Interplay of heavy chain introns influences efficient transcript splicing and affects product quality of recombinant biotherapeutic antibodies from CHO cells

Figures & data

Figure 1. Heavy chain transgene engineering approaches to eliminate splice variants (a) a pictorial representation of the exon/intron arrangement in the HC expression cassette, the intron between the hinge and CH2 domain is highlighted as particularly susceptible to mis-splicing, SP = signal peptide (b) Depiction of the different HCCD intron arrangements generated in a single plasmid encoding both the HC and LC for mAb-B, the different HCCD formats were cDNA sequence optimized (cDNA-opt), gDNA non-sequence-optimized (gDNA) and gDNA sequence optimized with three mutations in the CH2 to lower the score of in silico predicted cryptic splice acceptors, illustrated by the red stars (gDNA-opt-mut). Plasmids were used to generate stable CHO pools which were subject to a fed-batch process, n = 3 pools for each construct, graph shows mean+SD. Terminal fed-batch titers represented as fold change compared to the cDNA sequence optimized plasmid, statistics analysis was performed using an unpaired t-test, *= P < 0.05, **= P < 0.005, ***=P < 0.005, ****=P < 0.0005. (c) Molecular mechanism of the variant transcript produced by mis-splicing using a cryptic acceptor site resulting in deletion of the first 54 nucleotides of the CH2 domain. (d) Molecular mechanism of the hinge-less splice variant transcript. Both variant transcripts resulted in in-frame shifts in coding sequence, * = Stop codon. (E) Non-reduced and reduced western blot analysis of mAb-B HC (top panels) and LC (lower panels) from transient expression supernatants, sampled at day 5 post transfection; mAb-1 to 5 are the five expression constructs in Figure 2a, present as positive controls, hinge-less mAb 1 and 2 are two transfection replicates of the same plasmid.

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(A). A pictorial representation of the exon/intron arrangement in the heavy-chain expression cassette, the intron between the hinge and CH2 domain, is highlighted as particularly susceptible to mis-splicing. There are five introns present in the heavy chain: one between the signal peptide exon and the VH exon, one between the VH and CH1 exons, one between the CH1-hinge (HCCD-Intron1), one between hinge-CH2 (HCCD-Intron2), and one between CH2 and CH3 (HCCD-Intron3). (b). Bar graph showing terminal fed-batch titers represented as fold change compared to cDNA. There are significantly higher titers in gDNA and gDNA-opt-mut constructs compared to the equivalent cDNA construct. (c). Molecular mechanism of the variant transcript produced by mis-splicing due to a cryptic acceptor site in the coding region of the CH2 resulting in deletion of the first 54 nucleotides of the CH2 domain. Location of true (at the intron/exon junction) and cryptic (within the CH2 domain) splice acceptor sites are highlighted in the nucleotide sequence. The resulting post-splicing product with the translated in-frame shift sequence is shown. (d). Location of splice donor and acceptor sites are highlighted in the nucleotide sequence at the CH1-hinge-CH2 intron/exon junctions, and the resulting post-splicing hinge-less product is shown with the translated in-frame shift sequence below. (e). Non-reduced and reduced western blot analysis of heavy chain and light chain from transient expression supernatant samples. Represented as four blots, non-reduced on the left and reduced on the right, heavy-chain blots on the top panels and light-chain blots on the bottom panels. There is a clear accumulation of heavy-chain monomer and a distinct lack of fully formed antibody in the hinge-less lanes compared to wild-type controls.

Table 1. Heavy chain transgene engineering summary.

Heavy chain format	Splice variant transcript present?		% Transcript occurrence of splice variant		Description
	mAb-A	mAb-B	mAb-A	mAb-B
gDNA-opt	✓	✓	8.8	6.5	Original splice variant from Delmar et al, 2022^Citation10 (Fab+Ala)
cDNA-opt	×	×	NA	NA	No splice variants detected
gDNA-opt-mut	Not evaluated	✓	Not evaluated	1.1	Splice variant detected; 54 nucleotide deletion at 5’ of CH2
gDNA	Not evaluated	✓	Not evaluated	0.44	Splice variant detected; hinge-less transcript

Description of the plasmid engineering approaches taken to eliminate the Fab+Ala cryptic splice variant using expression plasmids depicted in Figure 1b, and the outcome of each approach. % transcript occurrence detected by RT-PCR using primers spanning the hinge-CH2 region followed by TOPO cloning and colony PCR of initially 96 colonies per format, a further 672 colonies were analyzed for gDNA format due to the detection of the hinge-less splice variant occurring at a frequency of less than 1%. PCR products were analyzed using Sanger sequencing. Opt = sequence optimized for expression in CHO, mut = triple mutation to lower in silico predicted score of cryptic splice acceptors.

