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. 2025 Sep 18;13(9):980.
doi: 10.3390/vaccines13090980.

GMP Manufacturing and Characterization of the HIV Booster Immunogen HxB2.WT.Core-C4b for Germline Targeting Vaccine Strategies

Sammaiah Pallerla  1 , Latha Kallur Siddaramaiah  2 , Philipp Mundsperger  3 , Dietmar Katinger  3 , Katharina Fauland  3 , Günter Kreismayr  3 , Robert Weik  3 , Onur Arslan  3 , Mingchao Shen  2 , Gabriel Ozorowski  4 , Wen-Hsin Lee  4 , Andrew B Ward  4 , Sabyasachi Baboo  4 , Jolene K Diedrich  4 , John R Yates 3rd  4 , James C Paulson  5 , Tracy Blumen  1 , Daniel Craig  1 , Ryan Swoyer  1 , Maoli Yuan  1 , Leonidas Stamatatos  2
Affiliations

GMP Manufacturing and Characterization of the HIV Booster Immunogen HxB2.WT.Core-C4b for Germline Targeting Vaccine Strategies

Sammaiah Pallerla et al. Vaccines (Basel). .

Abstract

Background/objectives: Despite progress in antiretroviral therapy, HIV remains a major global health challenge with over one million new infections annually. An effective vaccine is urgently needed. Germline-targeting immunogens show promise in initiating broadly neutralizing antibody (bNAb) precursors. This study developed a scalable, cGMP-compliant process to manufacture the HIV vaccine booster immunogen HxB2.WT.Core-C4b, a nanoparticle designed to direct bNAb precursor maturation after priming.

Methods: A CHO cell platform was established through single-cell cloning from a high-producing stable pool. Upstream and downstream processes were optimized for scalability and yield. Three scales were tested 10 L, 40 L, and 400 L. Key parameters (pH, temperature, feeding, metabolite profiles) were systematically refined. Analytical characterization included glycosylation profiling, electron microscopy, and antigenicity testing. Viral clearance was evaluated per ICH Q5A guidelines.

Results: Optimization ensured consistent yields above 130 mg/L, with titers up to 250 mg/L. The selected clone (4E22) demonstrated strong growth, viability, and reproducibility. Glycan occupancy at 18 N-linked sites, including bNAb epitopes (N276, N332), was stable across scales. Over 70% of self-assembling nanoparticle were fully assembled at the GMP level. Antigenicity and purity met cGMP release criteria. Viral clearance achieved >13-log reduction for enveloped and >7-log for non-enveloped viruses.

Conclusions: This work establishes a robust, scalable platform for HIV nanoparticle immunogens. Consistent quality and yield across scales support clinical development of HxB2.WT.Core-C4b and provide a model for other glycosylated nanoparticle vaccines. The immunogen is being evaluated in clinical study HVTN 320 (NCT06796686), enabling early testing of next-generation vaccines designed to elicit broadly neutralizing antibodies.

Keywords: GMP; HIV vaccines; clinical trial material.

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Conflict of interest statement

Author Philipp Mundsperger, Dietmar Katinger, Katharina Fauland, Günter Kreismayr, Robert Weik, Onur Arslan, was employed by the company Polymun Scientific Immunbiologische Forschung GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Schematic overview of the cGMP-compliant manufacturing process for HxB2.WT.Core-C4b immunogen production.
Figure 2
Figure 2
Fed batch performance of the top three subclones 4E22, 9K21, and 5C9, with 4E22 cultivated in three modes: 3 L fill-up simulation (F988), 8 L standard fed-batch (F986), and 8 L Glc-enriched feed (F987). (a) Titer, (b) pH levels, (c) lactate concentration, (d) glucose levels, and (e) ammonia levels.
Figure 2
Figure 2
Fed batch performance of the top three subclones 4E22, 9K21, and 5C9, with 4E22 cultivated in three modes: 3 L fill-up simulation (F988), 8 L standard fed-batch (F986), and 8 L Glc-enriched feed (F987). (a) Titer, (b) pH levels, (c) lactate concentration, (d) glucose levels, and (e) ammonia levels.
Figure 2
Figure 2
Fed batch performance of the top three subclones 4E22, 9K21, and 5C9, with 4E22 cultivated in three modes: 3 L fill-up simulation (F988), 8 L standard fed-batch (F986), and 8 L Glc-enriched feed (F987). (a) Titer, (b) pH levels, (c) lactate concentration, (d) glucose levels, and (e) ammonia levels.
Figure 3
Figure 3
Fed batch performance of the top clone MCBCHO/HXB2C/4E22/MCB cell line at small scale. (a) Product concentration, (b) pH level, (c) lactate levels, and (d) glucose. F994 was cultivated for an extended process with harvests at 11 and 14 days.
Figure 3
Figure 3
Fed batch performance of the top clone MCBCHO/HXB2C/4E22/MCB cell line at small scale. (a) Product concentration, (b) pH level, (c) lactate levels, and (d) glucose. F994 was cultivated for an extended process with harvests at 11 and 14 days.
Figure 4
Figure 4
Process performance for confirmation runs (10 L) and full-scale (400 L) production.
Figure 5
Figure 5
Yield overview (step yields and overall process yields) of the cGMP HxB2.WT.Core-C4b downstream purification sequence.
Figure 6
Figure 6
Site-specific N-glycosylation analysis. Bar graphs representing proportion of glycosylation and N-glycan types at each of the 18 N-glycosylation sites on the analyzed immunogen (HxB2.WT.Core-C4b) and its clones expressed in CHO cells. Color code represents proportion of glycosylation state: gray (unoccupancy), magenta (complex glycans), and green (high mannose or hybrid glycans). Error bars represent negative standard error of mean (SEM).
Figure 7
Figure 7
EM images of the material from 10 L and 400 L scale production batches (Clone CL-4E22).
Figure 8
Figure 8
BLI traces of top five clones CL-5C9, CL-3K21, CL-9K21, CL-3N15, CL-4E22, and L20 BDS binding to mVRC01, P1B5, and 179NC75 mAbs.
Figure 8
Figure 8
BLI traces of top five clones CL-5C9, CL-3K21, CL-9K21, CL-3N15, CL-4E22, and L20 BDS binding to mVRC01, P1B5, and 179NC75 mAbs.
Figure 9
Figure 9
BLI binding affinity (KD) comparison of top clones and L20 BDS against mVRC01, P1B5, and 179NC75 mAbs (*: p ≤ 0.05).
Figure 9
Figure 9
BLI binding affinity (KD) comparison of top clones and L20 BDS against mVRC01, P1B5, and 179NC75 mAbs (*: p ≤ 0.05).
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