Coronavirus disease in 2019 (COVID-19) has posed a continuous challenge to global public health since its outbreak [1]. To combat the pandemic, the global rollout of rapid diagnosis and vaccines is unprecedented [2]. In recent years, vaccine candidates employing various platforms have been developed, entered into clinical trials and authorized for use [[3], [4], [5], [6]]. However, virus variation has also led to a significant reduction in the potency of the commercial vaccines, especially forcirculating Omicron subvariants, resulting in continuous waves of infection among unvaccinated and vaccinated individuals [7,8]. Therefore, rapid-response development and manufacturing of effective vaccines against emerging variants have become urgent global priorities, especially in downstream processing. However, the variation, to a certain extent, has rendered the current downstream processing no longer suitable for the vaccine manufacturing of new variants. In this case, the refinement of existing operations and improvements in platform technology are always essential to ensure product quality and process compliance, leading to more mandatory process revalidation from the regulatory authority. In light of this dilemma, the advent of broad-spectrum affinity chromatography may provide a more powerful platform technology for vaccine manufacturing to respond rapidly to the emergence of new variants and address the unknown pandemic occurring in the future.

In recent decades, affinity chromatography has become the prevailing method of the choice for the purification of viral proteins and virus-like particles (VLPs) [[9], [10], [11], [12]]. The purification of adeno associated virus (AAV) vectors via AVB Sepharose gel is a paradigm of affinity chromatography for VLPs. In the mid-2000s, GE Healthcare, currently Cytiva, released AVB Sepharose as well as Capto AVB for the purification of AAV vectors via affinity ligands obtained from libraries of camelid single-chain antibody scaffolds [13]. Although AVB Sepharose can purify most AAV serotypes, it fails to purify other AAV serotypes, including AAV8, AAV9 and AAV11 [14,15]. Moreover, to prevent infection with Leishmania major promastigotes, an immunoreactive protein was identified by Webb et al. from the L. major amastigote cDNA expression library, and the recombinant protein containing an N-terminal six histidine (His) tag was purified by immobilized metal affinity chromatography (IMAC) for vaccination against leishmaniasis [16]. This strategy was also applied in the purification of the receptor binding domain (RBD) on COVID-19 spike (S) proteins containing a C-terminal eight His tag [17]. Navigo Proteins disclosed an artificial protein ligand for the purification of the RBD and TrimerS on the basis of Navigo’s proprietary Precision Capturing® technology [18]. Its use in the purification of inactivated COVID-19 vaccines was discouraged. Moreover, several high-affinity peptides targeting the wild-type (wt) RBD were identified from various peptide libraries generated via phage and mRNA display for the development of COVID-19 virus diagnosis [19,20]. In our group, several peptides and affibodies targeting the RBD were also screened via molecular docking and molecular dynamics (MD) simulations for the purification of the RBD and its variants [21,22]. Kadio et al. reported the use of sulfate pseudoaffinity chromatography for the purification of the COVID-19 virus from Vero cell culture fluid (VCCF), and the yield of the virus was 59 %, whereas that of commercial Cellufine Sulfate and Capto DeVirS chromatography was 11–17 % [23]. However, there are still few reports on the application of affinity chromatography for large-scale manufacturing of COVID-19 vaccines. On the other hand, continuously accumulated mutations in the RBDs of emerging variants pose a great challenge to the application of affinity chromatography, in which affinity ligands are designed to target the RBD [24]. To address virus variation, a set of dodecapeptides with broad-spectrum affinity was recently identified by Ma et al. via phage display screening in combination with next-generation sequencing (NGS) [25,26]. These peptide ligands included HWKAVNWLKPWT (L-HWK) [26] and HYRTSHWHHLLG (L-HYR) [25]. The results revealed that the affinity gels coupled with l-HWK or l-HYR performed well in the purification of wt-COVID-19 and Omicron variant vaccines without any operation optimization, and yields ranging from 86.2 to 90.2 % for the wt-COVID-19 vaccine and 87.6–93.0 % for the Omicron variant vaccine were obtained.

As a very appealing class of affinity ligands, peptides have been widely investigated in the purification of recombinant proteins, antibodies and VLP therapeutics [10,27,28]. In actual applications, however, naive peptides do not always meet the stability requirements for affinity purification because of degradation and short half-lives, especially in complex feedstocks such as VCCF and fermentation lysates, and need to be improved [[29], [30], [31]]. An important means to respond to this challenge is the substitution and cyclization of peptides and the incorporation of nonnatural amino acids, such as d-amino acids, 2-aminoisobutric acids, and β-amino acids [[31], [32], [33], [34], [35]]. d-amino acids are among the state-of-the-art approaches to confer proteolytic resistance due to their inability to interact with protease active sites [36]. However, the design and application of all d-amino acid containing peptides (all d-peptides) have substantial limitations. As reported previously, only 30 % of currently approved peptide drugs possessed a d-amino acid component and none were all d-peptides [37,38]. Recently, Juraszek et al. described a computational method for de novo design of all d-peptide ligands that target the hotspot residues on the hemagglutinin of influenza A [37]. On the other hand, Valiente et al. reported a set of novel all d-peptide inhibitors that target the human angiotensin-converting enzyme 2 (ACE2)-binding site by converting all l-amino acid-containing peptides to highly stable d-analogs [36,39]. This provides an important cue for the development of more stable peptide ligands for VLP purification.

In this work, we resynthesized l-HWK by substituting all the residues with their d-amino acid counterparts to prepare the d-enantiomer of l-HWK (D-HWK). The binding affinity and stability of d-HWK were evaluated via surface plasmon resonance (SPR) and proteolytic kinetics, and MD simulations were carried out to analyze the molecular mechanism of d-HWK binding with the RBD. By coupling d-HWK to agarose gels, the chromatographic performance of the as-prepared affinity gels was investigated to evaluate the operationality, applicability, broad-spectrum and stability of affinity chromatography in the purification of COVID-19 vaccines. These results demonstrated the great potential of all d-peptide ligands for the industrial manufacturing of wt-COVID-19 and Omicron vaccines.