Figure 2. Removal of individual introns in the HCCD does not affect titer. (a) Depiction of the different HCCD intron arrangements generated in a plasmid expressing mAb-B showing systematic removal of individual introns. Expression plasmids were evaluated using a stable CHO pool fed-batch process (b) Terminal fed-batch titers represented as fold change compared to gDNA (c) Average viable cell number (VCN) (x10⁶/mL) (d) Cell viability (%) (e) Integral of viable cells (IVC) (10⁹ cell hr/L) (f) Cell productivity (qP) (pg/(cell day)) represented as fold change compared to gDNA. All graphs show the mean ± SD, n = 3 in all cases, statistical analysis was performed using an unpaired t-test, ns= not significant, *= P < 0.05, **= P < 0.005.

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(A). Depiction of the different HCCD intron arrangements generated in a plasmid expressing mAb-B where individual introns have been removed. (b). Bar graph showing terminal fed-batch titers represented as a fold change compared to gDNA. The data show that there is no decrease in titer when individual introns are removed, but the cDNA construct has titers achieving approximately only 50% of the gDNA construct in comparison. (c). A line graph showing Viable Cell Number for all constructs is comparable over the 13-day fed-batch time-course, VCN peaks at day 9, and slowly decreases. (d). A line graph showing cell viability for all constructs is comparable over the 13-day fed-batch time-course, viabilities are close to 100% at day 3 and start to drop after day 9 to approximately 50% by day 11 and approximately 30% by day 13. (e). A line graph showing IVC is comparable for all constructs over the time-course reaching approximately 150x109 cell hr/L. (f). A bar graph showing specific productivity (qP) mirrors the titer results; there is no decrease in titer or qP when individual introns are removed but the cDNA construct has approximately 50% of the productivity in comparison.

Figure 3. The VH-CH1 intron is inhibitory to titer unless HCCD-Intron1 is present (a) Depiction of the different HCCD intron arrangements generated in a plasmid expressing mAb-B. Expression plasmids were evaluated using a stable CHO pool fed-batch process. (b) Terminal fed-batch titers represented as fold change compared to gDNA∆2. (c) Depiction of the different HCCD intron arrangements generated in plasmids expressing mAb-B. Expression plasmids were evaluated using a stable CHO pool fed-batch process (d) Terminal fed-batch titers represented as fold change compared to gDNA. The graphs show the mean + SD, n = 3 in all cases, statistics determined using an unpaired t-test, **= P < 0.005, ns=not significant.

(A). Depiction of the different HCCD intron arrangements generated in a plasmid expressing mAb-B where either intron2 and 3 have been removed simultaneously (cDNA+1) or intron1 and 2 have been removed simultaneously (cDNA+3), cDNA (containing no introns in the HCCD) and gDNA containing introns 1 and 3 but without intron2 (gDNA∆2) are present as controls. (b). A bar graph showing terminal fed-batch titers represented as a fold change compared to gDNA without intron2. The data show that there is no significant decrease in titer when introns 2 and 3 are removed simultaneously, but there is a significant decrease of approximately 50% when introns 1 and 2 are removed simultaneously, comparable to cDNA titers. (c). Depiction of the different HCCD intron arrangements generated in a plasmid expressing mAb-B including gDNA (containing all five introns), cDNA+1 (containing intron 1 only in the constant domain and the SP and VH-CH1 intron), cDNA (containing the SP and VH-CH1 intron only), HC+SP (containing the SP intron only), HCΔI (the HC containing no introns) and HC+1 (containing Intron1 only in the constant domain). (d). A bar graph showing terminal fed-batch titers represented as fold change compared to gDNA. The data show a significant increase in titer for HC+SP (the HC containing the signal peptide intron only) compared to cDNA (containing no introns in the constant domain) and no significant difference between gDNA (containing all five introns) or cDNA+1 (containing intron 1 only in the constant domain and the SP and VH-CH1 intron) and HCΔI (the HC containing no introns).

Figure 4. VH-CH1 intron is retained in the absence of HCCD-intron1 (a) Depiction of the different HCCD intron arrangements evaluated in plasmids expressing four different mAbs; mAb-A, mAb-B, mAb-C and mAb-D. Stable CHO pool were generated and evaluated using a fed-batch process. (b) Terminal fed-batch titers represented as fold change compared to gDNA for each molecule. The graphs show the mean + SD, n = 3 in all cases, statistical analysis was performed using an unpaired t-test, **= P < 0.005, ***=P < 0.005, ****=P < 0.0005, ns=not significant. Intron retention data from (c) is summarized beneath each construct tested, + = high level intron retention detection, - = little/no intron retention detected. (c) Evaluation of cDNA in a PCR based VH-CH1 intron retention assay using primers designed to anneal to each unique IgG variable HC and a common reverse primer in the CH1 constant region. PCR products were resolved and visualized on a 1% agarose gel. Molecular marker is Bioline HyperLadder 1kb. Table describes expected band sizes for spliced or unspliced species.

