The effects of Antibody Engineering CH and CL in Trastuzumab and Pertuzumab recombinant models: Impact on antibody production and antigen-binding

Current therapeutic antibodies such as Trastuzumab, are typically of the blood circulatory IgG1 class (Cκ/ CHγ1). Due to the binding to Her2 also present on normal cell surfaces, side effects such as cardiac failure can sometimes be associated with such targeted therapy. Using antibody isotype swapping, it may be possible to reduce systemic circulation through increased tissue localization, thereby minimising unwanted side effects. However, the effects of such modifications have yet to be fully characterized, particularly with regards to their biophysical properties in antigen binding. To do this, we produced all light and heavy chain human isotypes/subtypes recombinant versions of Trastuzumab and Pertuzumab, and studied them with respect to recombinant production and Her2 binding. Our findings show that while the light chain constant region changes have no major effects on production or Her2 binding, some heavy chain isotypes, in particularly, IgM and IgD isotypes, can modulate antigen binding. This study thus provides the groundwork for such isotype modifications to be performed in the future to yield therapeutics of higher efficacy and efficiency.


Size Exclusion Chromatography fractionations
The calibration of the size exclusion chromatography experiments were performed using the Superdex 200pg 16/600 (GE Healthcare) of the AKTA PURE system. The calibrations were carried out using the Gel filtration Low Molecular Weight (LWM) and High Molecular Weight (HMW) calibration kits (Catalogue: 28-4038-41 & 28-4038-42). The graph, generated using Unicorn v6.3 (GE Healthcare) was plotted as per manufacturer's recommendations.
Certain protein standards in the LMW and HMW overlapped, and were used to generate the full spectrum of the elution times of the various molecular weights ( Figure S1a). Figure S1a: Calibration curve of the various molecular weight standards in kDa.
Calibration of size exclusion chromatography on Superdex 200pg 16/600 (GE Healthcare) was performed using the AKTA PURE system with the Gel filtration Low Molecular Weight (LWM) and High Molecular Weight (HMW) calibration kits (Catalogue: 28-4038-41 & 28-4038-42). The calibration graph, generated using Unicorn v6.3 (GE Healthcare) was plotted as per manufacturer's recommendations. Both the spectrums from the protein standards in the LMW and HMW were pieced together to generate the full spectrum of the elution times of the various molecular weights. X-Axis: 40-100 ml time scale. Y-axis: mAU absorption as determined by UV detection.

1) Trastuzumab variants
For IgM, fraction 2 was collected while for IgA, IgE, and IgD, fractions 3 and 4 were collected. For other IgGs, the various fractions are shown below. Figure S1 and S2 are used for figure 1 while figure S3 and S4 are used for figure 4.  Figure S1f: Trastuzumab IgE Figure S1g: Trastuzumab IgG1 Figure S1f and S1g shows the size exclusion chromatogram profiles of Trastuzumab IgE and IgG1. Only the fractions corresponding to the ~150 kDa monomeric fractions were collected as indicated by the bold red line on the X-axis.
X-Axis: 40-100 ml time scale. Y-axis: mAU absorption as determined by UV detection. Figure S1h: Trastuzumab IgG2 Figure S1i: Trastuzumab IgG3 Figure S1h and S1i shows the size exclusion chromatogram profiles of Trastuzumab IgG2 and IgG3. Only the fractions corresponding to the ~150 kDa monomeric fractions were collected as indicated by the bold red line on the X-axis.
X-Axis: 40-100 ml time scale. Y-axis: mAU absorption as determined by UV detection.        Fraction numbers are indicated the X-axis with the bold red line and numbers. All samples were analysed using the same settings in AKTA Pure.
The x-axis is the ml (volume) of the elution segment of the SEC, based on 1ml/min flowrate on the column. Y-axis shows the mAU of the protein absorbance of UV detection.
All figures were exported and generated from the Unicorn v6 software.

SDS-PAGE analysis of the monomeric fractions of the IgA, D, G, E and oligomeric fraction of the IgM variants of Trastuzumab and Pertuzumab
SDS-PAGE gel photos of the concentrated isolated desired fractions of the antibodies.
The proteins were reduced and boiled before loading on 10%SDS-PAGE. The band sizes were determined using GelApp (Nature Methods Application Notes. doi:10.1038/an9643 ) with the calculated size (KDa) shown. Sections 1 and 2 show Gelapp screenshots for heavy chain variants of Trastuzumab and Pertuzumab (corresponding to figure 1 of the manuscript) while sections 3 and 4 are the λ light chain variants of Trastuzumab and Pertuzumab (corresponding to Figure 4 of the manuscript).

