Improving product quality and productivity of bispecific molecules through the application of continuous perfusion principles.

Bispecific protein scaffolds can be more complex than traditional monoclonal antibodies (MAbs) because two different sites/domains for epitope binding are needed. Because of this increased molecular complexity, bispecific molecules are difficult to express and can be more prone to physical and chemical degradation compared to MAbs, leading to higher levels of protein aggregates, clipped species, or modified residues in cell culture. In this study, we investigated cell culture performance for the production of three types of bispecific molecules developed at Amgen. In particular, we cultured a total of six CHO cell lines in both an approximately 12-day fed-batch process and an approximately 40-day high-density perfusion process. Harvested cell culture fluid from each process was purified and analyzed for product quality attributes including aggregate levels, clipped species, charge variants, individual amino acid modifications and host cell protein (HCP) content. Our studies showed that in average, the intensified perfusion process increased 15-fold the integrated viable cell density and the total harvested product (and fivefold the daily volumetric productivity) compared to fed-batch. Furthermore, bispecific product quality improved in perfusion culture (as analyzed in affinity-capture pools) with reduction in levels of aggregates (up to 72% decrease), clipped species (up to 75% decrease), acidic variants (up to 76% decrease), deamidated/isomerized species in complementarity-determining regions, and HCP (up to 84% decrease). In summary, the intensified perfusion process exhibited better productivity and product quality, highlighting the potential to use it as part of a continuous manufacturing process for bispecific scaffolds.

Culture temperature modulates half antibody and aggregate formation in a Chinese hamster ovary cell line expressing a bispecific antibody.

Therapeutic bispecific antibodies are formed by assembly of multichain polypeptides. In general, a bispecific antibody has two different light chains and two different heavy chains that fold and correctly pair via engineered interchain interactions. Because of some incorrect assembly, product-related impurities can be prevalent (e.g., half molecules, mispaired light chains, homodimers), requiring its removal during subsequent purification. In this study, we investigated the modulation of impurity levels in a stable Chinese hamster ovary cell line X expressing a bispecific antibody A formed by two light chains (LC1 and LC2) and two heavy chains (HC1 and HC2) that assembled intracellularly into a heterodimer (LC1-HC1 + LC2-HC2) via engineered charged residues. Cell line X exhibited the best volumetric productivity, growth, and viability in culture compared with other clones but also showed higher levels of half antibody species (>10%); therefore, to minimize process yield loss, better understanding, and control of impurity formation was pursued. We found this cell line decreased half antibody levels from 16% to 1% when temperature changed from 36°C to 32.5°C or 31.5°C. However, lower temperature also increased high-molecular-weight (HMW) species from 4% to 12%. To determine the impurity species composition, we characterized enriched fractions with half antibody or HMW. Intact mass spectrometry analysis revealed half antibody was LC2-HC2, whereas HMW was a mixture with ~50% as LC1-HC1 homodimer. Results suggested LC2-HC2 was easily folded and could be secreted as half antibody, especially at 36°C. On the contrary, LC1-HC1 was more susceptible to misfold or aggregate, a phenomenon more acute for cell line X at lower culture temperature because of 60% increased LC1 and HC1 messenger RNA levels. Although temperature modulation was cell line X-specific, the propensity of LC2-HC2 to form half antibodies and LC1-HC1 to aggregate appeared in other cell lines also expressing bispecific antibody A, suggesting an amino-acid sequence-dependent mechanism. In summary, impurity formation in cell line X was temperature-dependent and was influenced by different molecule characteristics between the LC1-HC1 and LC2-HC2 parts. Ultimately, we selected a biphasic cell culture process with a growth phase followed by a lower temperature phase to improve product quality and purification yield.

HspX vaccination and role in virulence in the guinea pig model of tuberculosis.

Mycobacterium tuberculosis (Mtb) currently infects billions of people; many of whom are latently infection and at risk for reactivation. Mycobacterium bovis Bacille Calmette-Guerin (BCG) while approved as a vaccine, is unable to prevent reactivation of latent tuberculosis infection (LTBI). Subunit vaccines boosting BCG or given alone are being tested for efficacy in LTBI models. Alpha-crystallin (Acr, HspX), is a latency associated protein and subunit vaccine candidate. In this report, three HspX formulas (native and two recombinant variants) were used as vaccines in the guinea pig model of tuberculosis; none were protective during challenge with WT Mtb. However, recombinant HspX was protective in animals challenged with a strain of Mtb lacking hspX (X4-19), indicating protection was driven by molecules co-purifying with HspX or an adjuvant effect of recombinant HspX in this system. Mtb X4-19 was significantly less virulent than WT Mtb. Quantitative PCR and whole genome sequencing identified several genes (Rv2030c-Rv2032, Rv1062, Rv1771, Rv1907, and Rv3479) with altered expression that may contribute to loss of virulence. Physiological differences required for the establishment of Mtb infection in different hosts may affect the potential of subunit vaccines to elicit protection, supporting the need for rigorous biochemical and modeling analyses when developing tuberculosis vaccines.

HspX-mediated protection against tuberculosis depends on its chaperoning of a mycobacterial molecule.

New approaches consisting of 'multistage' vaccines against (TB) are emerging that combine early antigenic proteins with latency-associated antigens. In this study, HspX was tested for its potential to elicit both short- and long-term protective immune responses. HspX is a logical component in vaccine strategies targeting protective immune responses against primary infection, as well as against reactivation of latent infection, because as previously shown, it is produced during latency, and as our studies show, it elicits protection within 30 days of infection. Recent studies have shown that the current TB vaccine, bacilli Calmette-Guerin (BCG), does not induce strong interferon-γ T-cell responses to latency-associated antigens like HspX, which may be in part why BCG fails to protect against reactivation disease. We therefore tested HspX protein alone as a prophylactic vaccine and as a boost to BCG vaccination, and found that HspX purified from M. tuberculosis cell lysates protected mice against aerosol challenge and improved the protective efficacy of BCG when used as a booster vaccine. Native HspX was highly immunogenic and protective, in a dose-dependent manner, in both short- and long-term infection models. Based on these promising findings, HspX was produced as a recombinant protein in E. coli, as this would enable facile purification; however, recombinant HspX (rHspX) alone consistently failed to protect against aerosol challenge. Incubation of rHspX with mycobacterial cell lysate and re-purification following incubation restored the capacity of the protein to confer protection. These data suggest the possibility that the native form may chaperone an immunogenic and protective antigen that is mycobacteria-specific.