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(A). Depiction of the different HCCD intron arrangements evaluated in plasmids expressing four different mAbs: mAb-A, mAb-B, mAb-C, and mAb-D, each present in gDNA, cDNA+1, and cDNA formats. (b). Four bar graphs showing terminal fed-batch titers represented as fold change compared to gDNA for mAb-A, mAb-B, mAb-C, and mAb-D. For all four mAbs, there is no significant difference in the titers between gDNA and cDNA+1 but there is a significant reduction between the cDNA+1 and cDNA. Below the x-axis of each graph is a plus or minus symbol to represent VH-CH1 intron retention was present in the intron retention assay in C. (c). PCR gel images of the products from the intron retention assay. For all four mAbs, there are two bands present in all the cDNA lanes, one corresponding to a fully spliced product and another higher molecular weight band corresponding to the product with VH-CH1 intron retained. There is a single high-intensity band in gDNA and cDNA+1 lanes corresponding to the correct molecular weight for the fully spliced product, and there is also a faint band for the intron-retained product. It is likely that the faint bands present in the gDNA and cDNA+1 constructs correspond to pre-processed mRNA as a consequence of the total cellular cDNA synthesis method used, but clearly the intron-retained species is markedly increased in the cDNA construct. The table describes the expected band sizes for spliced or unspliced species.

Figure 5. HCCD-intron1 can alleviate VH-CH1 intron retention when moved within the HCCD (A) Depiction of the different HC intron arrangements used in plasmids expressing mAb-B. Expression plasmids were evaluated using a stable CHO pool fed-batch process. SM= sequence modified by 1-nucleotide to increase splice donor strength (b) Fed-batch titers represented as fold change compared to gDNA. The mean + SD is shown, n = 3 in all cases, statistical analysis was determined using an unpaired t-test, ns=not significant. Intron retention data from (c) is summarized beneath each construct tested, + = high level intron retention detection, - = little/no intron retention detected. (c) Evaluation of the expression plasmids in a VH-CH1 intron retention assay. Total RNA was extracted from the CHO pool cells, followed by cDNA synthesis. PCR was carried out with primers designed to anneal to each unique variable HC and a common reverse primer in the constant region. PCR products were resolved and visualized on a 1% agarose gel. Molecular marker is Bioline HyperLadder 1kb. Table describes expected band sizes for spliced or unspliced species.

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(A). Depiction of the different heavy-chain intron arrangements used in plasmids expressing mAb-B; gDNA (containing all five introns), cDNA+1 (containing the SP, VH-CH1, and intron 1 in the constant domain), cDNA+3 (containing the SP, VH-CH1, and intron 3 in the constant domain), cDNA (containing the SP, VH-CH1 only), cDNA+3in1 (containing the SP, VH-CH1 introns, and Intron3 replacing intron 1 between the CH1 and hinge within HCCD), cDNA+3in1-SM (as for cDNA+3in1 with sequence mutated), cDNA+1in3 (containing the SP, VH-CH1 introns, and Intron1 replacing intron 3 between the CH2 and CH3 within HCCD). (b). A bar graph showing fed-batch titers represented as fold change compared to gDNA. The data show that there is no significant difference between the cDNA+1in3 (Intron1 in position 3 within HCCD) and the gDNA or cDNA+1. All other arrangements have approximately 50% lower titers, comparable to cDNA. Below the x-axis of the graph is a plus or minus symbol to represent if intron retention was present in the intron retention assay in C. (c). PCR gel image of the products from the intron retention assay. For gDNA, cDNA+1, and cDNA+1in3 (Intron1 in position 3 within HCCD), there is a single band corresponding to the correct molecular weight for the fully spliced product. For cDNA+3, cDNA, cDNA+3in1 (Intron3 in position 1 within HCCD), cDNA+3in1-SM (sequence mutated), there are two bands, one corresponding to a fully spliced product and another higher molecular weight band corresponding to the product with intron retained. The table describes the expected band sizes for spliced or unspliced species.

Delmar JA, Harris C, Grassi L, Macapagal N, Wang J, Hatton D, Xu W. Monoclonal antibody sequence variants disguised as fragments: identification, characterization, and their removal by purification process optimization. J Pharm Sci. 2022;111(11):3009–16. doi:10.1016/j.xphs.2022.08.002.

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