Heterologous recombinant expression of non-originator NISTmAb

Figures & data

Figure 1. The amino acid sequences of the signal peptides encoded in the non-originator cell lines for the light and heavy chains.

A. The signal peptide of the light chain is shown. The red arrow shows the expected cleavage site and the blue arrow shows the alternate cleavage site, resulting in an additional Ser residue on a small proportion of the mAb. B. The signal peptide of the heavy chain is shown. The red arrow shows the expected cleavage site.

Figure 2. SDS-PAGE analysis of NISTmAb RM 8671, NS0-59, NS0-60, and NS0-66.

SDS-PAGE analysis was used to evaluate the RM 8671, NS0-59, NS0-60 and NS0-66 antibodies in the presence (lanes 3, 5, 7, and 9) or absence (lanes 2, 4, 6, and 8) of 700 mmol/L BME. Protein samples (1.25 μg) were separated on a 4–20% gradient SDS-PAGE followed by staining with GelCode Blue Safe Protein Stain. Lane 1, molecular weight marker (kDa); lanes 2 and 3, RM 8671; lanes 4 and 5, NS0-59; lanes 6 and 7, NS0-60; lanes 8 and 9, NS0-66. mAb, full length antibody; HC, heavy chain; LC, light chain.

Figure 3. Size-exclusion chromatograms of non-originator and RM 8671. A, NS0-59; B, NS0-60; C, NS0-66; D, RM 8671. HMW, high molecular weight; LMW, low molecular weight.

Figure 4. ¹H-¹³C gs-HSQC methyl spectra of PS 8670, NS0-59, NS0-60, and NS0-66.

NMR data were collected at 50°C on a 900 MHz spectrometer with a cryogenically cooled HCN triple resonance probe equipped with a z-axis gradient system as described in Materials and Methods. A, PS 8670; B, NS0-59; C, NS0-60; D, NS0-66.

Figure 5. Deconvoluted intact mass spectra of non-originator NISTmAbs and RM 8671.

Peaks marked with a * indicated the presence of a C-terminal lysine on the previous glycoform. Peaks with a number of 1-5 indicate the presence of a partially uncleaved Ser of the signal peptide (see Figure 1). A, NS0-59; B, NS0-60; C, NS0-66; D, RM 8671.

Table 1. Measured molecular mass values for NISTmAb RM 8671 and non-originator lots NS0-59, NS0-60, and NS0-66.

		Observed Mass (Da)
Proteoform	Theoretical Mass (Da)^a	RM 8671	NS0-59	NS0-60	NS0-66
G0F/G0F – GlcNAc	147833.9	147838.0	147841.0	ND	147841.8
G0F/G1F – GlcNAc	147996.1	148000.4	ND	ND	ND
G0F/G0F	148037.2	148044.1	148043.9	148044.9^b	148042.9
G0F/G0F + S1	148124.2	ND	148128.2	148124.5	148127.7
G0F/G0F + K*	148165.3	148164.0	148166.0	148173.1^b	148170.3
G0F/G1F	148199.3	148205.4	148207.7^b	148205.7	148205.0
G0F/G1F + S2	148286.4	ND	148298.0^b	148298.8^b	148299.8^b
G0F/G1F + K*	148327.5	148324.2	148329.9	148329.6	148330.4
G1F/G1F	148361.4	148368.3	148370.0^b	148367.6	148367.5
G1F/G1F + S3	148448.5	ND	148461.1^b	148456.8^b	148460.3^b
G1F/G1F + K*	148489.6	148485.6	148491.6	148492.2	148488.3
G1F/G2F	148523.6	148528.9	148528.5	148527.8	148521.3
G1F/G2F + S4	148610.7	ND	148626.9^b	148620.9^b	148622.0^b
G1F/G2F + K*	148651.8	148657.1	ND	148652.0	148651.2
G2F/G2F	148685.7	148689.3	148688.6	148688.9	ND
G2F/G2F + S5	148772.8	ND	148788.0^b	148784.9^b	148788.0
G2F/G2F + Hex	148847.7	148847.4	ND	148848.2	ND

a. Theoretical values include 16 disulfide bonds, two N-terminal pyroglutamic acids, and no C-terminal Lys (K), unless otherwise noted. Theoretical values were calculated using the NIST Mass and Fragment calculator. Peaks labeled with a * indicate presence of a C-terminal lysine on the previous glycoform. Peaks labeled with a number of S1–5 indicate the presence of a partially uncleaved serine of the leader peptide. Glycan assignments were made based on putative compositions expected from the glycan biosynthetic pathway.

b. Peak assignments for these indicated peaks were made despite mass errors in excess of 50 ppm. See supplementary Table S2 for calculated mass errors. ND, not detected.

Table 2. In vitro SPR binding data for NISTmAb RM 8671 and non-originator lots NS0-59, NS0-60, and NS0-66. (n = 3).

Product	Peptidic epitope binding (Kd ± 1SD in nmol/L)	FcRn binding (Kd ± 1SD in nmol/L)
NISTmAb	66 ± 2	212 ± 5
NS0-59	80 ± 1	194 ± 5
NS0-60	84 ± 4	190 ± 6
NS0-66	87 ± 4	197 ± 6