) were incubated with the washed beads.

Techniques: Sequencing, Binding Assay

Journal: mAbs

Article Title: Reduction of therapeutic antibody self-association using yeast-display selections and machine learning

doi: 10.1080/19420862.2022.2146629

Figure Lengend Snippet: First round of evaluation of bococizumab variants with predicted co-optimized levels of high affinity and low self-association. (a) Logistic regression models predict ~0.3% of the bococizumab variants (out of ~44,000 variants) to display both high affinity and low self-association. (b) Amino acid frequencies of each CDR site for the variants predicted to be co-optimal in terms of high affinity and low self-association. (c) Antigen binding (1 nM PCSK9, blue) and AC-SINS (green) analysis for bococizumab variants as IgGs. In (c), AC-SINS measurements are normalized as described in , and mean values for both antigen binding and AC-SINS are reported along with the individual data points (n = 2).

Article Snippet: For single-point binding measurements, reagents [1 nM biotinylated-PCSK9 (Sino Biological 10692-H27H-B), 0.1 mg/mL SMP, 0.1 mg/mL ovalbumin) were incubated with the washed beads.

Techniques: Binding Assay

Journal: mAbs

Article Title: Reduction of therapeutic antibody self-association using yeast-display selections and machine learning

doi: 10.1080/19420862.2022.2146629

Figure Lengend Snippet: Second round of evaluation of bococizumab variants with predicted co-optimized levels of high affinity and low self-association. (a) Additional bococizumab variants identified either based on model predictions (green) or based on results from the first round of analysis (red). (b) Structural model of bococizumab Fv region with the mutated sites highlighted in blue and heavy chain CDR3 sites that were not mutated based on the first round of results highlighted in red. (c) Experimental measurements of self-association (AC-SINS) and antigen binding (1 nM PCSK9) for 23 bococizumab IgG variants. In (c), AC-SINS measurements are normalized as described in , and mean values for both antigen binding and AC-SINS are reported along with the individual data points (n = 2).

Article Snippet: For single-point binding measurements, reagents [1 nM biotinylated-PCSK9 (Sino Biological 10692-H27H-B), 0.1 mg/mL SMP, 0.1 mg/mL ovalbumin) were incubated with the washed beads.

Techniques: Binding Assay