Affinity Purification Chromatography fractionation
The affinity purification chromatography experiments were performed using the Protein G 5ml column (GE Healthcare) or Protein L 5ml column (GE Healthcare) in the AKTA PURE system. All IgGs were pull down using Protein G column while all other isotypes were pull down using Protein L column. The elutions (as shown in red bar) were directly captured to a superloop prior to size exclusion chromatogram. The graphs were generated using Unicorn v6.3 (GE Healthcare) and plotted as per manufacturer's recommendations. Figure S9a: Trastuzumab IgM Figure S9a shows the affinity purification chromatogram profiles of IgM using Protein L column. Only the fractions corresponding to mAU >20 were collected as indicated by the bold red line on the X-axis. X-Axis: ml time scale. Y-axis: mAU absorption as determined by UV detection. Figure S9b: Trastuzumab IgA1 Figure S9b shows the affinity purification chromatogram profiles of IgA1 using Protein L column. Only the fractions corresponding to mAU >20 were collected as indicated by the bold red line on the X-axis.
X-Axis: ml time scale. Y-axis: mAU absorption as determined by UV detection. Figure S9c: Trastuzumab IgA2 Figure S9c shows the affinity purification chromatogram profiles of IgA2 using Protein L column. Only the fractions corresponding to mAU >20 were collected as indicated by the bold red line on the X-axis.
X-Axis: ml time scale. Y-axis: mAU absorption as determined by UV detection. Figure S9d: Trastuzumab IgD Figure S9d shows the affinity purification chromatogram profiles of IgD using Protein L column. Only the fractions corresponding to mAU >20 were collected as indicated by the bold red line on the X-axis.
X-Axis: ml time scale. Y-axis: mAU absorption as determined by UV detection. Figure S9e: Trastuzumab IgE Figure S9e shows the affinity purification chromatogram profiles of IgE using Protein L column. Only the fractions corresponding to mAU >20 were collected as indicated by the bold red line on the X-axis.
X-Axis: ml time scale. Y-axis: mAU absorption as determined by UV detection. Figure S9f: Trastuzumab IgG1 Figure S9f shows the affinity purification chromatogram profiles of IgG1 using Protein G column. Only the fractions corresponding to mAU >20 were collected as indicated by the bold red line on the X-axis.
X-Axis: ml time scale. Y-axis: mAU absorption as determined by UV detection. Figure S9g: Trastuzumab IgG2 Figure S9g shows the affinity purification chromatogram profiles of IgG2 using Protein G column. Only the fractions corresponding to mAU >20 were collected as indicated by the bold red line on the X-axis.
X-Axis: ml time scale. Y-axis: mAU absorption as determined by UV detection. Figure S9h: Trastuzumab IgG3 Figure S9h shows the affinity purification chromatogram profiles of IgG3 using Protein G column. Only the fractions corresponding to mAU >20 were collected as indicated by the bold red line on the X-axis.
X-Axis: ml time scale. Y-axis: mAU absorption as determined by UV detection. Figure S9i: Trastuzumab IgG4 Figure S9i shows the affinity purification chromatogram profiles of IgG4 using Protein G column. Only the fractions corresponding to mAU >20 were collected as indicated by the bold red line on the X-axis.
X-Axis: ml time scale. Y-axis: mAU absorption as determined by UV detection. Figure S9j: Pertuzumab IgM Figure S9j shows the affinity purification chromatogram profiles of IgM using Protein L column. Only the fractions corresponding to mAU >20 were collected as indicated by the bold red line on the X-axis.
X-Axis: ml time scale. Y-axis: mAU absorption as determined by UV detection. Figure S9k: Pertuzumab IgA1 Figure S9k shows the affinity purification chromatogram profiles of IgA1v using Protein L column. Only the fractions corresponding to mAU >20 were collected as indicated by the bold red line on the X-axis.
X-Axis: ml time scale. Y-axis: mAU absorption as determined by UV detection. Figure S9l: Pertuzumab IgA2 Figure S9l shows the affinity purification chromatogram profiles of IgA2 using Protein L column. Only the fractions corresponding to mAU >20 were collected as indicated by the bold red line on the X-axis.
X-Axis: ml time scale. Y-axis: mAU absorption as determined by UV detection. Figure S9m: Pertuzumab IgD Figure S9m shows the affinity purification chromatogram profiles of IgD using Protein L column. Only the fractions corresponding to mAU >20 were collected as indicated by the bold red line on the X-axis.
X-Axis: ml time scale. Y-axis: mAU absorption as determined by UV detection. Figure S9n: Pertuzumab IgE Figure S9n shows the affinity purification chromatogram profiles of IgE using Protein L column. Only the fractions corresponding to mAU >20 were collected as indicated by the bold red line on the X-axis.
X-Axis: ml time scale. Y-axis: mAU absorption as determined by UV detection. Figure S9o: Pertuzumab IgG1 Figure S9o shows the affinity purification chromatogram profiles of IgG1 using Protein G column. Only the fractions corresponding to mAU >20 were collected as indicated by the bold red line on the X-axis.
X-Axis: ml time scale. Y-axis: mAU absorption as determined by UV detection. Figure S9p: Pertuzumab IgG2 Figure S9p shows the affinity purification chromatogram profiles of IgG2 using Protein G column. Only the fractions corresponding to mAU >20 were collected as indicated by the bold red line on the X-axis.
X-Axis: ml time scale. Y-axis: mAU absorption as determined by UV detection. Figure S9q: Pertuzumab IgG3 Figure S9q shows the affinity purification chromatogram profiles of IgG3 using Protein G column. Only the fractions corresponding to mAU >20 were collected as indicated by the bold red line on the X-axis.
X-Axis: ml time scale. Y-axis: mAU absorption as determined by UV detection. Figure S9r: Pertuzumab IgG4 Figure S9r shows the affinity purification chromatogram profiles of IgG4 using Protein G column. Only the fractions corresponding to mAU >20 were collected as indicated by the bold red line on the X-axis.
X-Axis: ml time scale. Y-axis: mAU absorption as determined by UV detection. Figure S10a: Binding kinetics of Trastuzumab and Pertuzumab isotype variants using Blitz (with fitting).
The graphs showing the binding kinetics of Trastuzumab and Pertuzumab to Her2 (loaded on NTA sensor) using Blitz (ForteBio). The fitting (black line overlay) is as calculated by the Blitz software. Figure S10b: Binding measurements of Trastuzumab and Pertuzumab isotype variant using Octet (with fitting).
Graphs showing the binding kinetics of Her2 to Trastuzumab or Pertuzumab isotype variants (loaded on ProL sensor) using Octet (ForteBio). Fitting (red line overlay) is as calculated using the Octet software.